Split (1)

train · 133k rows

text stringlengths 0 868k
A 47-year - old female presented to our institution with diminished vision on the left side and red eye for 2 months . Her past history was not significant, except for partial penetrating keratoplasty of the left eye 18 months prior to treat a corneal ulcer . She experienced one episode of graft rejection 12 months prior to presentation, and since then had been using eyedrops containing levofloxacin (cravit; taejoon pharm, seoul, korea) and 1% rimexolone (vexol; alcon laby inc . After the rejection, a 22 mm, round - shaped corneal haziness associated with mild superficial infiltration had been present at the suture site in the 2 o / c area . We thought that this was a suture - related infection, so we removed the suture 6 months prior to presentation . After removal of the suture, the corneal haziness improved . In order to prevent additional rejection, we recommended the use of eyedrops containing levofloxacin and 1% rimexolone, and artificial tears 4 times a day . When the patient visited us with the above symptoms, eyelid examination revealed a greasy appearance and meibomian gland plugs that were determined to be posterior blepharitis on the left upper and lower eyelids . On slit lamp examination, a 43 mm, round - shaped corneal haziness and epithelial defect associated with mild superficial stromal infiltration was found at the junction between the graft and donor site at the 2 to 3 o / c area . The corneal ulcer had progressed slowly and seemed to be chronic, so we performed a corneal gram stain and culture . We recommended warm massage and scrubbing of the upper lid and prescribed a lid cleanser (blephasol; samil pharm, seoul, korea) in an effort to improve the eyelid hygiene . We also recommended use of eyedrops containing 5% moxifloxacin (a 4th generation fluoroquinolone) every 2 hours and 0.15% amphotericin 4 times a day, because we were unsure whether the infection was fungal or bacterial . We also recommended reducing the use of vexol, a steroid eyedrop she had been using, to once a day . There was no improvement after 4 weeks with this treatment . Eventually, the corneal culture grew mrsa (ofloxacin resistance was also shown). According to the antibiotic sensitivity testing, we prescribed fortified 2.5% vancomycin - containing eyedrops 6 times a day . After 4 weeks, the corneal ulcer decreased to 23 mm in size, and we recommended reducing the use of the 2.5% vancomycin - containing eyedrops to 4 times a day . After 8 weeks, the corneal epithelium was healed completely and the corneal thickness returned to normal (581 m) (fig . 2). A 62-year - old man presented with red eye, ocular pain, and eyewax on his right eye . He had a history of brain surgery to treat a brain tumor 1 year prior to presentation . After the surgery, a right - sided facial nerve palsy was noted, which reduced his blinking reflex . Postoperative exposure keratitis developed, and he began to use eyedrops containing ofloxacin (ocuflox; samil pharm) and artificial tears 4 times a day . On examination of the eyelids, a greasy appearance and meibomian gland plugs, which resembled thick toothpaste upon compressing the upper lid, were found, and posterior blepharitis involving the right upper and lower eyelids was diagnosed . On slit lamp examination, mild ciliary injection at the conjunctiva, an 88 mm round - shaped corneal haziness and epithelial defect associated with mild superficial stromal infiltration, and a 1.5 mm high anterior chamber hypopyon we recommended warm massage and scrubbing the upper lid with a lid cleanser (blephasol) in order to improve the eyelid hygiene . We also recommended the use of eyedrops containing 5% moxifloxacin every 2 hours, alternating with 5% ceftazidime (a 3rd generation cephalosporin) every 2 hours . There was no improvement in the corneal ulcer or the hypopyon after 4 days of treatment . After this, as we believed the ulcer was neurotrophic and we decided to reduce the frequency of eyedrops to 6 times a day . Mrsa was eventually detected in the initial corneal culture, which took 1 week to grow (resistance to ofloxacin was also shown) (fig . 4). Based on the antibiotic sensitivity testing, we prescribed fortified 2.5% vancomycin - containing eyedrops 6 times a day . After 5 days, the corneal ulcer size, superficial stromal infiltration, and hypopyon all decreased (fig . We then recommended a reduction in the use of the eyedrops containing 2.5% vancomycin to 4 times a day . By the 2 week follow - up visit a 47-year - old female presented to our institution with diminished vision on the left side and red eye for 2 months . Her past history was not significant, except for partial penetrating keratoplasty of the left eye 18 months prior to treat a corneal ulcer . She experienced one episode of graft rejection 12 months prior to presentation, and since then had been using eyedrops containing levofloxacin (cravit; taejoon pharm, seoul, korea) and 1% rimexolone (vexol; alcon laby inc . After the rejection, a 22 mm, round - shaped corneal haziness associated with mild superficial infiltration had been present at the suture site in the 2 o / c area . We thought that this was a suture - related infection, so we removed the suture 6 months prior to presentation . After removal of the suture, the corneal haziness improved . In order to prevent additional rejection, we recommended the use of eyedrops containing levofloxacin and 1% rimexolone, and artificial tears 4 times a day . When the patient visited us with the above symptoms, eyelid examination revealed a greasy appearance and meibomian gland plugs that were determined to be posterior blepharitis on the left upper and lower eyelids . On slit lamp examination, a 43 mm, round - shaped corneal haziness and epithelial defect associated with mild superficial stromal infiltration was found at the junction between the graft and donor site at the 2 to 3 o / c area . The corneal ulcer had progressed slowly and seemed to be chronic, so we performed a corneal gram stain and culture . We recommended warm massage and scrubbing of the upper lid and prescribed a lid cleanser (blephasol; samil pharm, seoul, korea) in an effort to improve the eyelid hygiene . We also recommended use of eyedrops containing 5% moxifloxacin (a 4th generation fluoroquinolone) every 2 hours and 0.15% amphotericin 4 times a day, because we were unsure whether the infection was fungal or bacterial . We also recommended reducing the use of vexol, a steroid eyedrop she had been using, to once a day . There was no improvement after 4 weeks with this treatment . Eventually, the corneal culture grew mrsa (ofloxacin resistance was also shown). According to the antibiotic sensitivity testing, we prescribed fortified 2.5% vancomycin - containing eyedrops 6 times a day . After 4 weeks, the corneal ulcer decreased to 23 mm in size, and we recommended reducing the use of the 2.5% vancomycin - containing eyedrops to 4 times a day . After 8 weeks, the corneal epithelium was healed completely and the corneal thickness returned to normal (581 m) (fig . A 62-year - old man presented with red eye, ocular pain, and eyewax on his right eye . He had a history of brain surgery to treat a brain tumor 1 year prior to presentation . After the surgery, a right - sided facial nerve palsy was noted, which reduced his blinking reflex . Postoperative exposure keratitis developed, and he began to use eyedrops containing ofloxacin (ocuflox; samil pharm) and artificial tears 4 times a day . On examination of the eyelids, a greasy appearance and meibomian gland plugs, which resembled thick toothpaste upon compressing the upper lid, were found, and posterior blepharitis involving the right upper and lower eyelids was diagnosed . On slit lamp examination, mild ciliary injection at the conjunctiva, an 88 mm round - shaped corneal haziness and epithelial defect associated with mild superficial stromal infiltration, and a 1.5 mm high anterior chamber hypopyon were found (fig . We recommended warm massage and scrubbing the upper lid with a lid cleanser (blephasol) in order to improve the eyelid hygiene . We also recommended the use of eyedrops containing 5% moxifloxacin every 2 hours, alternating with 5% ceftazidime (a 3rd generation cephalosporin) every 2 hours . There was no improvement in the corneal ulcer or the hypopyon after 4 days of treatment . After this, as we believed the ulcer was neurotrophic and we decided to reduce the frequency of eyedrops to 6 times a day . Mrsa was eventually detected in the initial corneal culture, which took 1 week to grow (resistance to ofloxacin was also shown) (fig . 4). Based on the antibiotic sensitivity testing, we prescribed fortified 2.5% vancomycin - containing eyedrops 6 times a day . After 5 days, the corneal ulcer size, superficial stromal infiltration, and hypopyon all decreased (fig . We then recommended a reduction in the use of the eyedrops containing 2.5% vancomycin to 4 times a day . By the 2 week follow - up visit the prevalence of mrsa differs depending on the group which is being studied; for example, the prevalence of mrsa was reported to be 0.1% in geneva's hospital, 2.8% in a san francisco nosocomial community in 2000, and 6.8% in a homeless group in san francisco in 2003 . The prevalence of mrsa infection is quite different among societies, but it is almost universally increasing . Mrsa infections have many different appearances in different sites, and also show multiple resistances to antibiotics making treatment quite difficult . Infections in immunocompromised patients can cause severe life - threatening problems, such as necrotizing cellulitis, pneumonia, and sepsis . In hospital infections, mrsa can be transmitted from patient - to - patient, via clinical materials, physician's hands, and hospital instruments . Ocular mrsa infections manifest as blepharitis, conjunctivitis, keratitis, dacryocystitis, endophthalmitis, and orbital cellulitis . Generally, mrsa ocular conditions do not respond to commercially - used eyedrops, which can cause a delay in treatment and can lead to severe complications, including permanent loss of vision [21 - 26]. According to the study by sotozono et al . The first step is the asymptomatic carrier state or the patient who shows only conjunctivitis . The second step is the patient who shows a corneal epithelial defect with superficial infiltration . The third step is the patient who shows superficial keratitis with a corneal ulcer which progresses slowly and causes corneal stromal infiltration . Thus, mrsa corneal ulcers have many manifestations, and can be treated with vancomycin - containing eyedrops or ointment according to each step, while intravenous vancomycin can be used in severe cases . According to kato and hayasaka, mrsa exists as normal flora in the conjunctiva in 1.3% of the population . High - risk factors for ocular mrsa infection are known to be previous long - term use of antibiotic - containing eyedrops, long - term use of steroids, long - term hospitalization, systemic disease (respiratory failure, diabetes, cancer, and hepatic failure), ocular immunocompromised states (dry eyes, ocular surface diseases, and stevens - johnson syndrome), long - term use of contact lenses, and previous ocular surgery (partial penetrating keratoplasty and refractive surgery). Patients who have had a keratoplasty, as in case 1, have multiple risk factors . Not only do they have a history of previous ocular surgery, but they also likely have a history of long - term use of antibiotic- and steroid - containing eyedrops to prevent rejection and infection . Donor rejection must be excluded when corneal irritation symptoms develop, and consequently proper treatment can be delayed . The clinical findings of the cornea in rejection are quite different when compared to bacterial keratitis, including round superficial stromal infiltration and chronicity . The use of steroid or antifungal eyedrops can aggravate the corneal ulcer and cause corneal toxicity, which can lead to severe complications, including corneal perforation . Therefore, if a patient has a slowly progressive chronic corneal ulcer and has risk factors for a mrsa infection, they should be examined and a mrsa corneal ulcer should be suspected . Corneal culture and antibiotic sensitivity testing must be performed to establish the exact diagnosis . In patients who have been hospitalized for a long time, as in case 2, it is important to consider that mrsa could be a nosocomial infection . Nosocomial mrsa infections can cause worsening of underlying diseases, and the prevention of nosocomial mrsa spread is becoming increasingly important . Most transmission of mrsa in hospital inpatients is thought to be via transiently - colonized healthcare workers . Found that 73% of hospital rooms with patients infected with mrsa and 69% of rooms with patients colonized with mrsa have some environmental contamination . In the same study, nurses contaminated their gloves 42% of the time in rooms with mrsa patients despite only touching environmental contamination . Other potential environmental reservoirs include computer keyboards used by clinicians, blood pressure cuffs, showers, and bathtubs . . Found that mrsa can survive on the external surfaces of sterile goods packaged for over 38 weeks . Therefore, hand hygiene may be the single most important measure for controlling transmission of mrsa . For that reason, in mrsa patients confirmed by bacterial culture and antibiotic sensitivity testing, isolation and sterilization of the patient's medical materials and handwashing by healthcare workers are of the utmost importance . Although mrsa keratoconjunctivitis can be treated with vancomycin - containing eyedrops or ointments, mrsa infections are of great concern to ophthalmologists because these ocular infections, including blepharitis, conjunctivitis, keratitis, or endophthalmitis, respond poorly to conventional antibiotic treatments and are sometimes sight - threatening . Consequently they have to be prepared directly in the hospital, which means they can be easily contaminated, may have high toxicity to the corneal epithelium, and may have unstable acidity which makes long - term storage difficult . Unfortunately, with the high rate that bacteria are now developing antibiotic resistance, we expect that vancomycin resistant staphylococcus will also become a problem . We should therefore choose antibiotics carefully using the results of bacterial culture and antibiotic sensitivity testing . In corneal ulcers with a round - shaped, mild superficial infiltration that have a slow progression and in lesions that do not improve with -lactam antibiotics, mrsa infection should be considered, especially in high - risk groups . These high - risk groups include patients who have had penetrating keratoplasty and exposure keratitis . In such cases, bacterial culture and antibiotic sensitivity testing must be performed and treatment decisions should be based on the results of these tests . Also, healthcare workers should practice serial hand washing and careful isolation of medical materials from known mrsa patients in order to prevent transmission of mrsa . The prevalence and transmission of mrsa in the community are increasing and preventing mrsa infection is becoming an increasingly important consideration.
Gastric cancer is the second most frequent cause of cancer - related death in the world and almost two - thirds of the cases occur in asian countries, especially china and japan . Although current major therapies, including surgery and chemotherapy, have been widely used, the prognosis of gastric cancer is generally rather poor, with 5-year relative survival below 30% in most countries . However, the main cause of treatment failure in gastric cancer is the development of multidrug resistance to cytotoxic chemotherapies, which is at least in part related to the anti - apoptosis effect . It is well known that the resistance of cell death is one of the hallmarks of cancer cells . Although the relationship between autophagy and cancer is still unclear, especially in the regulation of cancer development and progression, there has been much important progress in our understanding that autophagy may have important roles in the treatment of gastric cancer cells . Autophagy is an evolutionarily conserved catabolic process by which damaged or long - lived cellular proteins and organelles are degraded . In the cancer cells it is still unclear if autophagy represents a survival mechanism or is involved in type ii programmed cell death (pcd), which is termed autophagic cell death . Autophagy is up - regulated when cells need to generate intracellular nutrients and energy, for example, during starvation, growth factor withdrawal, or high bioenergetic demands . Moreover, basal autophagy can serve as an important homeostatic cellular recycling mechanism responsible for degrading unnecessary or dysfunctional cellular organelles and proteins in all living cells; thus, autophagy can be viewed as a potent cytoprotective survival pathway in normal and cancer cells . Recently, accumulating evidence has indicated that autophagy is particularly activated during metabolic stress such as nutrient depletion and hypoxia, and has a special homeostatic role mediating removal of old or dysfunctional proteins and organelles, and is particularly important for cell survival during conditions of metabolic stress . Moreover, autophagy is the only mechanism for degrading large structures such as protein aggregates and damaged organelles . Although autophagy has been induced in many different cancer cell lines, including gastric cancer cells using different agents such as chemotherapeutics, the role of autophagy in tumor cell death or survival is still unclear . Autophagy has several adaptive roles in diverse human pathologies, including cancer and other diseases, and can act as a cytoprotective survival pathway . Thus, under many conditions, autophagy is induced in response to many different forms of stress, including nutrient and growth factor deprivation and chemotherapeutics, and is utilized as a protective mechanism against cell death in the hostile environment . In fact, more and more results have suggested that autophagy can provide a survival advantage to tumors treated with chemotherapeutic agents . In human hepatocellular carcinoma and colorectal carcinoma cells, moreover, inhibition of autophagy by 3-methyladenine (3-ma) can potentiate cisplatin - induced apoptosis in esophageal squamous cell carcinoma cells . In addition, many studies have revealed that inhibition of autophagy can augment cancer cell death through apoptosis, which indicates that autophagy may act as a protector in tumor cell survival . Furthermore, other studies have proved that induction of autophagy can enhance tumor resistance in different tumor cell lines . Hypoxia - induced autophagy can decrease hepatoma cell sensitization to chemotherapeutic agents that affect their apoptotic potential . In gastric cancer cells thus, results of many studies suggest that induction of autophagy can protect tumor cells against cancer treatment . Previous studies have shown that inhibition of autophagy can potentiate the cell death induced by anticancer drugs in gastric cancer cells [1921]. Therefore, we speculate that inhibition of autophagy may be a strategy for overcoming gastric cancer resistance to therapy . In recent years, the role of autophagy in cancer cells has been investigated extensively . Although the impact of autophagy on tumor cells is still unclear and more studies are needed to prove the effects of autophagy in gastric cancer, recent research results suggest that autophagy may be a new target for combatting resistance of gastric cancer to therapy.
In india, transmission of blood - borne viruses in unsafe health care is endemic. (1) poor sharps waste management and misconceptions about injection safety both contribute to injection equipment reuse in india . Recent outbreak investigations suggest that many injection providers believe it is safe to reuse a syringe after changing the needle, that it is safe to reuse injection equipment to access an iv line, or that it is safe to reuse injection equipment on the same patient when reconstituting from a multidose vial, without sterilization. (2) these misconceptions are still reported in high - income developed countries and are thought to be prevalent in the developing world. (3) even more troubling, recycling of sharps waste for repackaging and resale is practiced on a large scale in india, uncovered recently in the investigation of a deadly hepatitis b outbreak. (4) the goal of this review is to evaluate the incremental cost - benefit of using the auto - disable syringe for all medical injections in india, in terms of prevented disability and mortality from hepatitis b, hepatitis c, and hiv infections . India's ministry of health issued an advisory to 24 governors and state health ministers to introduce auto - disable syringes for all medical injections in 2008 to prevent injection equipment reuse in health care settings . Most states have not implemented this policy for curative injections, although auto - disable syringes are now used nationwide for immunization injections . Most injections in india are not immunization injections, as injectable drugs are widely preferred over oral formulations. (5) the benefit of using only auto - disable syringes in a country with a low prevalence of blood - borne viruses has not been previously assessed . The total number of human immunodeficiency virus (hiv), hepatitis b virus (hbv) and hepatitis c virus (hcv) infections that would result from unsafe medical injections in india in 2010, barring the introduction of auto - disable syringes for all medical injections, is estimated following the mass action model used to develop the world health organization global burden of disease estimates. (6) the incidence of each blood - borne virus iv is a product of the size of the susceptible population ps, the probability of transmission in an unsafe medical injection for each virus ptv, and the number of contaminated medical injections performed nc, as given in equation (1). Based on a systematic review of studies of needlestick accidents in health workers, the who estimates that the probability of hiv transmission in an unsafe medical injection is 1.2%, the probability of hbv transmission is 6% for hbeag - negative source patients and 30% for hbeag - positive patients, and the probability of hcv transmission is 1.8%. (6) i = psv ptv nc (1) the number of contaminated injections nc is calculated from the unsafe reuse rate pr, the prevalence of each virus in the general population pv (or the adjusted probability that used injection equipment will be contaminated with the virus), and the number of injections per person - year n, as given in equation (2). Nc = pr pv n (2) because india's hiv epidemic is concentrated, the model for hiv is adjusted for associations between unsafe injection frequency and hiv prevalence across geographic and demographic segments of the population . The 2004 inclen injection safety report evaluated unsafe injection frequency in states and territories with varying hiv prevalence, and the national aids control organization of india reports hiv prevalence by state and territory. (78) the 20052006 demographic and health survey for india is used to relate unsterile injection frequency to hiv prevalence across age groups, gender and marital status, ethnic and religious groups, rural or urban residence, wealth and education level . Risk factor analyses investigating an association between injections and hepatitis b, hepatitis c and incident hiv infections in india were identified in a systematic search using pub med . The population attributable fractions from these risk factor analyses are used to validate the model . The incremental cost per disability adjusted life year (daly) from each prevented hepatitis b, hepatitis c and hiv infection is calculated in a cost benefit assessment of the use of the auto - disable syringe, priced at $0.0425 in india . In a nationwide survey in 2004, the estimated injection frequency varied by recall interval from 2.9 to 5.8 injections per person - year, of which 32% had the potential to transmit blood - borne viruses. (7) the demographic and health survey for india indicates that 0.71% of unsafe injections given to adults were potentially contaminated with blood from an hiv - positive patient, before adjusting for associations between unsafe injection frequency and hiv prevalence . Hiv prevalence in children under age 15 (29% of the population) is only 0.00028%. (9) in ecological regression, rural unsterile injection frequency is associated with hiv prevalence (p = 0.036, r = 0.30, = 1.4). The selected demographic predictors of hiv prevalence are also associated with unsafe injection frequency in adults (p = 0.010, r = 0.23, = 0.32). Equation (3) reports the adjustments for the probability of an unsafe injection given to a susceptible patient being contaminated with hiv, where 1 is the adjustment factor for geographic region, 2 is the adjustment factor for demographic group, pa is the probability the injection is being given in an adult patient care setting and pc is the probability that the injection is being given to a child . The estimated probability that an unsafe injection given to an hiv - negative patient will be contaminated with blood from an hiv - positive patient is 0.85% . Pv = 0.71 (1 + 2) pa + 0.00028 (1 + 2) pc (3) three studies of sexually transmitted disease (std) patients in india investigated an association between medical injections and incident hiv infections, with a median population attributable fraction of 12% . The modeled annual incidence of hiv from unsafe medical injections is 1021 cases per 100,000 . In south india, the adult incidence of hiv was 50 cases per 100,000 in 2008. (10) thus, this mass action model attributes 2042% of hiv transmission in india to unsafe medical injections, a far larger fraction than that supported by risk factor analyses in patients with competing risks . Hiv incidence in these std clinic attendees was far higher (5.18.2 per 100 person - years at risk) than the population average, suggesting that competing risks were greater in study participants than in the general population . The median population attributable fraction for chronic hepatitis b across five risk factor analyses to look at injection risks is 46% . This discrepancy suggests that either the modeled transmission efficiency of hepatitis b infections is a conservative estimate or the populations in which injections have been investigated as a risk factor for hepatitis b infection are at increased risk for unsafe injections . Population attributable fraction of hepatitis infections linked to injections in india the median fraction of hepatitis c infections attributed to injections across five risk factor analyses, also shown in table 1, is 38% . The average annual incidence suggested by the age distribution of hepatitis c infection in india is 0.1%. (11) for hepatitis c, the modeled incidence is 0.030.05%, or 3050% of prevalent infections . Following the age - adjusted global burden of disease model for south asia, these estimates predict that 352,000646,000 dalys from hepatitis b infection, 48,70081,200 dalys from hepatitis c infection and 2,605,0005,210,000 dalys from hiv infection would result which can be prevented by the use of the auto - disable syringe in 2010 . Estimating hiv, hbv and hcv incidence from the median population attributable fractions in epidemiological investigations instead, the incremental cost may be higher or lower at $3979 per daly . Unsafe medical injections carry a risk of blood - borne virus transmission (hiv, hbv, hcv) when the injection equipment is reused without sterilization. (22) this has been shown in case control studies indicating an association between blood - borne virus infection and receipt of medical injections,(2325) with many of these studies demonstrating a dose response relationship(2628) corroborating evidence is available from blood - borne virus epidemics in populations of injection drug users(2931) and from the outcomes of accidental needlesticks in health workers. (3234) phylogenetic analysis of outbreak strains of nosocomial hiv, hbv and hcv have further confirmed the biological plausibility of unsafe medical injections as a vehicle for transmission. (3537) continuing unsafe injection practices in india have been well documented and remain a serious threat to public health. (53839) the present model of blood - borne virus transmission in india through medical injection equipment reuse has several important limitations . Risk factor analyses investigating medical injection risks in india have not controlled for the full range of confounding variables thought to influence the association between medical injections and hiv, also likely to influence the association with hbv . Model - based cost - benefit assessments of the introduction of auto - disable syringes for all medical injections are as uncertain as the model parameters, and the modeled patient mixing patterns only indirectly capture differences in patient populations at facilities with high and low levels of injection equipment reuse . Taken together, however, model - based and empirical evidence of iatrogenic hiv, hbv and hcv transmission in india supports the introduction of auto - disable syringes as a cost - effective intervention . The estimated cost per daly averted is a fraction of average earned annual income in india by either estimation method, indicating that the introduction of auto - disable syringes for all medical injections is a cost - effective national policy for a low - income country with low prevalence of blood - borne viruses . This is an improvement from the incremental cost per daly averted estimated for south asia in 2003. (40) this review indicates that unsafe injections are associated with hiv at the individual level and at the population level in india . Unsterile injections may serve as a bridge for hiv transmission between high - risk groups and the general population, as has been documented on a tragic scale in the town of jalal pur in pakistan. (41) among patients at std clinics, cyclic hiv transmission dynamics are possible, and high rates of hepatitis c infection in female sex workers are suggestive of a role for blood exposures in concentrated hiv epidemics. (42) nevertheless, given the mismatch between modeled and epidemiological estimates of hiv transmission from unsafe medical injections, the true extent of iatrogenic hiv transmission remains unclear . The most uncertain parameter in the model of blood - borne virus transmission is the transmission efficiency of each virus in a medical injection . The probability of hiv, hbv or hcv transmission in a contaminated medical injection may be similar to the risk from a needlestick accident . However, descriptive studies of needlestick accidents are few and the ranges of transmission efficiency estimates across these studies are wide . Recently, it has been argued that rinsing injection equipment eliminates the blood - borne virus transmission risk. (43) however, a comparison of the inoculum volume in a needlestick injury (in which the needle is inserted but the plunger of the syringe is not depressed) and in an injection shows that after adjustment for both rinsing the syringe and injecting the remaining contents of the syringe, the transmission efficiency in an unsafe injection is probably of the same order of magnitude as the transmission efficiency in a needlestick accident. (44) some other model parameter estimates are less uncertain . The 95% confidence intervals around the injection frequency estimates on 2 weeks and 3 months recall intervals were 5.36.3 injections and 2.83.2 injections per person per year. (7) the 95% confidence interval around the proportion of medical injections with the potential to transmit blood - borne viruses was 2934%. (7) on the other hand, the adjustment for patient mixing patterns accounting for the association between hiv prevalence and exposure to unsafe medical injections was not empiric in the sense that demographic groups do not necessarily mix assortatively in clinical settings . Moreover, the adjustments did not account for the population distribution of the demographic characteristics that predict unsafe injection frequency and hiv prevalence . The average registered medical practitioner reuses disposable syringes three times without sterilization, but this model assumes that blood - borne viruses are only transmitted from the first patient to the second. (1) multidose vial contamination when an injection needle is used to reconstitute medication is also prevented by the introduction of auto - disable syringes, but this has not been modeled . In these respects, the model does not account for deaths from septicemia, a common complication of unsterile injecting among injection drug users that results from bacterial contamination. (45) septicemia can result from injection equipment reuse irrespective of whether injection equipment has been used on a patient previously infected . In hospitalized patients with a bacterial culture positive intravenous catheter, 61% had a concomitant bacterial infection. (46) injection equipment prepared for reuse by boiling is frequently culture positive (e.g., 33% contamination rate in one assay in tanzania), and by implication injection equipment reused without sterilization is likely to be infectious. (47) in countries with a low prevalence of blood - borne viruses, the primary benefit of preventing injection equipment reuse may be the prevention of potentially serious bacterial infections . Today, auto - disable syringes are required for all medical injections in india, burkina faso, the democratic republic of congo, nigeria, tanzania, and uganda, and the importation of syringes that are not auto - disable syringes is restricted in burkina faso and tanzania . These national injection safety policies address a patient safety crisis recognized even in high - income developed countries the widespread practice of injection equipment reuse without sterilization. (4849) the who recommends the use of auto - disable syringes for all immunization injections, and united nations children's fund (unicef) recommends that auto - disable syringes also be used for reconstitution . More extensive use of auto - disable syringes may be needed in countries with generalized hiv epidemics to prevent these nosocomial infections . In india, implementation of this policy still faces serious obstacles, as state ministries of health have not embraced the national government's advisory and the new minister of health has not pressed the issue . In kerala, karnataka and madhya pradesh, the policy has been opposed, although 2547% of injections in government health facilities in these states carry a risk of blood - borne virus transmission from patient to patient . Only eight states and the central government owned hospitals have partially introduced auto - disable syringes for curative injections . Hiv surveillance in india does not investigate iatrogenic risks, and awareness of medical injections as an important secondary hiv transmission route is not widespread. (50) as other developing countries move toward a zero - tolerance policy for iatrogenic hiv transmission, medical injection equipment reuse continues to contribute to widespread undetected blood - borne virus transmission in india . Implementation of the national advisory to use auto - disable syringes for all medical injections could radically improve patient safety and reduce the burden of disease from chronic and stigmatizing infections in the most vulnerable segments of society.
Nearly 233,000 new cases were estimated to be diagnosed in 2014 in the united states, representing 14% of all new cancer cases . Although metastatic bc (mbc) is diagnosed in only 5% of cases, nearly 30% of bc patients with earlier stage tumors eventually develop metastases . This advanced disease is associated with worse prognosis than early stage bc, with 5-year survival rates around 25% . Most bc samples overexpress hormone receptors (hr), including estrogen receptor (er) and/or progesterone receptor (pr) [4, 5], whereas human epidermal growth factor receptor 2 (her2) overexpression only occurs in 2030% of cases; thus, the most common bc subtype is hr+/her2 . Postmenopausal women, in particular, are more likely to have hr+/her2 tumors, as hr overexpression increases with age . The national comprehensive cancer network (nccn) treatment guidelines for hr+/her2 mbc recommend the use of endocrine therapy, particularly a nonsteroidal aromatase inhibitor (ai), as first - line treatment in postmenopausal women . Since most patients eventually develop resistance to these therapies, the nccn guidelines recommend another endocrine agent when the first therapy fails . After the failure of three sequential endocrine therapies, if symptomatic visceral disease is present or if the cancer is rapidly progressing or immediately life - threatening chemotherapy is recommended, either as monotherapy with an anthracycline, taxane, antimetabolite, or other microtubule inhibitors or as combination treatment . However, observed real - world treatment patterns are not consistent with nccn guidelines, showing that many hr+/her2 mbc patients only receive one line of endocrine therapy before switching to chemotherapy for second - line treatment [9, 10]. Chemotherapy is often accompanied by serious treatment side effects (grade 3/4), some of which have severe impact on the patients' health - related quality - of - life (qol) [11, 12]. Therefore, there is an unmet need for efficacious but more tolerable alternatives for the treatment of hr+/her2 mbc . A novel targeted agent, everolimus, was approved in july 2012 to be used in combination with endocrine therapy exemestane for the treatment of mbc in patients who failed nonsteroidal ai . The efficacy of everolimus / exemestane combinational therapy was demonstrated in the phase iii, double - blind, randomized, bolero-2 trial with significantly improved progression - free survival (pfs) compared to exemestane monotherapy [1315]. The efficacy of other everolimus - based therapies, such as combinational therapy of everolimus and tamoxifen, has also been examined in other clinical studies . Currently, there is limited evidence regarding the comparative effectiveness of everolimus - based therapy and chemotherapy, two common treatment options for hr+/her2 mbc after initial failure of nonsteroidal ai . Several recent studies presented in oncology conferences have indicated that everolimus / exemestane combinational therapy was associated with significantly longer survival compared to chemotherapies [17, 18], but these studies were based on small samples of patients or physician surveys . A network meta - analysis of previous mbc trials also found that everolimus / exemestane combinational therapy was associated with comparable or better pfs compared to some commonly - used chemotherapy; however, findings in clinical trial settings may not represent the real - world comparative effectiveness . In addition, this analysis also had inherent limitations due to indirect comparisons of treatments from different studies with heterogeneous designs and patient populations . The present study aims to address this knowledge gap and to compare the real - world effectiveness of everolimus - based therapy versus chemotherapy in treating hr+/her2 mbc patients in community - based oncology practices in the us . The everolimus - based therapy group includes everolimus monotherapy and combination therapy of everolimus and either endocrine therapy or chemotherapy; the chemotherapy group includes chemotherapy monotherapy, combinational therapy of chemotherapy agents, and combinational therapy of chemotherapy and endocrine therapy (excluding combination of chemotherapy and everolimus). The clinical outcomes of interest include time on treatment (tot), overall survival (os), and pfs . Community - based oncologists / hematologists who treated postmenopausal women with hr+/her2 stage iv mbc were invited from a nationwide online panel of over 9,500 oncologists / hematologists to participate in the chart review study . Physicians were eligible for participation if they had treated one or more postmenopausal hr+/her2 mbc patients who met all the patient selection criteria described below . Each physician was asked to provide data for up to 10 patients, selected at random from their list of eligible patients . Stratified sampling was used to ensure sufficient sample size in each treatment group (everolimus - based therapy or chemotherapy) and by line of therapy (first line, second line, third line, and above). A standardized electronic case report form (ecrf) the ecrf was extensively tested for logic and consistency and was pilot tested by three community based oncologists for clarity and understandability . The identity of physicians was blind to the authors and study sponsor, and vice versa . Patient medical records were selected for abstraction if the patient was a postmenopausal woman who had bc recurrence or progression on or after a nonsteroidal ai in an adjuvant or metastatic setting and subsequently initiated an everolimus - based therapy or chemotherapy in any line of treatment for mbc between 07/01/2012 and 04/15/2013 (the first treatment initiated during this time period that met the aforementioned criteria was defined as the index therapy). Patients who received everolimus - based therapies before their index treatment were excluded from the current analysis for both treatment groups . Furthermore, physicians were required to have access to their patients' mbc - related medical records from the first mbc diagnosis to the last follow - up (or death), while patients were required to not be enrolled in any clinical trials and to not have a history of primary malignancy of other nonbreast cancers (with the exception of nonmelanoma skin cancer and carcinoma in situ of the uterine cervix) within 3 years prior to the first mbc diagnosis date . Tot was defined as the time from initiation of index therapy to either death or discontinuation of index therapy, whichever occurred first . Patients without recorded death or discontinuation of the index therapy were censored at the last follow - up date . Os was defined as the time from initiation of the index therapy to death from any cause . Pfs was defined as time from initiation of index therapy to disease progression or death, whichever occurred first . Progression was determined by the participating physicians with radiographic evidence or tests, physical exams, or assessment of symptoms or through the use of other methods . Patients' baseline characteristics at either the first mbc diagnosis or the initiation of index therapy were summarized . They included age, race, insurance type, disease status (de novo, recurrent with adjuvant endocrine therapy, recurrent without adjuvant endocrine therapy), adjusted charlson comorbidity index (cci) (excluding a score of 6 for metastatic cancer), eastern cooperative oncology group (ecog) performance status, number and sites of metastases, physician - classified tumor volume, prior chemotherapy in the mbc setting, and time from initiation of last adjuvant endocrine therapy to the first mbc diagnosis . Patient baseline characteristics were compared between the everolimus - based therapy and chemotherapy groups using wilcoxon rank - sum tests for continuous variables and chi - square tests for categorical variables . Patients treated with everolimus - based therapy and chemotherapy were compared for all study outcomes (tot, os, and pfs) using kaplan - meier (k - m) analyses and cox proportional hazards models . Unadjusted comparisons between everolimus - based therapy and chemotherapy included (1) k - m curves generated for each study outcome and log - rank tests; (2) median estimates obtained for patients who were not censored for tot and pfs (e.g., for tot, medians were assessed among those patients who had completed their index treatment); (3) cox models used to compare each outcome using two approaches, one included treatment group assuming homogeneous comparative effectiveness across lines of therapy, and the other one included an interaction term between line of therapy and treatment group allowing heterogeneous comparative effectiveness across lines of therapy . Adjusted comparisons between everolimus - based therapy and chemotherapy were conducted using multivariate cox models, controlling for patient baseline characteristics including age, race, insurance type, index therapy line, disease status, adjusted cci, sites of metastases, ecog performance status, prior chemotherapy in the mbc setting, and time from initiation of last adjuvant endocrine therapy to first mbc diagnosis . Similar to the unadjusted cox regression analyses, one set of models adjusted for treatment group and the other adjusted for an interaction term between the line of therapy and treatment group . A total of 234 patients were included for the everolimus - based therapy group and 137 for the chemotherapy group . Both groups had similar comorbidity burden, insurance coverage, ecog performance status, prior chemotherapy in the mbc setting, and time from initiation of last adjuvant endocrine therapy to first mbc diagnosis . Patients treated with everolimus - based therapy were older (64 years versus 62 years, p = 0.050) and more likely to be caucasian than patients treated with chemotherapy (64.1% versus 50.4%, p = 0.009). Compared to the chemotherapy group, everolimus - based therapy group had a lower proportion of liver, lung, and visceral metastases, a smaller number of metastatic sites, and a lower proportion of high-/medium - volume tumors (all p <0.05). Overall, patients treated with everolimus - based therapy appeared to have less aggressive mbc than those treated with chemotherapy . Everolimus - based therapy was associated with significantly longer tot than chemotherapy (log - rank test p <0.001; unadjusted hazard ratio (hr) = 0.36, 95% confidence interval (ci): 0.270.47, p <0.001; table 2). Median tot among patients who completed their index treatment was 8.6 months for everolimus - based therapy patients and 6.1 months for chemotherapy patients . Multivariate - adjusted cox regression results showed that tot was significantly longer for everolimus - based therapy patients compared to chemotherapy patients (adjusted hr = 0.34, 95% ci: 0.250.45, p <0.001; table 2). When further adjusted by the interaction between line of therapy and treatment group, tot was longer in patients who received everolimus - based therapy in all lines of therapy than patients who received chemotherapy in the same lines (adjusted first - line hr = 0.30, 95% ci: 0.200.46, p <0.001; adjusted second - line hr = 0.30, 95% ci: 0.170.52, p <0.001; adjusted third - line and above hr = 0.45, 95% ci: 0.260.78, p = 0.004; table 3). Everolimus - based therapy was associated with significantly longer os than chemotherapy (log - rank test p = 0.002; unadjusted hr = 0.49, 95% ci: 0.300.78, p = 0.003; table 2). Multivariate - adjusted cox model results showed that os was significantly longer for everolimus - based therapy patients compared to chemotherapy patients (adjusted hr = 0.37, 95% ci: 0.220.63, p <0.001; table 2). When further adjusted by the interaction between line of therapy and treatment group, os was significantly longer in patients who received everolimus - based therapy in first - line or third - line and above than patients who received chemotherapy in the same lines (adjusted first - line hr = 0.35, 95% ci: 0.160.79, p = 0.011; adjusted third - line and above hr = 0.29, 95% ci: 0.120.75, p = 0.010; table 3). Everolimus - based therapy was associated with numerically longer pfs than chemotherapy, although the difference was only marginally significant (log - rank test p = 0.057; unadjusted hr = 0.74, 95% ci: 0.551.01, p = 0.058; table 2). Median pfs among patients who completed their index treatment was 8.5 months for everolimus - based therapy patients and 7.1 months for chemotherapy patients . Multivariate - adjusted cox regression results showed that pfs was significantly longer for everolimus - based therapy patients compared to chemotherapy patients (adjusted hr = 0.70, 95% ci: 0.500.97, p = 0.033; table 2). When further adjusted by the interaction between line of therapy and treatment group, pfs was longer in patients who received everolimus - based therapy in third - line and above than patients who received chemotherapy in third - line and above, although the difference was marginally significant (adjusted hr = 0.56, 95% ci: 0.301.02, p = 0.059; table 3). For the treatment of hr+/her2 mbc, the nccn guidelines recommend three consecutive lines of endocrine therapy (including everolimus / exemestane combinational therapy for patients who meet the eligibility criteria for the bolero-2 trial) before chemotherapy . However, real - world studies report that many patients start chemotherapy earlier [9, 10], possibly due to concerns about endocrine resistance or visceral symptoms . As newer targeted therapies become available for hr+/her2 mbc, evidence of the comparative effectiveness of these treatments versus chemotherapy is important for the decision - making process of physicians and payers . The current retrospective chart review showed that in hr+/her2 postmenopausal women with mbc, patients receiving everolimus - based therapy tended to have less aggressive mbc, in particular visceral metastases, than patients receiving chemotherapy . Everolimus - based therapy was associated with significantly longer os, pfs, and tot than chemotherapy after adjusting for the observed baseline characteristics; and the findings were largely consistent across lines of therapy . The present comparative effectiveness findings are consistent with recent studies showing that hr+/her2 mbc patients treated with everolimus - based therapy tended to have better os [17, 18] and pfs than those treated with chemotherapy . For example, using a small sample of hr+/her2 mbc patients, pouget et al . Showed that everolimus plus endocrine therapy resulted in significantly longer os than chemotherapy for patients pretreated with two or fewer lines of therapies for mbc [17, 18]. (2013) conducted a network meta - analysis of available mbc clinical trials and concluded that despite differences in patient characteristics across studies, everolimus / exemestane combinational therapy was associated with the longer mean pfs until 20 months compared to commonly - used chemotherapies such as capecitabine, doxorubicin, paclitaxel, and vinorelbine . Future head - to - head clinical trial evidence will help further assess the comparative efficacy of everolimus - based therapy compared to chemotherapy . A phase ii bolero-6 trial is actively recruiting patients and aims to compare the efficacy of chemotherapy (capecitabine monotherapy) with everolimus - based therapy in er+ mbc patients after recurrence or progression on prior nonsteroidal ai . Primary findings of the study are expected in early 2016 . While chemotherapy is recommended for more aggressive cancers, for the majority of hr+/her2 mbc patients who present a more manageable course of disease endocrine therapy presents a more favorable risk - benefit profile, particularly due to the treatment's similar efficacy but milder toxicity relative to chemotherapy . The current nccn guidelines recognize this and support subsequent treatment to prolong the benefits of endocrine therapies for as long as possible before initiating chemotherapy . Together with recent studies [17, 18], the current findings suggest that everolimus - based therapy may be a more effective alternative to chemotherapy after initial failure of nonsteroidal ais . Furthermore, previous studies have shown that while everolimus may result in some moderate toxicity [23, 24], the adverse events are generally manageable and the patient's health - related qol is similar to that of patients on endocrine therapy . This may make everolimus more preferable over chemotherapy, which is often accompanied by severe tolerability issues that result in worse health - related qol while on treatment [11, 12, 22]. The observed shorter tot among patients treated with chemotherapy may be due to oncologists' preference of only prescribing a limited cycle of chemotherapy in order to avoid cumulative toxicity . Future studies can compare real - world safety outcomes between the two treatments to better inform treatment decisions . First, inherent to observation studies, the findings may be subject to bias if important confounding factors are not identified and adjusted for in the study's analyses [27, 28]. In the current multivariable analyses, we adjusted for patient characteristics commonly recorded in medical charts and known to be prognostic for outcomes in mbc . These included characteristics such as age, race, insurance type, index therapy line, disease status, cci, sites of metastatic disease, ecog, prior chemotherapy in the mbc setting, and time from initiation of the last adjuvant endocrine therapy to the first stage iv mbc diagnosis . However, if patients treated with everolimus - based therapy are healthier based on unobserved measures of disease severity or have better coping skills, the results are likely to be biased in favor of everolimus . Second, the frequency of patient follow - up could be different between the two treatment groups . The group with more frequent visits to oncologists was more likely to be identified to have an event (such as discontinuation and progression). These limitations can only be addressed with a well - conducted randomized - controlled trial . Nonetheless, observational studies constitute a valuable and rich source of data, as they allow researchers to examine treatment effectiveness across patient groups in a large sample set and are directly reflective of (and applicable to) real - world clinical practice . In this retrospective review of hr+/her2 mbc patients from community - based oncology practices in the us, patients treated with everolimus - based therapy tended to have less aggressive mbc than patients treated with chemotherapy . After controlling for the observed baseline characteristics, everolimus - based therapy was associated with significantly longer os, pfs, and tot than chemotherapy . As this is an observational study, unobserved patient characteristics may affect study findings.
The management of parkinson's disease (pd) is often enhanced by complementary rehabilitation strategies, such as exercise . For example, studies of resistance [13] and endurance [47] exercise training have improved balance, gait, postural stability, and physical function and reduced falling in people with pd . Tai chi, a traditional chinese martial art that involves meditation and slow, graceful movements, is often recommended to reduce stress, improve mood, flexibility, physical function, and balance [810]. Studies of tai chi in people with chronic disease including parkinson's disease and old people have supported the potential for benefit, with gains in the quality of life, postural stability, gait, physical function, immune function, cardiometabolic disease risk factors, and other health - related parameters [4, 1119]. However, research on the effectiveness of tai chi is contradictory due to inconsistencies in the implementation of the tai chi movements, limited samples, and the lack of randomized control trials . The purpose of this study is to investigate the effects of a randomized control trial of therapeutic tai chi training on improving the motor function and physical function of parkinson's disease patients . All participants gave informed consent in accordance with the procedures of the parkinson's disease center and the sports medicine center of the asan medical center . The participants of this study were 24 clinically stable patients with diagnosed idiopathic parkinson's disease recruited from the parkinson's disease center in asan medical center in seoul, republic korea . Eligible participants met the following inclusion criteria: (1) hoehn - yahr stage 1 or 2 and (2) stable drug regimen . Volunteers were excluded from the study if they had (1) severe cognitive impairment, (2) concomitant severe neurologic, cardiopulmonary, or orthopedic disorders, (3) specific contraindications to exercise, or (4) they had recently participated in any physiotherapy or rehabilitation program . The volunteers were screened for inclusion and exclusion criteria based upon the medical history and physical examination . Participants were randomized to either a twelve - week intervention of therapeutic tai chi (ttc) or a non - exercise control group (see figure 1). The ttc group visited the clinic 2 times a week and performed home - based activity 1 time per week for 12 weeks . Each ttc session started with a 10-minute stretching exercise warm - up, followed by 30 minutes of tai chi exercises, and ending with 10 minutes of meditation and 10 minutes of stretching exercise cool - down . The exercise was performed within the intensity ranges of 11 to 15 (light to somewhat hard) on the borg ratings of perceived exertion (rpe) scale . Study outcome measures were obtained at baseline (prerandomization; one week prior to the start of the program) and within one week following the end of the 12-week intervention . The outcome measures included several test of physical function (lateral stance, agility, tandem gait, timed up and go, and six - minute walk) and the unified parkinson's disease rating scale (updrs) sections 13 . All the tests were performed in the same order, at the same time of day, and when the participant felt best . The participant stands with hands on hips with eyes closed; when ready, the participant stands on the foot of choice for as long as possible, with the knee of the other leg flexed to 90 degrees . When a light comes on, they jump up as fast as possible in response to a single light stimulus . The tandem gait test was used to assess balance, appendicular coordination, and gait, which involves multiple sensory and motor systems . The participant walks in a straight line while touching the heel of one foot to the toe of the other with each step . For the timed up and go test, the participant was seated in an arm chair (45 cm high) with their back against the chair . On a signal, they stand up, walk 3 meters as quickly and safely as possible, turn around, walk back to the chair, and sit down with their back against the chair . The six - minute walk test was applied as a test of cardiorespiratory endurance for daily physical activities . This test measures the distance that can be walked at a self - paced velocity on a flat hard surface over a period of six minutes . An analysis of variance (anova) for repeated measures with one between factor (treatment group; ttc versus control) and one within factor (time; pre- and postintervention) was used to evaluate the effects of the intervention . A total of 11 participants were randomized to the ttc and 11 participants to the control condition . Two subjects in the control group did not complete the study for personal reasons unrelated to the study . As shown in table 3, significant changes were observed in the mentation, behavior, and mood subscale and the motor subscale of the updrs, with no significant main effects on the activities of daily living scale (adl), balance, or reaction time . However, there were significant interactions between time and intervention group on the updrs activities of daily living subscale (figure 2). However, as shown in figures 3 and 4, there were significant (p <0.05) interaction effects for balance (one - leg standing test) and reaction time (light stimulus). In this study, we investigated the motor and nonmotor effects of therapeutic tai chi exercise training on participants with pd . The findings of our study showed modest effectiveness of tai chi training on people with parkinson's disease, with significant interaction effects for balance, reaction time, and adls . These findings support that tai chi is effective in improving some aspects of motor function, most notably reaction time and balance . In addition, pd participants reported an improvement in their ability to engage in their activities of daily living . Exercise therapy has a major role in the rehabilitation of the patients with parkinson's disease [26, 27]. Regular physical activity may increase the functional ability and enhance the capacity for independent living by decreasing the need for assistance with the activities of daily life . In addition, exercise may achieve the goal of neuroprotection, slow disease progression, and postpone disabilities [28, 29]. Several cohort studies have described the functional achievements of moderately affected parkinson's disease participants who have undergone intensive standardized exercise training [3033]. However, burini et al . Reported in a study with a crossover design that a 7-week tai - chi and aerobic exercise training did not affect the severity of neurological signs and symptoms as assessed by the updrs or have any significant impact of training on the attendant disability . . Showed that physical therapy is mainly focused on mobility rather than various neurologic symptoms such as rigidity and tremor . Reported that some aspects of the mobility and balance functions were positively affected by tai chi exercise . However, assessing the mobility and balance function by only one item, such as the walking function, is not very representative of the spectrum of the motor signs and symptoms . Furthermore, there is little attention toward non - motor symptoms such as fatigue mood and health quality of life, all of which may be altered by exercise [27, 3740]. In a recently published clinical trial, li et al . Found beneficial effects of tai chi exercise on balance, physical function, and falls, suggesting that tai chi is an appropriate physical activity for patients with parkinson's disease and it might be useful as a therapeutic exercise . The ability to balance is related to the control of the center of gravity within the base of support . Koller and huber found that the balance impairment in older adults with a longer duration of pd does not usually respond to levodopa; 38% of the persons with pd experience fall, 13% fall more than once per week, and some studies have reported that pd patients fall repeatedly throughout the day . Persons with pd are 5 times more likely to suffer falls - related injuries such as hip fractures than healthy older adults . Reaction time for agility was measured in the present study and improved with ttc training . Ttc training has benefits for postural stability in elderly people, including patients with pd, likely by acting on a number of sensorimotor systems that contribute to postural control [12, 43, 44]. This improvement combined with the observed improved agility performance confirms the separate observations of previous studies showing that ttc training results in better balance capacity, proprioception function, and muscle strength [11, 4547]. While our study and other studies have shown enhanced balance and agility resulting from tai chi, further work is needed to confirm that these improvements in fall risk factors actually result in fewer falls, as there are somewhat contradictory findings in the literature [19, 45, 48]. Physical function refers to the assessment of the capability to complete a specific task rather than assessing the physiologic - derived attributes, and it is believed to reflect the ability to carry out activities of daily living . In the present study, we assessed the physical function with the timed up and go, tandem gait, and six - minute walk tests . The timed up and go test showed no significant change after training, which is in contrast to the findings of li et al . . These differences may be due to differences in the programs of tai chi, the wider range of disease severity in the study by li et al ., or the smaller numbers of participants in our study . The timed up and go test largely depends on the muscular power and strength of the quadriceps and the hip extensor muscles to arise from a chair [49, 50]. It may be that our version of tai chi does not substantially improve lower extremity power . The results of the tandem gait test and the six - minute walk test were also not changed after 12 weeks of ttc training . Poor performance on the tandem gait and six - minute walk could result from several potential factors, including poor cardiorespiratory fitness and gait or balance abnormalities [23, 51]. Many of the pd participants likely had poor cardiorespiratory fitness and inadequate levels of physical activity, as shown by garber and friedman . Consistent with this finding, all of the participants in this study had below normal results on the six - minute walk test, suggesting that poor cardiorespiratory fitness, gait, and/or balance may have been an important determinant of performance . Other studies of tai chi have not shown an improvement in cardiorespiratory fitness, and our results are consistent with these previous findings . This study investigated motor function and the severity of motor and non - motor symptoms and signs . These motor and nonmotor symptoms and signs were not improved after treatment, although self - reported engagement in activities of daily living was enhanced by tai chi exercise . Ttc training involves splitting up complex movements into simple motor tasks and incorporating simultaneous movements, which is also beneficial for parkinson disease patients . Thus, this study lends additional evidence for tai chi as a complementary treatment for people with parkinson's disease one that will allow them to engage more fully in their activities of daily living . Further studies are needed that will consider the effects of various components of a tai chi program and also to help to identify the intensity, duration, and frequency of tai chi exercise to attain optimal benefits . Further, the influence of various medication and dietary factors may moderate the effects of exercise, and this has also not been studied.
Intracellular protein degradation is important for the regulation of many fundamental cellular processes for both plants and animals . The ubiquitin - proteasome - dependent proteolytic pathway that has been found in all eukaryotes is one of the nonlysosomal protein degradation systems . The 26s proteasome is a multicatalytic proteinase complex that acts as the atp - dependent degradation system and digests vast majority of intracellular proteins tagged with a multiubiquitin chain as a degradation signal . In all eukaryotes the ubiquitin - proteasome system (ups) degradation determines the turnover rate for a wide range of proteins including long- and short - lived ones as well as regulatory proteins e.g. Metabolic key enzymes, transcription factors, cyclins, inhibitors of cyclin dependent kinases as well as regulators of dna replication, repair and apoptosis . Proteasomal proteolysis is engaged in regulation of nuclear structure and functions such as replication, transcription and splicing . Cell cycle - dependent changes in the localization of the proteasomes suggest that it may have a regulatory function related to the cell cycle . Ubiquitin and proteasomes are intimately involved in chromatin structure modifications and gene control that is accompanied with histone ubiquitination . Degradation, by the proteasomal system, of oxidatively modified proteins plays an important role in the antioxidant defenses . This system provides for cellular quality control since it prevents accumulation of misfolded or damaged proteins . In plants the ups has been implicated in the regulation of almost every developmental process from embryogenesis to floral organ production . This pathway participates in the regulation of such processes as differentiation of vascular bundles, auxin signal transduction pathway, apoptosis, selective ion uptake through plasma membrane proton pomp, nitrogen recycling . The ups is involved in the replacement of histones with protamine - like proteins during spermatogenesis in algae . The role of the plant ups has been established in the defense against pathogen and in disease resistance, against abiotic stresses including heat shock and wounding as well as in organ senescence . The ubiquitin - proteasome pathway is also involved in the regulation of programmed cell death (pcd). However, the highest amount was observed in the root and shoot apical meristems, in leaf primordia and vascular tissue of young sunflower and rice plants suggesting that proteolysis mediated by the 26s proteasomes may be involved in organ formation . Hence, an enhanced level of ubiquitin and proteasomes can be observed at the onset of organogenesis . Generally in animal cells the proteasomes are more abundant in cytosol than in nucleoplasm, although the ups is an active component of the cell nucleus and this proportion varies with the cell type and growth conditions . Nuclear distribution of proteasomes is species - specific and differs in higher versus lower eukaryotes . An important function of the nuclear ups is to control the supply of ribosomal proteins for the assembly of new ribosomal subunits in the nucleolus . Ubiquitin and ubiquitin - like proteins play a role in ribosome biosynthesis and control the flow of materials to and from a nucleolus . It was shown that complexes associated with pre - rrna processing factors were ubiquitinated and immunofluorescence revealed that ubiquitin was present within nucleoli . Inhibition of proteasome activity resulted in nucleolar morphology disruption and accumulation of 90s preribosome, it also affected the distribution of rrna - processing proteins in nuclei, resulting in impaired rrna processing . Moreover, fibrillarin and nucleophosmin, main nucleolar proteins participating in early and late rrna processing, respectively, undergo ubiquitination . That is why the aim of the present work was to check, by immunofluorescence, the presence of ubiquitin and proteasomes within plant root meristematic cell nucleoli . To date, localization of the ups elements has been reported neither in animal nor in plant normal nucleoli, except in the above mentioned examples . Root meristematic cells of soybean seedlings seem to be an attractive object to study plant nucleoli . Nucleoli of soybean, a plant from subtropical climate, clearly responded to chilling showing differences in ultrastructure and considerably reduced transcriptional activity . Moreover, in the nucleoli of plants subjected to recovery after chilling stress transcriptional activity and transport of ribosomal subunits to the cytoplasm were even more intensive than in the control and then characteristic, single, huge, and centrally located nucleolar vacuoles appeared in them . It seems that the ups contributes indirectly to changes of nucleolar activity and structure in soybean cells quickly responding to environmental conditions . Current studies showed that immunosignals against 20s proteasomes and ubiquitin were present in nucleolar vacuole territories . Aldana (obtained from plant breeding and acclimatization institute in radzikw, poland) were germinated for three days at 25c in darkness in petri dishes on filter paper moistened with distilled water (control). To obtain nucleoli with vacuoles the control seedlings were treated with chilling (10c) for four days, and then the plants were transferred to 25c for one day . Root apical meristems were cut off from the seedlings and fixed in 4% paraformaldehyde in 0.01 m phosphate buffered saline (pbs), ph 7.4 for 45 min at room temperature (rt) and then washed in pbs . Next the material was treated with a digestion mixture (1% pectinase, 1% cellulase, 1% pectolyase in pbs) for 30 min at rt . After washing in pbs slides were air - dried and then incubated in 5% bsa with 0.5% triton x-100 in pbs for 60 min at rt, washed in pbs with 0.5% triton x-100 . The preparations were covered with the mouse monoclonal antibody to 20s proteasomes (10 g / ml diluted in pbs with 0.1% triton) or with the rabbit polyclonal antibody to ubiquitin (1:300 diluted in dako solution; dako, chemniec, poland) and both left for 12 h at 4c then washed five times in pbs with triton . The secondary antibodies conjugated with fitc - anti - mouse ig (1:30; sigma aldrich, poznan, poland) and anti - rabbit ig (1:80; sigma) to proteasomes and ubiquitin, respectively, were incubated for 2 h in darkness at rt . After this, the slides were washed in pbs with 0.5% triton and once only in pbs . Dried preparations were covered with pbs: glycerol mixture (9:1) with 2.3% dabko (sigma). Observations were performed using optiphot-2 epifluorescence microscope equipped with b-2a filter (blue light; =470 nm) for fitc . . Moreover, the control tests with other antibodies, i.e. Polyclonal rabbit tri - methyl histone h3 (lys4) antibody (h3k4met3) and rabbit polyclonal acetyl histone h3 (lys9) antibody (h3k9acetyl) (cell signaling technology, inc . Root tips of each variant were fixed in 2% glutaraldehyde in 1% cacodylate buffer (ph 7.27.4) for 3 h at 4c . After dehydration in the ethanol series, the material was embedded in a medium containing the mixture of epon 812 and spurr's resin . Nucleolar vacuoles were observed by means of light microscope jenamed-2 (carl zeiss, jena, germany) and ccd camera mtv - 1801 cb connected to a microscope and computer - aided imal-512 system . Meristematic cells with nucleoli showing high activity, i.e. Those from the control and post - chilling recovered plants were taken into account to check both the number of vacuolated nucleoli and for immunofluorescent studies . Five root tips for each experimental variant and 150 cells from each root tip were examined . The number of cells with vacuolated nucleoli was counted on semi - thin sections, while the number of cells with nucleoli with immunosignals was estimated on squashed preparations . Significance of difference between vacuolated and immunolabeled vacuolated nucleoli as well as between nucleoli immunolabeled with the antibodies against proteasome and ubiquitin was estimated for the control and recovered seedlings . Statistical significance of differences were done using the student's t - test (p<0.05). Aldana (obtained from plant breeding and acclimatization institute in radzikw, poland) were germinated for three days at 25c in darkness in petri dishes on filter paper moistened with distilled water (control). To obtain nucleoli with vacuoles the control seedlings were treated with chilling (10c) for four days, and then the plants were transferred to 25c for one day . Root apical meristems were cut off from the seedlings and fixed in 4% paraformaldehyde in 0.01 m phosphate buffered saline (pbs), ph 7.4 for 45 min at room temperature (rt) and then washed in pbs . Next the material was treated with a digestion mixture (1% pectinase, 1% cellulase, 1% pectolyase in pbs) for 30 min at rt . After washing in pbs slides were air - dried and then incubated in 5% bsa with 0.5% triton x-100 in pbs for 60 min at rt, washed in pbs with 0.5% triton x-100 . The preparations were covered with the mouse monoclonal antibody to 20s proteasomes (10 g / ml diluted in pbs with 0.1% triton) or with the rabbit polyclonal antibody to ubiquitin (1:300 diluted in dako solution; dako, chemniec, poland) and both left for 12 h at 4c then washed five times in pbs with triton . The secondary antibodies conjugated with fitc - anti - mouse ig (1:30; sigma aldrich, poznan, poland) and anti - rabbit ig (1:80; sigma) to proteasomes and ubiquitin, respectively, were incubated for 2 h in darkness at rt . After this, the slides were washed in pbs with 0.5% triton and once only in pbs . Dried preparations were covered with pbs: glycerol mixture (9:1) with 2.3% dabko (sigma). Observations were performed using optiphot-2 epifluorescence microscope equipped with b-2a filter (blue light; =470 nm) for fitc . Moreover, the control tests with other antibodies, i.e. Polyclonal rabbit tri - methyl histone h3 (lys4) antibody (h3k4met3) and rabbit polyclonal acetyl histone h3 (lys9) antibody (h3k9acetyl) (cell signaling technology, inc ., danvers, ma, usa) were carried out . Root tips of each variant were fixed in 2% glutaraldehyde in 1% cacodylate buffer (ph 7.27.4) for 3 h at 4c . After dehydration in the ethanol series, the material was embedded in a medium containing the mixture of epon 812 and spurr's resin . Nucleolar vacuoles were observed by means of light microscope jenamed-2 (carl zeiss, jena, germany) and ccd camera mtv - 1801 cb connected to a microscope and computer - aided imal-512 system . Meristematic cells with nucleoli showing high activity, i.e. Those from the control and post - chilling recovered plants were taken into account to check both the number of vacuolated nucleoli and for immunofluorescent studies . Five root tips for each experimental variant and 150 cells from each root tip were examined . The number of cells with vacuolated nucleoli was counted on semi - thin sections, while the number of cells with nucleoli with immunosignals was estimated on squashed preparations . Significance of difference between vacuolated and immunolabeled vacuolated nucleoli as well as between nucleoli immunolabeled with the antibodies against proteasome and ubiquitin was estimated for the control and recovered seedlings . Statistical significance of differences were done using the student's t - test (p<0.05). Genes encoding subunits of the ubiquitin - proteasome system (ups) are present in organisms ranging from some prokaryotes to all studied eukaryotes, and appear to have been highly conserved throughout evolution, hence in the current work the antibodies to core subunits of the human 20s proteasomes and to the human ubiquitin were used to identify proteasomes and ubiquitin in the plant material . In this study the immunocytofluorescent microscopy showed nuclear and cytoplasmic labeling with the antibodies against 20s proteasomes and ubiquitin in all studied cells of soybean root meristems suggesting that both elements of the ups were present in the nucleus as well as in the cytoplasm . In other eukaryotes both components of the ups are also found in the cytoplasm and within the nucleus . Proteasomes alone are found in the cytoplasm, both free and attached to the cytoplasmic reticulum, and in golgi apparatus, associated with cytoskeletal elements and in the nuclei of eukaryotic cells . In the cell nucleus proteasomes are present throughout nucleoplasm, concentrating in subnuclear structures such as splicing speckles, promyelocytic leukemia (pml) nuclear bodies and clastosomes . In all mammalian cells analysed so far endogenous proteasomes were distributed throughout the cytoplasm and nucleoplasm, however they were detectable neither in the nuclear envelop nor in nucleoli . It is interesting that in these studies immunopositive signals were also present in the nucleoli of soybean cells . However, immunostaining was not observed in every nucleolus and those which were stained were labeled only partially . Immunosignals for proteasomes and ubiquitin were present solely in these nucleoli which most probably contained nucleolar vacuoles . The nucleoli without vacuoles were excluded from immunostaining (figure 1a, a'). Observations showed that the distribution and pattern of labeling for both proteasomes and ubiquitin in the soybean root meristematic cells were almost identical (figure 1 a, a',b, b',c, c',d, d'). Dimensions of the labeled areas in particular nucleoli were various depending, as we suppose, on sizes of nucleolar vacuoles . The labeling was most frequently located in the central part of a nucleolus and occupied relatively big area (figure 1 b, b'). There were also immunosignals which occupied single, small areas (figure 1 c, c') and a few small foci in one nucleolus (figure 1 d, d'). The sizes of immunosignal areas and their location were in agreement with the sizes and location of nucleolar vacuoles (figure 2 a, b, c, d). In order to show that occurrence of cells with nucleolar vaculoles as well as labeling of these vacuoles are not rare or accidental the rows of neighbouring cells, as they occur in root meristems, were demonstrated in figure 3 a, b, c . Earlier and present studies showed that in the cells of control plants there were 1826% vacuolated nucleoli while in the cells of plants recovered after 4-day chilling there were many more nucleoli with vacuoles, 4054% . In most cases the nucleolar vacuoles were huge, single and centrally located in those nucleoli (table 1). Current studies showed that higher number of nucleoli with immunosignals was observed in the cells of plants subjected to recovery in comparison to the control plants . These experiments, on the basis of sizes and localization of immunosignals, indeed, would confirm the fact that immunopositive reactions were localised to nucleolar vacuole territories . In this paper only nucleoli of the recovered plants were demonstrated in figures, although nucleoli of the control plants also contained nucleolar vacuoles with immunosignals, but these were less spectacular than those of the recovered plants . Figure 1immunofluorescent detection of proteasomes (a, b, c, d) and ubiquitin (a', b', c', d') in root meristematic cells of soybean; a, a'), nucleoli free of immunosignals; b, b'), nucleoli with one big, centrally located labeled area; c, c'), nucleoli with one small labelled area; d, d'), nucleoli with a few small labelled areas . Immunofluorescent detection of proteasomes (a, b, c, d) and ubiquitin (a', b', c', d') in root meristematic cells of soybean; a, a'), nucleoli free of immunosignals; b, b'), nucleoli with one big, centrally located labeled area; c, c'), nucleoli with one small labelled area; d, d'), nucleoli with a few small labelled areas . A), nucleoli without vacuoles; b), nucleoli with one, big, centrally located vacuole, characteristic of plants recovered after chilling stress; c), nucleolus with a small vacuole; d), nucleolus with a few small vacuoles . A), nucleoli without vacuoles; b), nucleoli with one, big, centrally located vacuole, characteristic of plants recovered after chilling stress; c), nucleolus with a small vacuole; d), nucleolus with a few small vacuoles . Figure 3rows of neighbouring cells in soybean root meristems with: immunodetection of proteasomes (a) and ubiquitin (b) as well as a semithin section of cells with nucleolar vacuoles (c). Rows of neighbouring cells in soybean root meristems with: immunodetection of proteasomes (a) and ubiquitin (b) as well as a semithin section of cells with nucleolar vacuoles (c). Table 1frequency of vacuolated and of immunolabeled vacuolated nucleoli in control and recovered soybean seedlings.growth conditionsfrequency (%) ofvacuolated nucleolivacuolated nucleoli immunolabeled with antibody againstproteasomeubiquitincontrol (3d/25c)26.33.123.83.625.13.9recovery(3d/25c + 4d/10c + 1d/25c)53.75.949.36.251.55.8mean values sd; there is no significance of difference between vacuolated and immunolabeled vacuolated nucleoli as well as between nucleoli immunolabeled with antibody to proteasome and ubiquitin for both control and recovered experimental variants, p<0.05 . Mean values sd; there is no significance of difference between vacuolated and immunolabeled vacuolated nucleoli as well as between nucleoli immunolabeled with antibody to proteasome and ubiquitin for both control and recovered experimental variants, p<0.05 . Although the number of vacuolated nucleoli was somewhat higher than of those with immunosignals in both experimental variants, the statistical analysis showed that there was no significance of difference between them (table 1). Similar situation occurred in the case of the number of nucleoli labeled with the antibodies to proteasome and ubiquitin (table 1). On the basis of the obtained results a conclusion could be drawn that all vacuolated nucleoli contained immunosignals for proteasomes or ubiquitin . Some experimental procedures, especially fixation, may induce artefact formation . In order to check that immunosignals present in nucleolar vacuoles were not artifacts some control tests were carried out . The negative controls without primary antibodies did not show any specific immunosignals at the whole cell areas (figure 4a). Tests with the use of antibodies to nuclear proteins, h3k4met3 and h3k9acetyl, and the same procedure as in the case of proteasome and ubiquitin, did not show any immunosignals at the nucleolar territory . Figure 4control tests: a), without primary antibody, scale bar: 15 m; b), with nuclear antibodies h3k4met2 and c), with h4k12acetyl, scale bar: 10 m . Control tests: a), without primary antibody, scale bar: 15 m; b), with nuclear antibodies h3k4met2 and c), with h4k12acetyl, scale bar: 10 m . The nucleoli themselves are generally thought to be devoid of proteasomes under normal growth condition as detailed proteomic analysis of human nucleoli showed and the role of nucleolus in the proteasomal activity in degradation of the nucleolar proteins has not been documented to date, but ubiquitin is present in normal nucleoli . Moreover, sumoylation acting in coordination with the ups regulates the maintenance of nucleolar integrity . A nucleolus is also the key organelle in which sumo-1 conjugates accumulate in response to proteasome inhibition . In some cases, under particular conditions for example, proteasomes and their potential substrates accumulated in nucleoli during inhibition of proteasomal protein degradation by mg 132 in human cells, during increased myc expression and when altered localization of the nuclear protein pml occurred, suggesting that nucleoli are the site of protein degradation . Proteasome inhibition exerts specific effects on certain components of nucleolar architecture and on early and late processing factors of pre - ribosome biosynthesis, as well as it disrupts multiple processes in ribosome biogenesis and substantially influences pol i transcription . Even in the soybean seedlings treated with epoxomycin, an inhibitor of the proteolytic activity of proteasomes, alterations in nucleolar ultrastructure can be seen (stpiski, unpublished data). This protein is required for early pre - rrna processing and for release of pre-60s from the nucleolus into the nucleoplasm . Additionally the functions of ubiquitin and nucleoli seem to be connected . A possible role for ubiquitin in nucleolar disassembly also ubiquitin ligase is known to regulate the processing and nuclear export of trna, mrna as well as rrna in yeast . Ubiquitin has been localized within both the nucleolus and the pre - ribosomal complexes of mammalian cells . Since biosynthesis of ribosomal precursors is regulated at multiple levels including the transcription of ribosomal genes, phosphorylation, methylation and acetylation of nucleolar factors, the levels of these factors could be controlled by the ups, hence, it is suggested that the ups is required for multiple steps of ribosome biogenesis . More speculatively, one could believe that presence of both components of the ups, ubiquitin and proteasomes in a nucleolus establishes a functional degradation pathway of nucleolar and ribosomal proteins in this organelle so their transport to distinct subnuclear domains or to the cytoplasm would not be necessary . However, the nucleolar protein, fibrillarin, was described to be degraded in nucleoplasmic foci under conditions that specifically inhibit nucleolar transcription . It means that nucleolar proteins are degraded out of nucleoli . In mammalian cells upon inhibition of proteasome - dependent proteolysis, nucleolar proteins, fibrillarin and ubf, did not accumulate so it is suggested that they do not represent proteasomal substrates and they generally do not co - localize with protein degradation foci in interphase nuclei . However, more recent data show that usp36, a deubiquitinat ing enzyme, plays an essential role in regulating nucleolar structure and function and specifically localizes to nucleoli together with its substrates, namely fibrillarin and nucleophosmin, nucleolar proteins which undergo deubiquitination . Moreover, nucleoli of all eukaryotic organisms, including plants, play the same general function, i.e. Ribosome subunit production, and contain almost identical set of factors / proteins to act the function, hence, it is possible that components of ups might be present also in nucleoli of plant cells . It is interesting and intriguing that ubiquitin and proteasomes have been localised exclusively in nucleoli possessing vacuoles . This might be the reason why ubiquitin and proteasomes have not been identified in other objects so far . In the case of soybean, vacuolated nucleoli appeared in plants grown at optimal temperature of 25c as well as in those subjected to 4-day chilling stress (when rrna transcription considerably slowed down) and subsequently recovered at optimal temperature . In the latter case the nucleoli with big vacuoles appeared and rrna transcription proceeded even more intensively than in the control . So, presence of the ups components solely in the vacuolated nucleoli proves the fact that the ubiquitin - proteasome pathway is present in nucleoli with intensified activity . It cannot be excluded that nucleolar and ribosomal proteins predestined to be degraded in poorly active, non - vacuolated nucleoli, are transported to the extranucleolar nucleoplasm or to the cytoplasm and their proteolysis takes place there . On the other hand in very active, vacuolated nucleoli, where there is no time for transport of proteins predestined for degradation out of nucleoli the whole machinery of the ups is brought to nucleoli and proteolysis occurs inside nucleolar vacuoles . Under normal conditions synthesis and import of ribosomal proteins is in excess in relation to needs . The more so as the excess of such proteins may occur in the most active cells, i.e. In plants subjected to recovery . Ribosomal proteins that have failed to assemble into ribosomal subunits in nucleoli and excessive ribosomal proteins are also degraded by the proteasome . Moreover, in mammalian nucleoli the structures called nucleolar aggresomes, which contain proteins and other compounds are formed . Aggresomes are also formed under various forms of stress as well as when cells are treated with poteasome inhibitors . Most proteins accumulating in nucleolar aggrosomes are degraded in proteasomes but a question whether this process takes place in nucleoli remains opened . Increased nuclear load of proteins which are destined to be degraded enhances aggresome formation . The more so as such a situation could take place in soybean plants under chilling stress during which damage of proteins could appear . Moreover, communication between ribosome assembly factors and proteasome activity could be an important mechanism to synchronize ribosome synthesis with cell growth and division . Especially it might refer to the cells with active, vacuolated nucleoli where metabolic processes proceed with high speed and the activity of the factors controlling biochemical processes has to be quickly regulated also by the ups . It is accepted that nucleolar vacuoles appear when transport of ribosomal subunits exceeds their synthesis . However, the ups may help form or enlarge nucleolar vacuoles by digestion of nucleolar matrix proteins at the site of nucleolar vacuole arising . On the one hand, these observations can suggest that the ups may act within the nucleolus and may provide additional level of regulation of intracellular proteolysis via compartment - specific activities of its components . Possibly, the localization of proteasomes and ubiquitin is associated with the need for enhanced proteolysis in individual cell compartments, in this case in a very active nucleolus, to regulate nucleolar processes as well as to clean up unwanted proteins produced as the consequence of stress . Stress conditions may create situations in which massive amounts of ribosomal proteins become unemployed and thus become potential substrates for the nuclear ups . On the other hand, it is worth noticing that immunopositive signals for ubiquitin and proteasomes present both in nucleolus and in the other cellular compartments refer to free ubiquitin and its various conjugates . Proteins modified with ubiquitin are not necessarily predestined for degradation but their activity and localization can be also regulated through ubiquitylation . That is why the presence of ubiquitin in a nucleolus does not necessarily mean that degradation of ubiquitinated proteins takes place in a nucleolus, however it may indicate that nucleoli, precisely the nucleolar vacuoles, may not merely be useless empty space formed in active nucleoli as a consequence of intensive transport of pre - ribosomal particles out of the nucleoli, but they could be the site of temporary sequestration or storage of the ups components or ubiquitin conjugates as well as other biochemical cellular components in plant cells, similarly as different discrete, focal regions in animal nucleoli are used as storage compartments for certain enzymatic and regulatory proteins, which can be released in a regulated manner to exert their function elsewhere.
Several classic studies have shown that gh deficiency (ghd) in adults is associated with abnormalities in body composition, metabolic derangements, and suboptimal physical performances; these impairments improve with gh replacement therapy [13]. Hypopituitarism is associated with increased cardiovascular mortality, women are disproportionally affected, and gh deficiency has been considered a contributory factor [46]. The increased mortality is reflected in elevated cardiovascular risk markers [79] and gh replacement results in improvement in these markers [1014]. In obesity there is a markedly decreased gh secretion . For both children and adults, the greater the body mass index (bmi), the lower the gh response to provocative stimuli [15, 16], including the response to gh - releasing hormone (ghrh) [17, 18]. The altered somatotroph function of obesity is not permanent; it can be reversed by a return to normal weight or by short - term calorie restriction . The most striking example of reversibility appeared when obese subjects were treated with ghrh plus gh - releasing peptide-6 (ghrp-6) both at saturating doses, which resulted in a massive gh response for obese subjects . This relative gh deficiency may contribute to develop or maintain the obese state and gh treatment has been employed in obesity [2326]. A recent meta - analysis of recombinant human gh as therapy for obesity in adults suggests that gh therapy leads to a decrease in visceral adiposity and increase in lean body mass as well as beneficial changes in lipid profile in obese adults, without inducing weight loss . However, little is known about whether the association between decreased gh secretion and increased cardiovascular risk markers observed in patients with ghd and hypopituitarism occurs in obese patients without organic hypothalamic or pituitary disease . There are some recent interesting studies that have evaluated the relationship between gh secretion, evaluated by ghrh - arginine testing and cardiovascular risk markers or central adiposity measures . Found that the gh response to ghrh - arginine testing is reduced in both overweight and obese men and negatively associated with indices of central abdominal obesity including waist circumference, trunk fat, and visceral adipose tissue . They also found that the use of waist circumference adds predictive information to the determination of gh response, independent of bmi . Utz et al . Found that hdl was lower, high - sensitivity c - reactive protein (hscrp) and tumour necrosis factor- (tnf-) receptor i higher, in young overweight or obese women meeting ghd criteria than in women with gh sufficiency . These differences remained significant after controlling for age plus bmi, waist circumference, or trunk fat; there were no differences in measures of insulin resistance . Carmichael et al . Found that peak gh response to ghrh - arginine, in a population of adults aged 5090 years, was significantly related to fasting glucose, insulin, bmi, hdl cholesterol, triglycerides, trunk fat, and abdominal subregion fat, with fasting glucose ranking first by multiple regression analysis . The most clearly altered gh secretion test in obesity is ghrh - induced gh secretion [18, 21, 30, 31], due to this markedly decreased gh secretion; it should be especially attractive to study its relationship with different cardiovascular risk factors in a homogenous population . We studied women of reproductive age because data in this group are particularly lacking, and it is very important to identify risk factors for future cardiovascular disease in this population . The aim of the present study was to evaluate the relationship between ghrh - induced gh secretion in obese premenopausal women and cardiovascular risk markers or insulin resistance . Forty eight premenopausal obese patients, aged 3552 years (32.6 10.3 year), with a bmi of 36.0 6.4 none of the obese patients had diabetes mellitus or other medical problems nor were they taking any drugs . The subjects had been eating a weight - maintaining diet for several weeks prior to the study . All subjects provided informed consent and approval for this study was obtained from the hospital committee . Between 08:30 and 09:00 am, after an overnight fast and while seated, a peripheral venous line was obtained . Fifteen minutes later ghrh (100 micrograms, iv, 0 minutes) was administered . We obtained blood samples for gh at baseline (0 minutes) and then at times 15, 30, 60, 90, and 120 minutes . Fasting blood was drawn for igf - i, serum cardiovascular risk markers, and hormonal determinations . Mid - waist circumference was measured as the midpoint between the iliac crest and the lowest rib, with the patient in the upright position . Measurement of the hip circumference was performed at the widest point, also with the subject in an upright position . Bia is based on measurement of the transmission speed of a 1/4 volt electrical pulse between electrodes at the feet and hands . Because fat - free mass is comprised of water, proteins, and electrolytes, and resistance and reactance are used to estimate total body water, and by extension, fat mass and lean mass, with the latter including bone . / l) was measured by a solid - phase, two - site chemiluminescent enzyme immunometric assay (immulite, euro / dpc) with a sensitivity of 0.01 g / l and with intrassay coefficients of variation of 5.3%, 6.0%, and 6.5% for low, medium, and high plasma gh levels, respectively; and with interassay coefficients of variation of 6.5%, 5.5%, and 6.6% for low, medium, and high plasma gh levels, respectively . Igf - i (ng / ml) was determined by a chemiluminescence assay (nichols institute, san clemente, ca, usa) and with intrassay coefficients of variation of 4.8%, 5.2%, and 4.4% for low, medium and high plasma igf - i levels, respectively, and with interassay coefficients of variation of 7.7%, 7.4%, and 4.7% for low, medium, and high plasma igf - i levels, respectively . Insulin (u / ml) was measured with a solid - phase two - site chemiluminescent immunometric assay (immulite 2000 insulin, dpc, los angeles, ca, usa) and with intrassay coefficients of variation of 5.5%, 3.3%, and 3.7% for low, medium and high plasma insulin levels, respectively, and with interassay coefficients of variation of 7.3%, 4.1%, and 5.3% for low, medium, and high plasma insulin levels, respectively . Leptin (ng / ml) was measured by radioimmunoassay (mediagnost, tubigen, germany) and with intrasay and interassay coefficients of variation of 5.3% and 13.6%, respectively . Glucose, total, hdl, and ldl cholesterol, triglycerides, glutamic oxalacetic transferase (got), glutamic pyruvic transferase (gpt), and gamma glutamyl transferase (ggt) were measured using standard laboratory previously described methods . All samples from a given subject were analysed in the same assay run . The area under the secretory curve (auc) was calculated by a trapezoidal method . Insulin resistance was calculated on the basis of fasting values of plasma glucose and insulin, according to the homeostasis model assessment (homa - ir) method as follows: homa - ir = fasting insulin levels fasting glucose levels/22.5, where basal insulin levels is in u / ml, and glucose is in mmol / l . The results are presented as mean values sem . We use mean values sem . The spss software 12.0 (chicago, il, usa) clinical characteristics, cardiovascular risk markers, and hepatic aminotransferases are shown in table 1 . The age range of study participants was 3248 years, and the bmi ranged from to 30.4 to 52.2 kg / m . Subjects were arbitrarialy classified as meeting the criteria for gh deficiency (ghd) when peak gh after stimulation with ghrh was 3 ng / ml; although ghrh alone is not a well - validated test for the diagnosis of gh deficiency, this cutoff point is based on standard criteria used to diagnose adults with hypopituitarism after various stimuli [37, 38]. Gh secretion after stimulation with ghrh at the different time points in the entire group is shown on figure 1 . Clinical characteristics, cardiovascular risk markers, and hepatic aminotransferases in gh - deficient and gh - sufficient obese women are presented in table 2 . 23% of obese women met our stablished criteria for ghd based on the response to ghrh . The group of the patients classified as meeting hypopituitary ghd criteria had a mean bmi of 45.5 6.3 kg / m, with a midwaist circumference of 117 9.2 cm, significantly higher than in obese patients with gh sufficiency (p = .007 for bmi and p = .039 for midwaist cicumference). / l had a tendency to a higher mean age than the group of patients with peak gh after stimulation> 3 g / l, 42.3 9.0 versus 30.4 9.4, respectively, although the difference was not significant . When the patients were divided in two groups, above or below bmi median, peak gh secretion was 15.2 2.0 and 5.6 1.8 (g / l) for patients above or below the median, respectively (p = .005) (figure 2). Bmi was strongly and inversely associated with peak gh after stimulation with ghrh (r = 0.721, p = .022). Peak gh secretion was strongly and inversely associated with waist circumference (r = 0.576, p = .020), (r = 0.748, p = .001), and trucal fat (r = 0.595, p = .019). Mean levels of cardiovascular risk markers, hepatic aminotransferases, and insulin resistance were compared between in ghd and gh - sufficient obese patients (table 2). Mean total and ldl cholesterol were higher, in subjects who would have been classified as ghd based on hypopitutary criteria compared with gh - sufficient subjects (p = .039 and p = .044, resp .) Got, gpt, and ggt, were all higher, in subjects who would have been classified as ghd based on hypopitutary criteria when compared with gh - sufficient subjects (p = .012, p = .022, and p = .007, resp .) (figure 4). Mean fasting insulin and homa - ir were higher, in the group of patients with peak gh after stimulation 3 g / l when compared with the group of patients with peak gh after stimulation> 3 g / l (p = .005 and p = .018, resp .) (figure 5). These differences were no longer significant after controlling for measures of adiposity whether bmi, mid - waist circumference, or trunk fat . Correlations between gh secretion or igf - i and cardiovascular risk markers or aminotransferases are shown in table 3 . Peak gh secretion after stimulation with ghrh was inversely associated with total (r = 0.532, p = .034) and ldl cholesterol (r = 0.692, p = .006) and positively associated with hdl cholesterol (r = 0.561, p = .037). Peak gh secretion after stimulation with ghrh was inversely associated with serum hepatic aminotransferase, got (r = 0.685, p = .020), gpt (r = 0.656, p = .011), and ggt (r = 0.642, p = .018). Peak gh secretion after stimulation with ghrh was inversely associated with fasting insulin (r = 0.650, p = .012) and homa - ir (r = 0.846, p = .001). These correlations were no longer significant after controlling for measures of adiposity whether bmi, mid - waist circumference, or trunk fat . Igf - i was strongly and inversely associated with bmi (r = 0.577, p = .024) and percentage of total body fat (r = 0.728, p = .002). Igf - i was strongly negatively associated with ldl cholesterol (r = 0.725, p = .005) and positively associated with hdl - cholesterol (r = 0.568, p = .043). Igf - i was strongly negatively associated with homa - ir (r = 0.613, p = .034). These correlations were no longer significant after controlling for measures of adiposity whether bmi, mid - waist circumference, or trunk fat . Homa - ir was strongly and positively associated with serum hepatic aminotransferases, got (r = 0.821, p = .023), gpt (r = 0.705, p = .023), and ggt (r = 0.693, p = .026). We have found a strong inverse relationship between bmi or indices of central adiposity and peak - stimulated gh levels . The presence of decreased gh secretion is associated with a deleterious cardiovascular marker risk profile, increases in total and ldl cholesterol . We report strong inverse associations of gh peak after stimulation by ghrh in a group of obese otherwise healthy premenopausal women with traditional lipoprotein cardiovascular risk markers . Our results are similar to those of utz et al ., although they found significant differences with other cardiovascular risk factors, mainly nontraditional, in that study hdl was lower, hscrp (high - sensitivity c - reactive protein), and tnf- receptor i higher, in young overweight or obese women meeting ghd criteria than in women with gh sufficiency . Data, in this study the increase in total and ldl cholesterol was no longer significant after controlling for measures of adiposity whether bmi, mid - waist circumference, or trunk fat . These differences could be due in part to the different stimulus employed or to the different patient group; we include only obese women without other disease, or to the use of indirect indices of visceral fat . We think that all these data raise the question of whether decreased endogenous gh secretion may contribute to an elevated risk of future cardiovascular events in young obese women . The primary cause of the impaired gh secretion of obesity could be an altered hypothalamus, abnormal pituitary function, or a perturbation of the peripheral signals acting at either the pituitary or hypothalamic level . There are different studies that suggest that the pathophysiological mechanism responsible for the gh hyposecretion of obesity is probably multifactorial; there is a chronic state of somatostatin hypersecretion, increased ffa and decreased ghrelin [3944]. Quite surprisingly, we found in the present study that decreased gh secretion was associated with increased levels of serum hepatic aminotransferases . We report a strong inverse association of gh peak after stimulation by ghrh in a group of obese otherwise healthy premenopausal women with increased hepatic aminotransferases . As far as we know, there has not been previously published such a relationship . Although there are some recent data that suggest that gh deficiency could participate in the pathogenesis of nonalcoholic fatty liver disease . We hypothesize that the mechanism of that correlation is a manifestation of hepatic insulin resistance . It has been found that the higher the bmi, the higher the prevalence of elevated liver enzymes, and the presence of insulin resistance was highly predictive of elevated hepatic enzymes, suggesting an important role for insulin resistance in nonalcoholic fatty liver disease . In our study the presence of decreased gh secretion is associated with increased serum insulin levels and insulin resistance indices . Moreover, we report a strong inverse association of gh peak after stimulation with ghrh and the insulin resistance indices, homa - ir . The importance of fasting insulin in gh secretion has been found in some studies [29, 47]. There is also clinical and experimental evidence suggesting an important role for insulin as a direct inhibitor of gh secretion . All these data suggest that insulin resistance could be another factor responsible for the altered gh secretion of obesity . In accordance with previous studies [50, 51] we have found that in the presence of decreased gh secretion, igf - i levels were normal, although igf - i was positively associated with peak gh secretion . But igf - i was strongly and inversely associated with bmi and insulin resistance (homa - ir). These data suggest, on one hand, that igf - i is still gh dependent in obesity, but that there are other factors such as insulin or others that regulate igf - i levels . On the other hand that the decreased gh secretion of obesity is not due to increased igf - i levels as previously suggested . There are many common features between subjects with ghd due to pituitary disease and subjects with a clustering of cardiovascular risk factors in the absence of pituitary disease, but it is not clear whether the low gh associated with these cardiovascular risk factors is cause or effect . Low gh in the setting of obesity has been shown to be reversed with weight reduction, implying that it is obesity that causes the low gh [19, 30]. By contrast, the observation that subjects with ghd due to pituitary or hypothalamic disease develop obesity and its metabolic consequences [13], and that some cardiovascular risk factors are inversely related to gh independently of obesity indices, suggests that gh plays a physiological role in the development of obesity and its cardiovascular complications . This distinction is important in our understanding of the role of gh in the development and maintenance of cardiovascular risk factors and may have therapeutic implications . Although our data do not differentiate between low gh being a cause or an effect of these cardiovascular risk factors, they indicate that the relationship between low gh and increased cardiovascular risk may be physiologically important in the absence of pituitary disease . Another limitation that should be considered in this study is the lack of a control lean group . These data in a group of obese young women showing a clear correlation of low gh to individual cardiovascular risk factors suggest an important role for gh in the clustering of these risk factors, whether or not the relationship is a causative one . Analogous to the gradual acceptance of a wide variety of what are now considered traditional cardiovascular risk factors, perhaps it is time to consider low gh as an independent marker for cardiovascular disease . In conclusion, we have found that obese premenopausal women with decreased ghrh - induced gh secretion when compared with obese patients with gh sufficiency, have increased total cholesterol, ldl cholesterol, liver enzymes, fasting insulin and homa - ir, when compared with obese patients with gh sufficiency . There was a significant negative correlation between peak gh secretion and total cholesterol, ldl cholesterol, liver enzymes, fasting insulin or homa - ir . There was a significant positive correlation between igf - i and peak gh and a negative correlation between igf and homa - ir, but igf - i levels we think that these data strongly suggest a role for insulin resistance in the decreased gh secretion of obesity, and the concept that the blunted gh secretion of central obesity could be the pituitary expression of the insulin resistance (metabolic) syndrome.
Prepare lb - agar by dissolving 2.5 g lb and 1.5 g agar in 100 ml dh2o . Autoclave and cool to an estimated 50 - 55 c, which as a rule of thumb, is when the flask can be held comfortably . Syringe filter and freeze unused aliquots . Add 100 l of amp stock (final concentration 50 g / ml) to the lb - agar from 1.1 . Swirl to mix and pour into 10 cm bacterial dishes (15 - 20 ml / dish). Allow it to solidify (15 - 30 min .) And store unused plates inverted at 4 c for 2 - 3 weeks . To transform e. coli, quickly thaw an aliquot of dh5 competent cells in an ice bath . Flick the tube to mix and incubate on ice for 30 minutes . Heat shock at 42 c for 45 seconds and place back on ice for 2 minutes . Add 900 l soc medium and grow for one hour at 37 c with shaking . Spread 50 - 100 l of the transformed bacteria on an lb - agar - amp plate using a bent sterile pasteur pipette . Incubate the plate right side up in a 37 c incubator for 5 minutes and then invert and grow overnight . A single colony will be picked from the plate for preparation of the gst - tagged protein (step 2.1). For future use, in addition, bacterial stocks can be prepared for more prolonged storage by growing individual colonies in 2 ml sterile lb - amp overnight at 37 c with shaking . Mix an aliquot with sterile 80% glycerol in a 1:1 ratio and freeze at -80 c . When cool, add 50 l amp from stock to 50 ml lb (50 g / ml final concentration). Inoculate with a well isolated colony of transformed bacteria and grow overnight at 37 c with agitation . When at full density (od600> 1.0) dilute with 450 ml lb - amp and grow for an additional 30 minutes at 37 c . Prepare a 100 mm stock solution of isopropyl b - d - thiogalactopyranoside (iptg) by dissolving 0.238 g in 10 ml dh2o . Induce bacteria to produce rho protein by adding 500 l 100 mm iptg to 500 ml culture (a final concentration of 100 m). If needed, the 500 ml culture can be divided into 50 ml tubes for centrifugation . Freeze pellet(s) for at least 1 hour (or preferably overnight) at -80 c . Prepare 200 ml lysis buffer containing 20 mm hepes (0.95 g)/ ph 7.5; 150 mm nacl (1.75 g); 5 mm mgcl2 (0.203 g); 1% tx-100 (2 ml). Prepare stock solutions of 1 m dtt (1.542 g in 10 ml dh2o) and 100 mm pmsf (0.174 g/10 ml etoh). To prepare lysis buffer +, supplement 10 ml with 1 mm dtt (10 l of stock) and 1 mm pmsf (100 l of stock) and one complete mini protease inhibitor tablet . Working on ice, spin the sonicated lysate at 15,000 - 20,000 g for 15 minutes at 4 c, and remove the clarified sonicate (supernatant) to a sterile capped 15 ml tube . Prepare the glutathione sepharose by gently mixing the original tube containing a 75% slurry and transfer 335 l into a 15 ml tube . Use a wide bore tip to pipette beads . Add 10 ml cold pbs, and spin 500 g for 5 minutes at 4 c . Discard the supernatant, add 1 ml lysis buffer+ to the beads and spin as for previous wash . Prepare 500 ml hbs containing 20 mm hepes (2.38 g)/ph 7.5 and 150 mm nacl (4.38 g) in dh2o . Prepare stock solutions of 1 m mgcl2 (0.952 g in 10 ml dh2o) and 1 m dtt (1.542 g in 10 ml dh2o). To prepare hbs+, supplement 100 ml just before use with 5 mm mgcl2 (50 l from stock) and 1 mm dtt (100 l from stock). Spin the beads from step 2.7 at 500 g for 5 minutes at 4 c . Discard the supernatant and wash beads 2x with 10 ml lysis buffer+, and 2x with 10 ml hbs+ . After the final wash, make a 50% slurry by resuspending the beads in hbs+ supplemented with bd baculogold protease inhibitor (20 l of 50x bd baculogold / ml). Dilute 10 l of the final beads preparation with 2x laemmli sample buffer containing -mercaptoethanol . Make bovine serum albumin (bsa) standards . Use a 2 mg / ml stock (0.02 g of bsa in 10 ml dh2o). Mix 10 l of stock with 10 l laemmli (20 g final); 5 l of stock with 5 l of dh2o and 10 l laemmli (10 g final); and 2.5 l stock with 7.5 l dh2o and 10 l laemmli (5 g final). Spin the bead sample and run supernatant with bsa standards and molecular weight markers on a 10% sds - polyacrylamide gel . Prepare the comassie blue stain (0.1 g in 10 ml acetic acid, 40 ml methanol and 50 ml dh2o) and the destain solution (500 ml dh2o, 400 ml methanol and 100 ml acetic acid). Store at room temperature . Stain the gel for 20 - 30 minutes, remove the dye (it can be reused multiple times) and rinse with destain solution twice . Continue to destain with gentle shaking for several hours until bands are clearly visible . Estimate the concentration of gst - rhoa(g17a) coupled to the beads using the bsa standards as a reference (fig 2). Aliquot an equal volume of beads containing ~10 - 15 g protein into 1.5 ml micro centrifuge tubes . Freeze at -80 c in hbs+/glycerol in a 3:1 ratio to use within a few days . Serum deprive for at least 3 hours and treat as required . Prepare lysis buffer+ as in step 2.4 . Prepare enough lysis buffer for 700 l / dish plus some extra amount to allow for pipetting errors . Working on ice, remove culture medium from the dishes and wash with ice - cold hbs . Swirl plates to cover all areas, scrape and collect lysates into numbered 1.5 ml tubes . If your cell number is equivalent in all dishes being tested, you can omit doing a protein assay, and move to step 3.5 . Otherwise measure the protein concentration of each supernatant using bio - rad quick protein assay and equalize the supernatant for volume and concentration . The amount of total protein depends on the cell types used (typically 1 - 1.5 mg protein for llc - pk1 cells). Remove 30 l of each supernatant and mix with 30 l 2x reducing laemmli sample buffer, boil and set aside for step 3.7 . Add remaining supernatants to aliquots of the gst - rhoa(g17a) beads from step 2.12 . Discard the supernatant and wash the beads 3x with lysis buffer, spinning in the same way between washes . Completely remove the final wash using a 1 cc syringe fitted with a 30 g needle and add 20 l 2x reducing laemmli sample buffer . Spin to pellet beads and either run the supernatant immediately (preferable) or store it at -80 c for later analysis . Run 20 l total cell lysates and all of the precipitated protein samples on the appropriate percentage sds - polyacrylamide gel for the size of gef you are studying . Part 1 and 2 of the protocol describes preparation of gst - rhoa(g17a) coupled to gsh - sepharose beads and its testing by sds - page (see outline of protocol on fig 1). The sample with the eluted protein should contain a single band at approximately 50 kda (fig 2, lane 6). The concentration of the protein can be estimated using the bsa reference samples . In the example on fig 2, the concentration of rhoa(g17a) the typical yield in our hand is 15 - 20 g protein from 10 ml bacterial lysis . Part 3 of the protocol describes the affinity precipitation assay (see overview on fig 1). A successful gef assay detecting activation of the exchange factor gef - h1 is shown on fig 3 . The rhoa(g17a) protein captured some gef - h1 from the control (untreated) cell lysates, suggesting that gef - h1 has basal activity . The amount precipitated however increases in cells treated with the inflammatory cytokine tumor necrosis factor - (tnf-), consistent with the notion that tnf- activates gef - h1 . Importantly, the total cell lysates show similar amounts of gef - h1 in the control and the treated sample, suggesting that the treatment did not alter gef - h1 levels and the input used in the assay is equal . Representative result of the bead preparation protocol . A coomassie stained gel with successful gst - rhoa(g17a) bead preparation is shown . Bead sample and bsa protein standards were separated by sds - page using a 10% acrylamide gel . To test the beads, 10 l of the final bead slurry containing gst - rhoa(g17a) the following samples were loaded: lane 1: molecular weight marker (mw) (froggabio blueye prestained protein ladder); lanes 2 - 4: 5, 10 and 20 g bovine serum albumin (bsa); lane 5 is empty; lane 6: 10 l of the freshly prepared rhoa(g17a) beads . After separation is completed, the gel is stained using coomassie blue and subsequently destained to reveal proteins . The molecular weight of the gst - rhoa(g17a) protein is roughly 50 kda and runs around the level of the 48 kda marker . The concentration of the gst - rho protein in this particular sample is estimated to be around 15 g/10 l slurry . Confluent llc - pk1 cells were treated with 10 ng / ml tnf- for 5 minutes . Following treatment the cells were lysed and active gefs were captured using gst - rhoa(g17a) bound beads . The presence of gef - h1 in the precipitated proteins (top blot) and total cell lysate samples (bottom blot) was detected using western blotting with an anti - gef - h1 antibody (cell signaling). Please note the increased amount of precipitated gef - h1 in tnf--treated versus non - treated cells relative to the equivalent inputs, indicating a successful result . Although the gef pulldown assay shown here resulted in some gef - h1 captured by the beads, the amounts precipitated from control and tnf--treated cells are the same . Thus tnf-, a know activator of gef - h1 in this case did not induce activation . In this particular experiment subsequent troubleshooting suggested that the tnf- used was not fresh enough and probably degraded . Prepare lb - agar by dissolving 2.5 g lb and 1.5 g agar in 100 ml dh2o . Autoclave and cool to an estimated 50 - 55 c, which as a rule of thumb, is when the flask can be held comfortably . Syringe filter and freeze unused aliquots . Add 100 l of amp stock (final concentration 50 g / ml) to the lb - agar from 1.1 . Swirl to mix and pour into 10 cm bacterial dishes (15 - 20 ml / dish). Allow it to solidify (15 - 30 min .) And store unused plates inverted at 4 c for 2 - 3 weeks . To transform e. coli, quickly thaw an aliquot of dh5 competent cells in an ice bath . Flick the tube to mix and incubate on ice for 30 minutes . Heat shock at 42 c for 45 seconds and place back on ice for 2 minutes . Add 900 l soc medium and grow for one hour at 37 c with shaking . Spread 50 - 100 l of the transformed bacteria on an lb - agar - amp plate using a bent sterile pasteur pipette . Incubate the plate right side up in a 37 c incubator for 5 minutes and then invert and grow overnight . A single colony will be picked from the plate for preparation of the gst - tagged protein (step 2.1). For future use, in addition, bacterial stocks can be prepared for more prolonged storage by growing individual colonies in 2 ml sterile lb - amp overnight at 37 c with shaking . Mix an aliquot with sterile 80% glycerol in a 1:1 ratio and freeze at -80 c . Prepare lb by adding 25 g lb to 1 l dh20 and autoclaving . When cool, add 50 l amp from stock to 50 ml lb (50 g / ml final concentration). Inoculate with a well isolated colony of transformed bacteria and grow overnight at 37 c with agitation . When at full density (od600> 1.0) dilute with 450 ml lb - amp and grow for an additional 30 minutes at 37 c . Prepare a 100 mm stock solution of isopropyl b - d - thiogalactopyranoside (iptg) by dissolving 0.238 g in 10 ml dh2o . Induce bacteria to produce rho protein by adding 500 l 100 mm iptg to 500 ml culture (a final concentration of 100 m). If needed, the 500 ml culture can be divided into 50 ml tubes for centrifugation . Freeze pellet(s) for at least 1 hour (or preferably overnight) at -80 c . Prepare 200 ml lysis buffer containing 20 mm hepes (0.95 g)/ ph 7.5; 150 mm nacl (1.75 g); 5 mm mgcl2 (0.203 g); 1% tx-100 (2 ml). Prepare stock solutions of 1 m dtt (1.542 g in 10 ml dh2o) and 100 mm pmsf (0.174 g/10 ml etoh). To prepare lysis buffer +, supplement 10 ml with 1 mm dtt (10 l of stock) and 1 mm pmsf (100 l of stock) and one complete mini protease inhibitor tablet . Working on ice, spin the sonicated lysate at 15,000 - 20,000 g for 15 minutes at 4 c, and remove the clarified sonicate (supernatant) to a sterile capped 15 ml tube . Prepare the glutathione sepharose by gently mixing the original tube containing a 75% slurry and transfer 335 l into a 15 ml tube . Use a wide bore tip to pipette beads . Add 10 ml cold pbs, and spin 500 g for 5 minutes at 4 c . Discard the supernatant, prepare 500 ml hbs containing 20 mm hepes (2.38 g)/ph 7.5 and 150 mm nacl (4.38 g) in dh2o . Prepare stock solutions of 1 m mgcl2 (0.952 g in 10 ml dh2o) and 1 m dtt (1.542 g in 10 ml dh2o). To prepare hbs+, supplement 100 ml just before use with 5 mm mgcl2 (50 l from stock) and 1 mm dtt (100 l from stock). Spin the beads from step 2.7 at 500 g for 5 minutes at 4 c . Discard the supernatant and wash beads 2x with 10 ml lysis buffer+, and 2x with 10 ml hbs+ . After the final wash, make a 50% slurry by resuspending the beads in hbs+ supplemented with bd baculogold protease inhibitor (20 l of 50x bd baculogold / ml). Dilute 10 l of the final beads preparation with 2x laemmli sample buffer containing -mercaptoethanol . Make bovine serum albumin (bsa) standards . Use a 2 mg / ml stock (0.02 g of bsa in 10 ml dh2o). Mix 10 l of stock with 10 l laemmli (20 g final); 5 l of stock with 5 l of dh2o and 10 l laemmli (10 g final); and 2.5 l stock with 7.5 l dh2o and 10 l laemmli (5 g final). Spin the bead sample and run supernatant with bsa standards and molecular weight markers on a 10% sds - polyacrylamide gel . Prepare the comassie blue stain (0.1 g in 10 ml acetic acid, 40 ml methanol and 50 ml dh2o) and the destain solution (500 ml dh2o, 400 ml methanol and 100 ml acetic acid). Store at room temperature . Stain the gel for 20 - 30 minutes, remove the dye (it can be reused multiple times) and rinse with destain solution twice . Continue to destain with gentle shaking for several hours until bands are clearly visible . Estimate the concentration of gst - rhoa(g17a) coupled to the beads using the bsa standards as a reference (fig 2). Aliquot an equal volume of beads containing ~10 - 15 g protein into 1.5 ml micro centrifuge tubes . Freeze at -80 c in hbs+/glycerol in a 3:1 ratio to use within a few days . Serum deprive for at least 3 hours and treat as required . Prepare lysis buffer+ as in step 2.4 . Prepare enough lysis buffer for 700 l / dish plus some extra amount to allow for pipetting errors . Working on ice, remove culture medium from the dishes and wash with ice - cold hbs . Swirl plates to cover all areas, scrape and collect lysates into numbered 1.5 ml tubes . If your cell number is equivalent in all dishes being tested, you can omit doing a protein assay, and move to step 3.5 . Otherwise measure the protein concentration of each supernatant using bio - rad quick protein assay and equalize the supernatant for volume and concentration . The amount of total protein depends on the cell types used (typically 1 - 1.5 mg protein for llc - pk1 cells). Remove 30 l of each supernatant and mix with 30 l 2x reducing laemmli sample buffer, boil and set aside for step 3.7 . Add remaining supernatants to aliquots of the gst - rhoa(g17a) beads from step 2.12 . Discard the supernatant and wash the beads 3x with lysis buffer, spinning in the same way between washes . Completely remove the final wash using a 1 cc syringe fitted with a 30 g needle and add 20 l 2x reducing laemmli sample buffer . Spin to pellet beads and either run the supernatant immediately (preferable) or store it at -80 c for later analysis . Run 20 l total cell lysates and all of the precipitated protein samples on the appropriate percentage sds - polyacrylamide gel for the size of gef you are studying . Part 1 and 2 of the protocol describes preparation of gst - rhoa(g17a) coupled to gsh - sepharose beads and its testing by sds - page (see outline of protocol on fig 1). The sample with the eluted protein should contain a single band at approximately 50 kda (fig 2, lane 6). The concentration of the protein can be estimated using the bsa reference samples . In the example on fig 2, the concentration of rhoa(g17a) the typical yield in our hand is 15 - 20 g protein from 10 ml bacterial lysis . Part 3 of the protocol describes the affinity precipitation assay (see overview on fig 1). A successful gef assay detecting activation of the exchange factor gef - h1 is shown on fig 3 . The rhoa(g17a) protein captured some gef - h1 from the control (untreated) cell lysates, suggesting that gef - h1 has basal activity . The amount precipitated however increases in cells treated with the inflammatory cytokine tumor necrosis factor - (tnf-), consistent with the notion that tnf- activates gef - h1 . Importantly, the total cell lysates show similar amounts of gef - h1 in the control and the treated sample, suggesting that the treatment did not alter gef - h1 levels and the input used in the assay is equal . Representative result of the bead preparation protocol . A coomassie stained gel with successful gst - rhoa(g17a) bead preparation is shown . Bead sample and bsa protein standards were separated by sds - page using a 10% acrylamide gel . To test the beads, 10 l of the final bead slurry containing gst - rhoa(g17a) is diluted 1:1 with reducing laemmli sample buffer and boiled for 5 minutes . The following samples were loaded: lane 1: molecular weight marker (mw) (froggabio blueye prestained protein ladder); lanes 2 - 4: 5, 10 and 20 g bovine serum albumin (bsa); lane 5 is empty; lane 6: 10 l of the freshly prepared rhoa(g17a) beads . After separation is completed, the gel is stained using coomassie blue and subsequently destained to reveal proteins . The molecular weight of the gst - rhoa(g17a) protein is roughly 50 kda and runs around the level of the 48 kda marker . The concentration of the gst - rho protein in this particular sample is estimated to be around 15 g/10 l slurry . Confluent llc - pk1 cells were treated with 10 ng / ml tnf- for 5 minutes . Following treatment the cells were lysed and active gefs were captured using gst - rhoa(g17a) bound beads . The presence of gef - h1 in the precipitated proteins (top blot) and total cell lysate samples (bottom blot) was detected using western blotting with an anti - gef - h1 antibody (cell signaling). Please note the increased amount of precipitated gef - h1 in tnf--treated versus non - treated cells relative to the equivalent inputs, indicating a successful result . Although the gef pulldown assay shown here resulted in some gef - h1 captured by the beads, the amounts precipitated from control and tnf--treated cells are the same . Thus tnf-, a know activator of gef - h1 in this case did not induce activation . In this particular experiment subsequent troubleshooting the method presented here is the only available non - radioactive activation assay for gefs that can follow the active pool of gefs in cells . The assay is similar to the precipitation assays used for following activation of small gtpases as well as gefs against rac and cdc42 . Those assays use different gst - tagged proteins and have slight differences from the one described here, however the basic steps are the same . Thus, this protocol can easily be adapted for other small gtpase and gef activation assays . The presented gef assay was recently modified for application for nuclear fractions . With further modifications, testing of gef activation in other subcellular compartments we use the presented method to study activation of gefs in epithelial cell lines . With some optimization when adapting to a specific cell type, find the optimal cell number, lysis buffer volume, and detection method for the gef to be tested (a good antibody for western blotting is important). For initial setup of the assay it is advisable to use a stimulus that is known to activate the gef of interest . When using an unknown stimulus, always use a positive control to verify that your assay is working . For this, captured gefs from control and stimulated samples should be analyzed on a coomassie - stained gel . Bands that appear only in stimulated samples might contain activated gefs and can be sent for identification by mass spectrometry (e.g.). Finally, a cautionary note . The assay is based on the assumption that the posttranslational modifications rendering gefs active are preserved after cell lysis . Indeed, this is clearly the case for a number of gefs, and since its establishments, this assay has been used by various groups to detect activation of different gefs, including gef - h1, p115rhogef and xpln (e.g.). Preservation of the active state however might not happen in the case of all gefs, and therefore it is conceivable that this assay will not work for all gefs . It also has to be noted, that many gefs exert activity towards more than one small gtpases . Thus, when a specific gef is studied, it is recommended to complement this assay with gef precipitation assays for other small gtpases, as well as functional studies examining rhoa, rac1 and cdc42 . Colonies of transformed bacteria should be picked from fresh, properly prepared plates to ensure adequate selection by amp, good outgrowth and yield . Transformation conditions for competent cells obtained from other sources may vary and should be consulted . All steps of the protein preparation protocol (from step 2.3) and the assay (from step 3.2) should be performed at 4c with cooled solutions and centrifuges . Bacterial lysis (step 2.5) should be thorough and complete in order to obtain a homogeneous suspension . When lysing the bacteria, vortex and pipette the lysate alternately, while maintaining it at 4c and ensure sonication is done on ice to prevent denaturing the protein . Incubation of sonicate with the beads should always be done at 4c on a rotator to ensure sufficient binding, and care should be taken to keep the timing consistent . Gst - rho mutants are somewhat unstable when expressed in bacteria, so it is best to use prepared beads right away or within a few days . The precipitation assay (part 3) is time and temperature sensitive, as active gefs can be easily lost from the cell lysate, so steps should be performed as quickly as possible . No or very low amount of mutant rho protein in the final bead preparation: this may be caused by inefficient induction, insufficient lysis of the bacteria, or a loss of the protein during the preparation process or storage . To help troubleshoot some of these possibilities, samples of bacteria can be analyzed before and after induction . If there is poor induction of the protein repeat the process using a colony from a freshly streaked plate or from re - transformed competent cells . If the lysis is insufficient (i.e. The protein remains in the pellet instead of the supernatant) varying salt and detergent concentrations in the lysis buffer can be tried . Alternate sonication times and settings should be considered, and samples before and after sonication can be checked by microscopy to determine efficiency of lysis . No precipitated gef, even though the gst - protein is present on the beads: this may be due to technical issues during the precipitation assay, or by a real absence of activation of the studied gef using the stimulus applied . Always use a known stimulus as a positive control to verify that your assay works . If the precipitation assay captures undetectable amounts of the gef studied in all conditions, verify that your gef is present and is well detectable in the supernatant after centrifugation that is to be used for the assay (step 3.3). Make sure all buffers and protease inhibitors are fresh, and perform all steps on ice as fast as possible . Increase the amount of input protein (e.g. By using lysates from 2 plates / sample). If the precipitation assay shows basal precipitation of your gef, but no difference is seen between the control and stimulated samples (fig 4), start troubleshooting by verifying that the applied stimulus worked using other known effects (e.g. By detecting activation of other signaling pathways). Rely on data from the literature reporting how your stimulus activates rho or other signaling to predict likely times and concentrations . When optimizing treatment time, use both short and long time points, as gef activation might be best detectable at a time point prior to well detectable rho activation . Finally, the same stimulus could result in a variable degree of activation due to a change in cell responsiveness caused by passage number, cell confluency, etc . Colonies of transformed bacteria should be picked from fresh, properly prepared plates to ensure adequate selection by amp, good outgrowth and yield . Transformation conditions for competent cells obtained from other sources may vary and should be consulted . All steps of the protein preparation protocol (from step 2.3) and the assay (from step 3.2) should be performed at 4c with cooled solutions and centrifuges . Bacterial lysis (step 2.5) should be thorough and complete in order to obtain a homogeneous suspension . When lysing the bacteria, vortex and pipette the lysate alternately, while maintaining it at 4c and ensure sonication is done on ice to prevent denaturing the protein . Incubation of sonicate with the beads should always be done at 4c on a rotator to ensure sufficient binding, and care should be taken to keep the timing consistent . Gst - rho mutants are somewhat unstable when expressed in bacteria, so it is best to use prepared beads right away or within a few days . The precipitation assay (part 3) is time and temperature sensitive, as active gefs can be easily lost from the cell lysate, so steps should be performed as quickly as possible . No or very low amount of mutant rho protein in the final bead preparation: this may be caused by inefficient induction, insufficient lysis of the bacteria, or a loss of the protein during the preparation process or storage . To help troubleshoot some of these possibilities, samples of bacteria can be analyzed before and after induction . If there is poor induction of the protein repeat the process using a colony from a freshly streaked plate or from re - transformed competent cells . If the lysis is insufficient (i.e. The protein remains in the pellet instead of the supernatant) varying salt and detergent concentrations in the lysis buffer can be tried . Alternate sonication times and settings should be considered, and samples before and after sonication can be checked by microscopy to determine efficiency of lysis . No precipitated gef, even though the gst - protein is present on the beads: this may be due to technical issues during the precipitation assay, or by a real absence of activation of the studied gef using the stimulus applied . Always use a known stimulus as a positive control to verify that your assay works . Use the prepared beads within a few days . If the precipitation assay captures undetectable amounts of the gef studied in all conditions, verify that your gef is present and is well detectable in the supernatant after centrifugation that is to be used for the assay (step 3.3). Make sure all buffers and protease inhibitors are fresh, and perform all steps on ice as fast as possible . Increase the amount of input protein (e.g. By using lysates from 2 plates / sample). If the precipitation assay shows basal precipitation of your gef, but no difference is seen between the control and stimulated samples (fig 4), start troubleshooting by verifying that the applied stimulus worked using other known effects (e.g. By detecting activation of other signaling pathways). Rely on data from the literature reporting how your stimulus activates rho or other signaling to predict likely times and concentrations . When optimizing treatment time, use both short and long time points, as gef activation might be best detectable at a time point prior to well detectable rho activation . Finally, the same stimulus could result in a variable degree of activation due to a change in cell responsiveness caused by passage number, cell confluency, etc.
We searched the pubmed, cinahl, web of science, and cochrane databases from inception to 2009 using mesh terms vasovagal syncope drug therapy adrenergic beta antagonists and individually using the key words neurocardiogenic syncope treatment metoprolol atenolol nebivolol bisoprolol acebutalol betaxolol and beta blocker . Hsrproj and national research register databases were used to identify any grey literature . The aim was to include randomized controlled studies that studied the clinical efficacy of oral b1 blockade compared to placebo in patients above 18 years of age with a diagnosis of neurocardiogenic syncope . Since there is no therapy with proven efficacy in this disorder, we did not include studies that compared b1 blockers with another drug . Due to the absence of a gold standard for the diagnosis of this condition, a diagnosis of neurocardiogenic syncope as determined by the study investigators was considered acceptable as case definition . A careful cardiovascular and neurological investigation was considered essential inclusion criteria before such a diagnosis could be made . Clinical recurrence was defined as occurrence of syncope while on therapy or a lack of perceived benefit . Four of these studies compared b1 blockade with placebo47 and one each with clonidine,8 propranolol,9 and no therapy.10 only studies that included a placebo arm were included in the analysis . In the study by brignole et al only the patients who underwent randomization were included in the analysis.4 perceived lack of improvement was the outcome studied by mahanonda et al6 while other studies reported rates of recurrence . Other than madrid et al,5 studies only included patients who had a positive head up tilt table test as part of the case definition . A score of 3 or more was felt to be criteria for inclusion . After discussion, consensus was reached and the studies included were felt to be of satisfactory quality . Review manager (revman version 5, oxford, england) was used for statistical calculations . Results of individual studies and overall result was expressed as an odds ratio (or) with 95% confidence intervals (ci). I test was used as a measure of heterogeneity and the random effects model was used for analysis when significant heterogeneity (defined as i> 50%) was present . A post - hoc sensitivity analysis was planned in case of significant heterogeneity including studies with similar outcome measures and mean follow up duration . Brignole and colleagues in 1992 randomized patients to drug therapy with various drugs including atenolol, dihydroergotamine, cafedrine, domperidone, and elastic compression stockings with or without drugs to placebo . 4 only the patients treated with atenolol alone (n = 7) were included and compared to the placebo group . The inclusion criteria required subjects to have two consecutive positive upright tilt table tests to be considered for the study . The study did not report a significant difference between any of the drug therapies compared to placebo . Atenolol was also compared to placebo in a randomized controlled study by mahanonda et al.5 patients with a history suggestive of vasovagal syncope were ruled out for structural heart disease and included in the study after a positive isoproterenol tilt table test . In addition to a statistically significant increase in patients reporting feeling better compared to placebo (p = 0.02), patients in the atenolol group had a drop in the number of episodes from 6 9.4/week to 0.6 1.6 per week (p = 0.025). However, in the study by madrid and colleagues, atenolol failed to decrease the recurrence of syncope compared to placebo.6 the median number of syncopal episodes during follow up was 2 in atenolol group and 0 in placebo group (p = 0.215). The largest trial and the only multicenter study to date was performed by sheldon et al who randomized patients with a history of syncope and a positive tilt table test to metoprolol versus placebo.7 metoprolol was no more efficacious than placebo in preventing recurrent syncope both in the intention to treat and on - treatment analysis . Sample size calculation with a study power of 80% was performed and reported in the latter three studies.57 we searched the pubmed, cinahl, web of science, and cochrane databases from inception to 2009 using mesh terms vasovagal syncope drug therapy adrenergic beta antagonists and individually using the key words neurocardiogenic syncope treatment metoprolol atenolol nebivolol bisoprolol acebutalol betaxolol and beta blocker . Hsrproj and national research register databases were used to identify any grey literature . The aim was to include randomized controlled studies that studied the clinical efficacy of oral b1 blockade compared to placebo in patients above 18 years of age with a diagnosis of neurocardiogenic syncope . Since there is no therapy with proven efficacy in this disorder, we did not include studies that compared b1 blockers with another drug . Due to the absence of a gold standard for the diagnosis of this condition, a diagnosis of neurocardiogenic syncope as determined by the study investigators was considered acceptable as case definition . A careful cardiovascular and neurological investigation was considered essential inclusion criteria before such a diagnosis could be made . Clinical recurrence was defined as occurrence of syncope while on therapy or a lack of perceived benefit . Four of these studies compared b1 blockade with placebo47 and one each with clonidine,8 propranolol,9 and no therapy.10 only studies that included a placebo arm were included in the analysis . In the study by brignole et al only the patients who underwent randomization were included in the analysis.4 perceived lack of improvement was the outcome studied by mahanonda et al6 while other studies reported rates of recurrence . Other than madrid et al,5 studies only included patients who had a positive head up tilt table test as part of the case definition . Efficacy data was extracted from the intention to treat analysis of all studies . The number of patients suffering adverse events was also identified . A score of 3 or more was felt to be criteria for inclusion . After discussion, consensus was reached and the studies included were felt to be of satisfactory quality . Review manager (revman version 5, oxford, england) was used for statistical calculations . Results of individual studies and overall result was expressed as an odds ratio (or) with 95% confidence intervals (ci). I test was used as a measure of heterogeneity and the random effects model was used for analysis when significant heterogeneity (defined as i> 50%) was present . A post - hoc sensitivity analysis was planned in case of significant heterogeneity including studies with similar outcome measures and mean follow up duration . Brignole and colleagues in 1992 randomized patients to drug therapy with various drugs including atenolol, dihydroergotamine, cafedrine, domperidone, and elastic compression stockings with or without drugs to placebo . 4 only the patients treated with atenolol alone (n = 7) were included and compared to the placebo group . The inclusion criteria required subjects to have two consecutive positive upright tilt table tests to be considered for the study . The study did not report a significant difference between any of the drug therapies compared to placebo . Atenolol was also compared to placebo in a randomized controlled study by mahanonda et al.5 patients with a history suggestive of vasovagal syncope were ruled out for structural heart disease and included in the study after a positive isoproterenol tilt table test . In addition to a statistically significant increase in patients reporting feeling better compared to placebo (p = 0.02), patients in the atenolol group had a drop in the number of episodes from 6 9.4/week to 0.6 1.6 per week (p = 0.025). However, in the study by madrid and colleagues, atenolol failed to decrease the recurrence of syncope compared to placebo.6 the median number of syncopal episodes during follow up was 2 in atenolol group and 0 in placebo group (p = 0.215). The largest trial and the only multicenter study to date was performed by sheldon et al who randomized patients with a history of syncope and a positive tilt table test to metoprolol versus placebo.7 metoprolol was no more efficacious than placebo in preventing recurrent syncope both in the intention to treat and on - treatment analysis . Sample size calculation with a study power of 80% was performed and reported in the latter three studies.57 there was no demonstrable efficacy of b1 blockers compared to placebo as demonstrated in figure 2 . When the analysis was repeated using a fixed effects model, there was still no statistically significant difference (or = 0.92, ci = 0.591.43, p = 0.7). Using a pre - specified sensitivity analysis, the study by mahanonda et al was excluded due to the shorter follow - up period (1 month), which yielded a i of 0% with an or = 1.18 (ci = 0.731.92, p = 0.5). Further sensitivity analyses not specified before the study were completed in view of these results showing no efficacy . Analysis performed by excluding the study with the lowest number of subjects (brignole et al4 which also did not report a sample size calculation) and by including only studies with atenolol (brignole et al, mahanonda et al and madrid et al) did not reveal any significant benefit from using b1 blockers compared to placebo (or = 0.75, ci = 0.232.40, p = 0.63 and or = 0.69, ci = 0.133.59, p = 0.66 respectively). Significant heterogeneity persisted in both of these analyses (i = 74 and 70% respectively). There were more adverse events in the beta blocker group (15 vs 8, or = 2.03, ci = 0.833.95, p = 0.12). Funnel plot analysis was not performed due to the small number of studies since such an analysis may be misleading.11 some have even questioned the existence of this disorder as a separate entity . In patients who are predisposed to this condition, reduced venous return from the lower extremities causes a decrease in the preload; the resultant decrease in cardiac output and blood pressure causes activation of the baroreceptor reflex.3 this causes an increase in ventricular contractility which is sensed by mechanoreceptors in the heart which project to the medullary dorsal vagal nucleus . A withdrawal of peripheral sympathetic tone and an increase in the vagal tone ensues resulting in vasodilation and bradycardia leading to clinical symptoms . This mechanism however discounts the importance of central mechanisms, which may contribute to syncope . There is some evidence of the importance of serotoninergic pathways which is supported by the evidence of the response of some patients to serotonin reuptake inhibitors.12 several drugs including non selective and selective beta blockers, clonidine, midodrine, fludrocortisone, and ssris have been studied in the treatment of this disorder.5 beta blockers (both selective and non selective) are commonly used medications in the treatment of patients diagnosed with neurocardiogenic syncope . We chose to study the rates of clinical recurrence of syncope while on b1 blocker therapy because of the practical clinical importance rather than study the response to tilt . We found no statistically significant improvement in clinical rates of recurrence of syncope with b1 blockers . The lack of clinical efficacy of b1 blockers may relate partly to the lack of understanding of the pathophysiology of neurocardiogenic syncope . The initial non randomized studies may simply have reflected the placebo nature of the administration of these medications . Adrenoreceptor stimulation in the heart may not play a major role in provoking syncope and thus selective b1 blockers may be ineffective . Non selective beta blockers such as propranolol may in fact be more effective by virtue of inhibition of the vasodilatory effect of activation of b2 receptor.13 in view of this difference; we did not include non selective blockers in this analysis as it would constitute an inhomogeneous treatment group . However, a randomized, placebo controlled study by flevari et al failed to show the efficacy of non selective beta blockers (propranolol and nadolol) compared to placebo.14 haghjoo et al reported similar efficacy of propranolol and metoprolol but the study lacked a placebo arm and the efficacy of these drugs may simply relate to a placebo effect.9 there may be a small subgroup of patients who may benefit from b1 blocker therapy but this has not been adequately represented in these trials . Sheldon et al studied two such subgroups in a prespecified analysis based on age (less than or greater than 42 years) and need for isoproterenol to provoke a positive tilt study and neither group appeared to benefit from b1 blocker therapy . There is evidence for a strong placebo effect in the treatment of neurocardiogenic syncope and the importance of including a placebo arm while studying this disorder has been described.15 this prompted the exclusion of one randomized study (ventura et al) which included a no treatment arm.10 this meta - analysis thus included only a small number of studies (n = 4). We also did not consider studies comparing b1 blockers to other medications (for example, non - selective -blockers) because the comparative drug may in fact be no better than placebo . There was significant heterogeneity among the studies, which resolved with the deletion of one study in the pre specified sensitivity analysis . Atenolol is a hydrophilic b1 blocker while metoprolol by virtue of its lipophilic nature may cross the blood another limitation common to all studies of neurocardiogenic syncope is the variable course of the disease with some patients experiencing long periods of remission between episodes of syncope.16 some patients may even experience spontaneous remission . This complicates the assessment of the clinical efficacy of a drug in the treatment of this condition . We performed this meta analysis to study the clinical efficacy of b1 adrenergic blockade in preventing clinical recurrence of syncope and to determine the rate of adverse events compared to placebo . Our results indicate a lack of clinical efficacy of these drugs and in fact a statistically non significant increase in adverse events . More randomized controlled studies are required to identify other effective therapies for this poorly understood disorder and a placebo arm should be a part of all such studies.
To ensure successful pain relief, in - depth knowledge of local anesthetic solutions and appropriate application of local anesthetic techniques is mandatory . The inferior alveolar nerve block is the most frequently used nerve block technique for anesthetizing mandibular teeth during endodontic procedures . In case of symptomatic irreversible pulpitis the success rate of mandibular anesthesia with the inferior alveolar nerve block has been reported to vary from 38 - 85% . Previous studies have shown success rates of only 19 - 56% for inferior alveolar nerve blocks in patients with irreversible pulpitis . It is evident from these studies that severely painful inflamed tooth is often difficult to manage conventionally and local anesthetic failure is a common occurrence . These include periodontal ligament injection, intrapulpal injection, intraosseous injection, and intraseptal injection . Supplemental injections are essential in patients with irreversible pulpitis as the local anesthetic injections had an eight - fold higher failure rate compared to normal patients . Needleless device uses pressure to force the anesthetic solution safely into oral tissues . In needleless jet injection, the anesthetic solution is immediately taken up by the myelin sheath of the nerve with an onset of action of approximately 1 ms . The x - tip intraosseous injection allows deposition of a local anesthetic solution directly into the cancellous bone adjacent to the tooth to be anesthetized . Success of these supplemental intraosseous injections in achieving pulpal anesthesia in patients with irreversible pulpitis has been reported to be 82 - 98% . The intraosseous systems available commercially in the market include the stabident system (fairfax dental inc ., miami), x - tip system (x - tip technologies, dentsply, maillefer), intraflow (intra vantage, plymouth, mn), and the quicksleeper (dht, cholet, france). Clinical trial studies have been published using stabident, x - tip sytem, intra flow, and the quicksleeper system . The x - tip anesthesia delivery system consists of a special hollow needle that serves as the drill and guide - sleeve component . The drill leads the guide sleeve through the cortical plate after which it is separated and withdrawn . The guide sleeve remains embedded in the bone and the anesthetic solution is delivered with the aid of a 27-gauge needle . There has been a continual search for safer and more profound local anesthetic compounds for attaining adequate pulpal anesthesia . Articaine, classified as an amide anesthetic, has increased liposolubility and potency because of the presence of a thiophene ring . Greater diffusion of articaine anesthetic solution to the teeth can be attributed to increased liposolubility and smaller size of thiophene ring of articaine in contrast to benzene ring of lignocaine . According to some authors, ability of articaine to diffuse into the bone can produce pulpal anesthesia in mandibular teeth even after infiltration anesthesia . Clinical studies comparing the success rate of 4% articaine with that of 2% lignocaine have shown that 4% articaine was superior to 2% lignocaine as a general - purpose anesthetic . The purpose of the study was to evaluate the anesthetic efficacy of x - tip intraosseous injection in patients with symptomatic irreversible pulpitis in mandibular posterior teeth, using 4% articaine with 1:100,000 adrenaline as local anesthetic when the conventional inferior alveolar nerve block proved ineffective . The purpose of the study was to evaluate the anesthetic efficacy of x - tip intraosseous injection in patients with symptomatic irreversible pulpitis in mandibular posterior teeth, using 4% articaine with 1:100,000 adrenaline as local anesthetic when the conventional inferior alveolar nerve block proved ineffective . Thirty patients, 15 males and 15 females 15 - 53 years of age with a diagnosis of symptomatic irreversible pulpitis of mandibular posterior teeth were included in this study . Approval for the study was obtained from institutional ethical committee and written informed consent was acquired from each patient . The diagnosis of irreversible pulpitis was confirmed by a chief complaint of spontaneous pain or pain at night, together with cold test and electric pulp testing showing an elevated and lingering pain response . Vitality of these teeth was first tested with cold pulp tester, dichlorodifluoromethane (endo - frost, roeko, langenau, germany) followed by electric pulp tester (digitest pulp tester). In all 30 patients selected, control teeth showed normal response and the inflamed teeth showed an elevated and prolonged pain response to these tests . Inferior alveolar nerve block injection was administered using 1.7 ml of 4% articaine with 1:100,000 adrenaline (septanest with adrenaline 1:100,000, septodont, france) with conventional long buccal injection . Before commencing access preparation, it was ensured that the control teeth did not respond to the maximum output of the electric pulp tester and the cold test . The patients were instructed to rate any discomfort during access using a heft - parker visual analogue scale (vas) [figure 1]. The vas scale consists of four categories . Mild pain was defined as greater than 0 mm and less than or equal to 54 mm . Supplemental intraosseous x - tip injection was administered in patients who had moderate or severe pain (vas rating greater than 54 mm) on access into dentin, when entering the pulp chamber or with initial file insertion . Heft - parker vas pain scale used for pain assessment the intraosseous injection with the x - tip system (dentsply maillefer, usa) was administered in the following manner: the perforation site was selected according to manufacturer's instructions, 2 - 4 mm apical to alveolar crestal bone level in the attached gingiva, at a site distal or mesial to the operating tooth . After determination of the perforation site, the perforator attached in micromotor handpiece was pushed through the attached gingiva until the x - tip contacted bone . After placing the drill at a 90 angle to the bone, the slow - speed air motor handpiece was activated and drilled at 90 to the bone . In 2 - 4 seconds, the drill perforated the cortical bone into the cancellous bone . The handpiece was always activated while the perforator was within the bone to prevent breakage that might occur if the perforator was allowed to stop rotating . After perforation, the drill was withdrawn from the guide sleeve, leaving the guide sleeve in place . The 27-gauge ultrashort x - tip needle was inserted into the guide sleeve to its hub, and 1.7 ml of 4% articaine with 1:100,000 adrenaline (septanest with adrenaline 1:100,000, septodont, france) was delivered over a 1-minute time period . The success of the supplemental x - tip technique is defined as the ability to access the pulp chamber, place initial files, and instrument the tooth without pain (vas score of zero) or mild pain (vas rating less than or equal to 54 mm). If the patient had moderate to severe pain (vas rating greater than 54 mm) during access or initial instrumentation, the x - tip technique was considered as a failure and an intrapulpal injection was administered . Out of the 30 patients who participated in the study, 25 patients experienced no pain or mild pain and were counted as success . Five patients who experienced moderate to severe pain during access or initial instrumentation after intraosseous injections were considered a failure . The various factors associated with anesthetic failure for conventional ian block include accessory innervations, accuracy of needle placement, anesthetic solution migration along the path of least resistance, tetrodotoxin - resistant class of sodium channels, which have been shown to be resistant to the action of local anesthetics, anxiety, psychological factors, and altered resting potentials and decreased excitability thresholds of nerves arising from inflamed tissue . Owing to these myriad limiting factors, supplemental injections become necessary in cases when inferior alveolar nerve block is not effective in providing satisfactory anesthesia . In needle - phobic patients, the time of onset of action for pressure anesthesia was less when compared to classic needle infiltration but the total duration of anesthesia was more for needle infiltration anesthesia . It can be used for restorative procedures of short duration and direct pulp injections . In this study, the anesthetic success using x - tip intraosseous injection was 83.33% . The results of this study shows that supplemental x - tip intraosseous injection helps in achieving successful pulpal anesthesia in patients with symptomatic irreversible pulpitis . None of the x - tip perforators broke, and the onset of anesthesia was immediate . The thickness of the cortical plate of posterior mandibular region condemns the use of infiltration injections . The intraosseous injection overcomes this problem by allowing direct access to the cancellous bone via perforation of the cortical bone . In this study, this fact is critical, as higher the lipid solubility, the higher is the potency and better will be the diffusion through the medium into which it is injected, and therefore, the greater is its ability to cross the lipid membranes of the epineuria this molecular configuration is subject to rapid hydrolysis by plasma cholinesterases after it is absorbed into the systemic circulation . Only the remaining 5 - 10% is subject to the slower, traditional hepatic metabolism . The allergen paraminobenzoic acid (paba), a frequent metabolite of ester metabolism, is not a by - product of the hydrolysis phase of articaine . Within the limitations of this study, we can conclude that when the inferior alveolar nerve block fails to provide profound pulpal anesthesia, the x - tip system using 4% articaine, was successful in achieving pulpal anesthesia in mandibular posterior teeth of patients presenting with irreversible pulpitis . Supplemental intraosseous injection with x - tip using 4% articaine with 1:100,000 adrenaline enabled soothing, swift, and sound anesthesia.
Mammalian memory is organized into dissociable neural systems that differ in terms of the type(s) of memory they mediate (white and mcdonald, 2002, squire, 2004, white et al ., 2013). Extensive evidence indicates that among these memory systems is a stimulus - response / habit system principally dependent on the integrity of the dorsolateral striatum (dls) (packard et al ., 1989, packard and mcgaugh, 1996, packard and knowlton, 2002, yin et al ., 2004, dls - dependent memory processes have been implicated in a variety of learning and memory tasks including response learning in the plus - maze, whereby animals acquire an egocentric turning response at the maze choice - point to receive reinforcement (packard and mcgaugh, 1996, chang and gold, 2004, yin and knowlton, 2004). Memory in dls - dependent maze tasks may be considered an exemplar of habit memory, given that the learned behavior in these tasks remains insensitive to reward devaluation (sage and knowlton, 2000, lin and liao, 2003, de leonibus et al . Stress influences a wide variety of learning and memory processes, and whether stress enhances or impairs memory partly depends on the type of memory being investigated (kim and diamond, 2002, mcgaugh, 2004, sandi and pinelo - nava, 2007, packard, 2009, roozendaal et al ., 2009, sandi, 2013, arnsten, 2015). Converging evidence indicates that dls - dependent habit memory in the plus - maze may be facilitated by the induction of emotional arousal through the exposure of animals to aversive unconditioned stimuli (packard, 2009, packard and goodman, 2012, packard and goodman, 2013, sandi, 2013, schwabe, 2013). For example, dls - dependent habit memory may be facilitated following chronic restraint stress, tail shock, exposure to predator odor, or administration of anxiogenic drugs (kim et al ., 2001, packard and wingard, 2004, wingard and packard, 2008, elliott and packard, 2008, schwabe et al ., 2012, leong and packard, 2014, taylor et al ., 2014, furthermore, some evidence suggests that, as observed with unconditioned emotional stimuli, exposure to emotionally arousing conditioned stimuli also modulates memory (holahan and white, 2002, holahan and white, 2004, hawley et al ., 2013, in particular, recent work from our laboratory revealed that exposing rats to shock - associated stimuli (i.e., a tone and context previously paired with footshock hereafter termed cs exposure) enhanced dls - dependent habit memory and biased animals toward the use of a response learning strategy in the plus - maze (leong et al . Noradrenergic activity, particularly within the basolateral complex of the amygdala (bla), plays a critical role in regulating emotional arousal and the emotional modulation of memory (mcgaugh, 2004, roozendaal et al ., additionally, the bla is required for the acquisition and expression of pavlovian fear conditioning (campeau and davis, 1995, maren et al ., 1996, ledoux, 2000, ledoux, 2003, maren, 2001a, maren, 2001b). Studies have found that noradrenaline administered directly into the bla modulates memory consolidation, whereas administration of a -adrenoceptor antagonist blocks the emotional modulation of memory (liang et al ., 1990, hatfield and mcgaugh, 1999). In addition, the memory modulatory effects of systemically administered adrenaline are also blocked after intra - bla administration of the -adrenoceptor antagonist, propranolol, across a range of learning and memory tasks (liang et al ., 1986; for review, see roozendaal et al ., 2009). Evidence from our laboratory indicates that similar neural mechanisms underlie the emotional enhancement of dls - dependent habit memory in the plus - maze . For example, administration of anxiogenic drugs directly into the bla is sufficient to enhance dls - dependent habit memory and the enhancement of habit memory produced by exposure to predator odor or systemic administration of anxiogenic drugs is blocked by neural inactivation of the bla (elliott and packard, 2008, wingard and packard, 2008, packard and gabriele, 2009, leong and packard, 2014). In view of this evidence, we hypothesized that the enhancement of dls - dependent habit memory consolidation after exposure to an aversive cs (leong et al ., 2015) may also be dependent on noradrenergic activity, particularly within the bla . In order to test this hypothesis, rats were first subjected to a standard fear conditioning paradigm (i.e., repeated tone - shock pairings). Rats were then trained in a response learning task in the water plus - maze that requires the use of dls - dependent habit memory . Following training sessions, rats were given systemic (experiment 1) or intra - bla (experiment 2) administration of propranolol immediately before cs exposure . Subjects were experimentally nave adult male long evans (blue spruce) rats, obtained from harlan laboratories (indianapolis, in), and weighing 275375 g at the time of training . Subjects were individually housed in clear plastic cages with sawdust bedding in a climate - controlled vivarium . Experimenters handled rats for 1 min per day for five days prior to the start of behavioral training or surgeries . For experiment 1, rats experienced a 12:12 light dark cycle (lights on at 7:00 a.m. and off at 7:00 p.m.). The institutional animal care and use committee at texas a&m university approved all experimental procedures . For experiment 1 and 2 the chambers (30 cm 24 cm 21 cm) are comprised of aluminum (side walls) and plexiglas (real wall, front door, and ceiling). The floor of each chamber consisted of 19 stainless steel rods (4 mm in diameter) spaced center to center at 1.5 cm apart . Footshock (2 s, 1 ma; unconditioned stimulus, us) was delivered via a shock source and solid - state grid scrambler (med associates). A speaker attached to each individual chamber provided the auditory conditioned stimulus (2 khz, 20 s, 80 db). Cameras mounted above the plexiglas ceiling of the chambers remotely recorded each animal's behavior . For the conditioning context, a small volume of 1.5% acetic acid odor was poured into the metal pan beneath the grid floor, the testing room lights remained on, and the cabinet doors were left open . The same contextual cues were used for both conditioning and cs exposure sessions . A load - cell platform beneath each chamber recorded chamber displacement (10 v to + 10 v) as a result of each animal's movement . Load - cell activity values were acquired and digitized at 5 hz with threshold activity software (med associates). Activity values were transformed offline into absolute values ranging from 0 to 100 (with lower values indicating less displacement of the chamber); rats were scored as freezing if absolute values were 10 for 1 s or more . Freezing was analyzed as a percentage of total time across each trial as described below . The water maze consisted of a clear plexiglas plus - maze (43 cm in height; each arm is 27 cm wide and 60 cm in length) that was inserted in a black circular tub (180 cm in diameter; 45 cm in height;, 2012, goodman and packard, 2014, leong and packard, 2014, leong et al ., 2015). For experiment 1 and 2, the maze was filled with water to a level of 21 cm; water temperature was 25 c (i.e., room temperature). A submerged clear plastic platform (15 cm 14 cm 20 cm) served as the hidden escape platform; the platform was about 1 cm below the water level throughout maze training . A movable piece of plexiglas (43 cm in height; 27 cm wide) blocked entry into the arm opposite to the start arm for each trial, creating a t - maze as necessary for the response learning task described below . Prior to behavioral training in experiment 2, rats were anesthetized with isoflurane and treated with atropine nitrate (0.4 mg / kg, i.p . ). Each rat was secured in a stereotaxic frame (david kopf instruments) and a small incision was made in the tissue above the skull; bregma and lambda of the skull were leveled on an even plane . Small holes were drilled in the skull and guide cannulae (10 mm, 26 gauge; small parts) were lowered to the following coordinates: 2.2 posterior to bregma; 5.0 medial / lateral to the midline; 6.0 ventral to dura (targeting the bla). Dental cement was used to anchor the guide cannulae to the screws in the skull . Stainless steel dummy cannulae (11 mm, 30 gauge) were inserted into the guide cannulae (extending 1 mm beyond the end of the guide cannulae into the bla). Rats were allowed 7 days of recovery from surgery before the start of behavioral training . Freezing behavior served as the index of fear for conditioning and during exposure to the conditioned fear stimuli . On the first day of behavioral training, rats (in squads of eight; counterbalanced by group assignments) were transported in black plastic containers from their homecages in the vivarium to the fear conditioning chambers in the laboratory . 3 min after being placed in the conditioning chambers, rats received three tone (2 khz, 20 s, 80 db)-footshock (2 s, 1 ma) pairings; the tone and shock co - terminated . Tone - footshock pairings were separated by 1-min interstimulus intervals; rats remained in the conditioning chambers for 1 min after the final tone - footshock pairing . Rats were immediately returned to the vivarium after conditioning . For experiment 1 and 2, training procedures for the response learning task were identical to the procedures employed in our previous studies (leong et al ., 2012, goodman and packard, 2014, leong et al ., 2015, wingard et al ., twenty - four hrs after fear conditioning, rats were individually transported from the vivarium to the room containing the water plus - maze . Rats were trained in the water maze across five consecutive days with six trials per day . For each trial, the subject was removed from the white transport container and gently placed into the water maze (facing the maze wall) in either the north (n) or south (s) arm; rats were allotted 1 min to swim to a hidden platform at the end of another arm (east or west). The arm opposite to the start arm would be blocked with the removable plastic wall . The location of the hidden platform was consistently in the arm in which a right body turn at the maze's choice point (i.e., at the middle of the maze) would lead to finding the platform . For instance, if a rat started in the north arm, the hidden escape platform would be in the west arm; if the rat started in the south arm, the hidden platform would be in the east arm . On the first, third, and fifth day of training (odd days), the sequence of the start arm was nssnns . If the rat did not locate the escape platform within 1 min, the experimenter would manually guide the rat to the escape platform . Once the rat climbed onto the platform, the rat would remain on the escape platform for 10 s before being returned to the white plastic container for a 30 s intertrial interval . If the subject made a full - body entry into the arm containing the hidden platform, then this response was scored as correct . If the rat made a full - body entry into the adjacent arm that did not contain the hidden platform, then this response was scored as incorrect . If the rat exited the start arm and made a full - body entry back into the start arm, then this was also scored as incorrect . Performance in the maze was analyzed as a percentage of correct responses for each day as described below . An overview of the designs for each experiment is depicted in fig . 1 . For experiment 1 (prior to behavioral training), rats were randomly assigned to drug (propranolol [prop] or vehicle [veh]) and exposure (fear or neutral) conditions, yielding the following groups: prop - fear (n = 8), prop - neutral (n = 8), veh - fear (n = 8), veh - neutral (n = 8). For experiment 1, prop and veh rats received systemic (i.p .) Administration of propranolol (3.0 mg / kg) or vehicle (respectively) immediately following maze training on the first three days . This dose of propranolol was selected based on previous evidence that this dose blocks the memory modulatory properties of glucocorticoid administration (roozendaal et al ., 2006b). Propranolol (sigma aldrich) was dissolved in saline and prepared fresh for each day's use . Systemic injection of propranolol was administered at a volume of 1 ml / kg . Immediately following propranolol or vehicle administration (on the first three days of training in the maze), rats were exposed to either the cs in the original conditioning chambers (fear rats) or to a clean blue plastic container enclosed in a separate room (neutral rats) for an equal duration . Fear rats received three non - reinforced conditioned tone presentations in the conditioning chambers (separated by 1 min interstimulus intervals in the chamber, with 3 min of baseline and 1 min following the final tone - alone presentation). Fear rats were transported to and from the fear conditioned chambers in the same black transport boxes used during conditioning . Neutral rats were transported in the white plastic containers used throughout their training in the maze . In experiment 2, rats were randomly assigned to drug (prop or veh) and exposure (fear or neutral) conditions, yielding the following groups: prop - fear (n = 6), prop - neutral (n = 9), veh - fear (n = 9), veh - neutral (n = 7). All apparatuses and procedures were identical to those in experiment 1, except that drugs were administered directly into the bla . For each infusion, propranolol (sigma aldrich) was dissolved in distilled water to a concentration of 1.0 g/l . Gas - tight syringes (hamilton co.) were secured to an automated syringe pump (kd scientific). Polyethylene tubing (pe-20; braintree scientific) was inserted over the gas - tight syringes . Internal injection needles (11 mm, 33 gauge; small parts) were fitted to the opposite end of the tubing . The stainless steel dummy cannulae were removed from within the guide cannulae and the injectors were inserted into the guides . Prop rats received bilateral infusions of propranolol at a rate of 0.5 l / min for 1 min, yielding a dose of 0.5 g of propranolol per hemisphere (veh rats received an equal volume of saline at an equal rate of infusion). This dose of propranolol was selected based on previous evidence that intra - bla infusions at this dose block the memory modulatory properties of glucocorticoid administration (roozendaal et al ., 2006b). Injectors remained in the guide cannulae for 1 min after infusion before being removed; clean dummy cannulae were inserted into the guides after these procedures . Rats from experiment 2 were overdosed on pentobarbital (0.5 ml, i.p .) And intracardially perfused with physiological saline and 10% formalin . Brains were extracted and stored in 10% formalin for twenty - four hrs then switched to a sucrose - formalin solution until sectioning . Only rats with injector tips localized within the bla (bilaterally) were included in the final analyses . Twenty - four hrs prior to training in the response learning task, rats reliably conditioned to the auditory tone (fig . 2a). Repeated measures anova revealed a main effect of trial [f(1,28) = 161.998; p <0.0001] such that rats significantly increased in freezing from baseline to the final tone at conditioning . As expected, rats did not differ based on drug or exposure assignments across conditioning trials [fs <2]. After maze training, cs exposure in the conditioning context reliably induced freezing behavior in fear rats (fig . . Collapsed across the three days of cs exposure, a main effect of trial revealed that rats significantly increased in mean freezing following the onset of the cs [f(1,14) = 30.194; p <0.0001], indicating robust cs - evoked fear . Peripheral administration of propranolol did not significantly alter freezing, as rats exposed to fear conditioned stimuli did not significantly differ across drug assignments during the retrieval phase [fs <1]. 2b . As illustrated, systemic propranolol administration prevented the memory enhancement produced by post - training exposure to the fear cs . This was confirmed in the anova by a significant drug exposure interaction for responding in the maze across days 25 of training [f(1,28) = 6.599; p <0.05]. Post hoc analyses revealed that veh - fear rats exhibited significantly more correct responses (%) across days 25 as compared to prop - fear [p <0.01] or veh - neutral [p <0.05] rats, whereas prop - fear, prop - neutral, and veh - neutral rats did not significantly differ from one another across training . A main effect of day indicated that performance in the maze improved for all groups across days 25 [f(3,84) = 13.733; p <0.0001]. Factorial anova of group performance on day 1 revealed no significant group differences [fs <2]. A trending but nonsignificant main effect of propranolol was detected across days 25 [f <3]. No other significant comparisons were detected [fs <1]. In sum, the data from experiment 1 reveal that post - training peripheral antagonism of -adrenoreceptors is sufficient to blunt the enhancement of habit memory as a result of exposure to fear css . A main effect of trial indicated that rats significantly increased in freezing from baseline to the final tone at conditioning [f(1,27) = 48.746; p <0.0001]. Groups did not significantly differ from baseline to the final conditioning trial [fs <2]. During post - maze fear exposure, the cs reliably induced freezing (fig . . Collapsed across the three days of cs exposure, a main effect of trial revealed that rats significantly increased in mean freezing following the onset of the cs [f(1,13) = 13.930; p <0.005]. Prop - fear and veh - fear rats did not significantly differ in their levels of freezing across the three days of fear cs exposure [fs <2] (similar to experiment 1). In contrast to experiment 1, anova of maze performance on the first day of maze training revealed a significant drug exposure interaction [f(1,27) = 5.395; p <0.05], indicating that the groups differed in baseline memory performance . This was unexpected, because all groups were treated equally before and during day 1 maze training . Drug administration and fear cs exposure did not occur until immediately after maze training on day 1 . Nevertheless, post hoc analyses revealed that veh - fear rats exhibited significantly fewer correct responses on the first day of maze training as compared to prop - fear rats [p <0.05]. On days 25, a main effect of day was observed [f(3,81) = 10.247; p <0.0001], but no other significant main effects or interactions were detected for% correct responses [fs <1.5]. Given that, in contrast to experiment 1, groups in experiment 2 displayed differences in day 1 baseline memory performance and that significant differences on the first day of training may influence differences in future performance, we normalized the responding of each rat in the maze to each rat's relative performance for the first day . Specifically, the percentages of correct responses of each rat for each day (25) were divided by the rat's percentage correct on day 1 (i.e., a value of 1 indicates an equal amount of correct responses as compared to day 1, a value of 2 indicates twice as many correct responses as compared to day 1, and so on). As such, we analyzed the relative rate of increase in habit memory expression in the maze as compared to the first day of training (i.e., before drugs were administered). Anova of percentage correct responses across training days 25 revealed a significant drug exposure interaction [f(1,27) = 5.413; p <0.05]. Additionally, a significant day drug exposure interaction was revealed [f(1,27) = 2.855; p <0.05]. A main effect of prop was trending, but not significant [f <2.5]. Post hoc tests revealed that veh - fear rats increased their performance in the maze at a faster rate as compared to prop - fear [p <0.05] and veh - neutral [p <0.05] rats . Conversely, prop - fear, prop - neutral, and veh - neutral rats did not significantly differ across days 25 of maze training . A main effect of day was observed [f(3,81) = 9.642; p <0.0001], indicating that rats in general significantly increased in their performance in the maze across days . No other main effects or interactions were detected [fs <1]. In sum, intra - bla infusions of propranolol prevented the relative increase in performance in the response learning water plus - maze task after exposure of rats to conditioned fear cues . The present findings indicate that the enhancement of dls - dependent habit memory produced by exposure of rats to fear css is blocked by systemic (experiment 1) or intra - bla (experiment 2) antagonism of -adrenoreceptors . The finding that post - training exposure to fear css, relative to exposure to neutral stimuli, enhanced habit memory is consistent with previous research from our laboratory (leong et al ., 2015). Given previous evidence indicating that delayed post - training cs exposure does not influence habit memory in the plus - maze (leong et al ., 2015), we assume that cs exposure influences maze performance by augmenting the initial consolidation phase of habit memory . In addition, previous evidence indicates that animals not given fear conditioning or animals given fear conditioning but no post - training cs exposure do not display enhanced habit memory in the plus - maze (leong et al ., 2015). This suggests that the enhancement of habit memory in the present study is specifically attributed to cs exposure (as opposed to the fear conditioning that transpired twenty - four hrs prior to maze training). Also, given the present finding that cs exposure was associated with conditioned freezing, it is plausible that post - training cs exposure enhanced habit memory by eliciting emotional arousal (i.e., fear). Attributing the present habit memory enhancement to emotional arousal concords with extensive previous evidence indicating that high emotional arousal produced by unconditioned stimuli enhances dls - dependent memory processes (packard, 2009, packard and goodman, 2012, schwabe, 2013). For example, systemic infusion of anxiogenic drugs such as -2-adrenoreceptor antagonists yohimbine or rs 79948 - 197 similarly enhances dls - dependent habit memory in the water plus - maze task (wingard and packard, 2008, packard and gabriele, 2009, leong et al ., 2012) and leads to the preferential use of dls - dependent learning in tasks that can be solved adequately using alternative strategies (packard and wingard, 2004, elliott and packard, 2008). Enhancements of dls - dependent habit memory are also observed after exposure to behavioral or ecologically valid stressors, such as chronic restraint, tail shock, or predator odor (kim et al ., 2001, notably, the stress / anxiety - induced enhancement of habit memory originally demonstrated in rodents (packard and wingard, 2004) has also been demonstrated in humans following administration of anxiogenic drugs (e.g., hydrocortisone) or exposure to psychological stressors (schwabe et al ., 2010b, schwabe et al ., 2013, schwabe and wolf, 2009, schwabe and wolf, 2010, guenzel et al ., 2014). The influence of emotional arousal on memory systems may involve the release of stress hormones and subsequent activation of glucocorticoid, mineralocorticoid, and adrenergic receptors in the brain (mcgaugh, 2004). Consistent with this suggestion, drug treatments increasing the activation of these receptors mimic the mnemonic effects of emotional arousal, whereas decreasing activation of these receptors through the use of selective antagonists prevents the effects of emotional arousal on memory (mcgaugh, 2004, roozendaal and mcgaugh, 2011). For example, administration of the -adrenoreceptor antagonist propranolol blocks the emotional enhancement of dls - dependent habit memory in humans (schwabe et al ., 2011b), and a similar blockade may be observed following administration of mineralocorticoid receptor antagonists in mice and humans (schwabe et al ., 2010a, schwabe et al ., 2013) in addition, propranolol administration blocked the fear cs - enhancement of habit memory in the present study, consistent with the hypothesis that noradrenergic activity also underlies the mnemonic benefit of exposure to aversive css . A modulatory role of the bla has also been implicated in the emotional enhancement of dls - dependent habit memory (packard, 2009, packard and goodman, 2012). Direct administration of anxiogenic drugs into the bla mimics the enhancement of habit memory produced by systemic administration of these drugs (elliott and packard, 2008, wingard and packard, 2008). In addition, the enhancement of habit memory after systemic administration of anxiogenic drugs or exposure to predator odor is blocked by reversible inactivation of the bla (packard and gabriele, 2009, leong and packard, 2014). The present finding that administration of the -adrenoreceptor antagonist propranolol directly into the bla blocks the fear - enhancement of habit memory suggests that the mnemonic effects of conditioned emotional stimuli might similarly depend on both the noradrenergic system and a modulatory role of the bla . Interestingly, this present finding suggests for the first time that noradrenergic activity specifically within the bla is required for emotional arousal to influence dls - dependent memory . Moreover, prior evidence indicates that increasing noradrenergic activity in the bla is sufficient to enhance memory in a task identical to the one employed in the present study (wingard and packard, 2008), and the present results suggests that this effect is likely mediated through -adrenergic receptors . Prior research indicates that exposure to fear css increases norepinephrine release in the amygdala (tanaka et al ., 2000, zhou et al ., 2015) and this increase in amygdala norepinephrine release may be responsible for the enhancement of habit memory observed in the present study . Future studies are necessary to examine whether increasing bla noradrenergic activity augments the enhancement of habit memory by fear css and whether bla norepinephrine levels correlates with these memory enhancements . Although we only analyzed data for rats having injectors within the bla, it is possible that drug had spread to other regions of the amygdala (e.g., the central nucleus [cea]). While previous evidence suggests that the bla, not cea, mediates the memory modulating capacity of the amygdala (see roozendaal and mcgaugh, 1996, quirarte et al ., 1997, roozendaal and mcgaugh, 1997, akirav and richter - levin, 2002), it is possible that the enhancement of habit memory in the present study may have been partially influenced by blockade of -adrenoreceptors in the cea . The cea may influence habit memory through an indirect cea - dorsal striatum pathway (ferreira et al ., 2008, lingawi and balleine, 2012). Whether the role of the cea in habit memory depends on -adrenoreceptor activity has yet to be examined . An additional consideration regarding the fear cs enhancement of habit memory is how the physiological processes during emotional arousal (e.g., stress hormone activity and bla function) lead to the enhancement of dls - dependent memory processes ., previous evidence indicates that systemic or direct administration of corticosterone into the dorsal striatum enhances memory consolidation in both the cued water maze and inhibitory avoidance task (medina et al . Thus, fear cs exposure may be associated with the release of stress hormones such as corticosterone that directly increase activity of the dls and consequently enhance habit memory consolidation in the plus - maze . Aside from this direct mechanism of enhancement, it is also reasonable to hypothesize that fear cs exposure may have enhanced habit memory indirectly through modulation of other brain regions . Extensive evidence indicates that in some learning situations, dls - dependent memory processes may be facilitated by lesion or inactivation of the hippocampal formation (packard et al ., 1989, mcdonald and white, 1993, schroeder et al ., 2002; for review, see poldrack and packard, 2003). Given that stress / anxiety is frequently associated with impaired hippocampus - dependent memory function (diamond et al ., 1996, de quervain et al ., 1998, conrad et al ., 2004, sandi et al ., 2005, park et al ., 2008, wingard and packard, 2008; for review, see sandi and pinelo - nava, 2007), cs exposure may have similarly impaired hippocampal function in the present study, thus indirectly enhancing dls - dependent habit memory . Consistent with this suggestion, previous evidence from our laboratory indicates that anxiogenic drug doses that impair hippocampus - dependent place learning also enhance dls - dependent response learning, and that these enhancing and impairing effects of anxiogenic drug administration critically depend on bla function (wingard and packard, 2008, packard and gabriele, 2009). Taking this indirect hypothesis a step further, it is tempting to speculate that propranolol administration in the present study might have blocked the cs enhancement of habit memory indirectly by preventing an impairment of hippocampal function . This hypothesis is consistent with some evidence indicating that propranolol might rescue the impairment of hippocampus - dependent memory produced by glucocorticoid administration (roozendaal et al ., 2004, in addition to memory impairments, previous evidence indicates that memory enhancements following corticosterone administration are also blocked by concurrent infusions of propranolol (quirarte et al . Notably, we have recently demonstrated that the corticosterone - induced enhancement of dls - dependent habit memory may also be blocked by concurrent propranolol administration (goodman et al . Thus, consistent with the view that glucocorticoid and noradrenergic mechanisms might interact to produce the emotional enhancement of habit memory, cs exposure in the present study would be expected to increase the release of glucocorticoids (goldstein et al ., 1996, cordero et al ., 1998, hagewoud et al ., 2011), whereas administration of propranolol might prevent glucocorticoids from enhancing habit memory (goodman et al ., another possible mechanism underlying the current results is that propranolol administration may have reduced fear expression (rodriguez - romaguera et al ., 2009, fitzgerald et al ., 2014, fitzgerald et al ., 2015, giustino et al ., 2016 however, in the current study, cs - evoked levels of freezing across retrieval were not significantly different between propranolol- and vehicle - treated animals in either experiment . Similarly, cain and colleagues (2004) reported no significant differences between mice treated (i.p .) With propranolol or vehicle in the early phases of massed auditory cs extinction or across a 60-min extinction session in a conditioned context (also, see fitzgerald et al ., 2015, zhou et al ., however, it is possible that floor effects might have competed with propranolol's effects on freezing in the current study . A higher dose of propranolol may also be required to significantly impact fear expression during post - maze cs exposure . Regardless, cs - evoked fear in the current study was sufficient to modulate performance in the maze for vehicle - treated animals . Finally, numerous investigators have suggested that enhancement of the dorsal striatum - dependent memory system might in part underlie the development of some neuropsychiatric disorders, in particular disorders with prominent habit - like behavioral features (white, 1996, everitt and robbins, 2005, schwabe et al ., 2011a, berner and marsh, 2014, gillan and robbins, 2014, goodman et al ., 2014, for instance, post - traumatic stress disorder (ptsd) is partly characterized by intractable avoidance behaviors that occur in response to trauma - related cues, and some investigators suggest that such avoidance symptoms may be a manifestation of enhanced dls - dependent habit memory following very high levels of emotional arousal (i.e., trauma; packard, 2009, schwabe et al . The fear cs enhancement of habit memory observed in the present study may be considered a putative animal model of this proposed mechanism, whereby the conditioned emotional stimuli represent the trauma - related cues that enhance dorsal striatum - dependent memory processes and lead to the development or expression of behavioral avoidance symptoms in ptsd . Clinical evidence indicates that -adrenoreceptor antagonists such as propranolol, when administered shortly after trauma or after ptsd has already developed, may be useful in treating some ptsd symptoms (famularo et al . 2016). Considering that propranolol blocked the fear cs enhancement of habit memory, propranolol administered in the acute aftermath of trauma
Diabetic retinopathy (dr) is a common complication of diabetes mellitus (dm) and is a leading cause of blindness worldwide . Diabetic macular ischemia (dmi) is an important category of diabetic retinopathy (dr) [2, 3]. During the imaging study of the normal macula, an important hallmark is the capillary - free region called foveal avascular zone (faz). It is recognized that the faz can enlarge and can become irregular in dr and seems to get larger as the stage of retinopathy advances [2, 3]. Dmi is characterized by the occlusion and loss of the macular capillary network or capillary dropout [4, 5]. This condition results in upregulation of growth factors, which contributes to the development of diabetic macular edema (dme), the most frequent sight - threatening disorder in individuals with dr . Dmi is an irreversible category of diabetic maculopathy, and its presence limits the potential benefits of treatments for dr [3, 5, 6]. The early treatment of diabetic retinopathy study (etdrs) established dmi standards that were determined using fluorescein angiography (fa). According to the etdrs report 11, clinically, there is a correlation between dmi and poor prognosis that varies according to the severity of the macular ischemia . The anatomy of the retina appears to be altered in dmi, with thinning of retinal nerve fibre layer (rnfl) and outer retina and thickening of outer choroid . In retinal microcirculation, blood supply is divided mainly into superficial capillary plexus (scp) and deep capillary plexus (dcp). Choroidal circulation appears to be the most important blood supply to the central macula, including photoreceptor inner segment (is) band, which is most likely the most important consumer of oxygen . It is likely that the dcp is responsible for up to 15% of the blood supply to the photoreceptors, especially during dark adaptation [8, 10, 11]. Fa has been the gold standard imaging modality since it was introduced in 1961 [7, 12, 13] however, it requires venipuncture, and reports of anaphylaxis and death related to contrast injections, despite being rare, have been documented . In addition, the technique is costly and time - consuming, requiring up to 10 minutes for framing acquisition [4, 8, 10, 11]. Spectral domain (sd) oct has emerged as a potential alternative for detecting macular nonperfusion in diabetic patients, but results are contradictory [1, 2, 13]. Macular ischemia may disrupt the normal flow of nutrients to the outer retina, but photoreceptor status on sd - oct remains controversial . In diabetic patients, the disruption of photoreceptors in sd - oct can indicate a manifestation of underlying dcp nonperfusion in patients with a relatively healthy macula [1416]. Oct angiography (octa) has been used for 3d mapping at microcirculation level [9, 1316]. It allows detection of retinal and choroidal structures via motion contrast imaging and high speed scanning, which detect blood flow by analysing signal decorrelations between scans [7, 11, 14, 15]. Both inner and outer retinal capillary plexuses are imaged in contradistinction in conventional angiography, which does not effectively image the outer plexus . This study aimed to compare the use of fluorescein angiography and octa in the diagnosis and quantification of dmi when applying a split - spectrum amplitude - decorrelation angiography algorithm (ssada) to improve the detection of flow signals in angiography with the purpose of offering a suitable sparing contrast alternative using 3 mm 3 mm octa for clinical investigations of diabetic patients [8, 1520]. This was a retrospective cross - sectional comparative study analysis conducted at federal university of goias and approved by ethics committee of the same institution . Signed informed consent was obtained from each subject prior to enrolment . Imaging data were collected from patients who underwent fa and octa on the same day in a tertiary referral retina center in the period between january 1, 2015, and july 30, 2015 . Exclusion criteria included significant cataract (without surgical indications at the time of examination), previous retinal arterial or venous occlusion, inherited macular dystrophy, posterior segment inflammation, and macular degeneration or scarring of any cause . Subjects that presented motion artifacts during octa or poor signal strength were also not included in this study . All subjects underwent a comprehensive ophthalmologic examination, including best - corrected visual acuity (bcva) on etdrs charts, + 78 d noncontact lens slit - lamp fundoscopy, color fundus photography, and fa and oct angiography, on a single day . Color fundus photographs were obtained and fa was performed using a digital retinal camera (topcon trc 50dx; topcon, paramus, nj). The central macular nonperfusion area, faz boundaries, and the maximum height and area were manually delineated . The faz area was measured using a single fa photograph, such as the octa angiogram of the scp and then divided into (superior to 0.32 mm) and small (inferior to 0.32 mm) groups . Group comprised both moderate and severe etdrs established grading groups [3, 19, 20]. The quantification of macular nonperfusion in both fa and oct angiography captured images was completed using imagej software (imagej, national institutes of health, bethesda, md). Octa instrument used was the rtvue xr avanti with angiovue software for octa (optovue, inc ., fremont, ca), and imaging data were obtained using the split - spectrum amplitude - decorrelation angiography (ssada) software . The algorithm was employed to improve the signal - to - noise ratio [8, 1521]. This instrument operates at ~840 nm wavelength, 70,000 a - scans per second, and a bandwidth of 50 nm . The tissue resolution is 5 m axially and there is a 15 m beam width . Each b - scan consisted of 304 a - scans for a total of 2 304 304 a - scans per acquisition, with a total acquisition time of approximately 3 seconds . The scanning area was captured in 3 mm 3 mm sections, and the acquired oct volumes were centered on the fovea [811]. A 3 mm 3 mm octa image was used primarily because this size is the one that provides images with higher resolution in the used octa device [10, 11]. The ssada algorithm was used to generate a volumetric rendering of blood flow from the internal limiting membrane (ilm) to the choroid, and it allowed the direct visualization of normal and abnormal blood circulation [1821]. For the octa angiograms, it was used the automatic segmentation of the retinal layers at the level of the scp generated by the angiovue software in an orthogonal view . In order to correct for automated segmentation error and projection artifacts, the segmentation slab was manually adjusted, using corresponding structural oct b - scans as a guide for the placement of 2 segmentation lines: the inner located at 3 m beneath the internal limiting membrane and the outer boundary at 15 m beneath the inner plexiform layer . This semiautomatic method permitted the readers to select images that pictured the largest extent of scp for subsequent quantitative analysis . The dcp image was segmented with an inner boundary 15 m beneath the inner plexiform layer and an outer boundary at 70 m beneath the inner plexiform layer . This section captured both layers of the outer capillary plexus, which sandwiched the inner nuclear layer . All statistical analyses were performed using statistical package for social science 22.0 (ibm spss; ibm, armonk, ny). Fa and octa images were independently reviewed by two independent masked readers retina specialists (jg and di) who reached an agreement regarding the area (mm) of macular nonperfusion that was obtained using both methods according to etdrs report 11 . Intraclass correlation coefficient (icc) was used to estimate the agreement between individual measurements from both readers . Since the icc was consistently> 0.9 between the 2 readers, nonparametric wilcoxon signed - rank test was used to compare area measurements performed by 1 reader on fa and octa 3 mm 3 mm scans . Thirty - four eyes from 34 patients, including 20 (58.82%) females and 14 (41.18%) males, were enrolled and separated according macular status (table 1). Twenty - four eyes from 24 patients were placed in the group of patients with dmi, including 15 (62.5%) females and 9 (37.5%) males . The mean (sd) age of the dmi population was 61.20 6.95 years . The group without dmi comprised 10 patients, including 5 (50%) females and 5 (50%) males . Four eyes from 2 patients were excluded from the study due to a high quantity of motion artifacts . Patients with dmi presented a mean fa of 0.68 0.53 mm (95% ic, 0.460.90; p <0.05). Octa angiogram analysis demonstrated a mean of 0.58 0.35 mm (95% ic, 0.430.73; p <0.05). The wilcoxon signed - rank test did not identify a significant difference between fa and octa in patients diagnosed with dmi (p = 0.1374). Patients without dmi presented a mean fa of 0.19 0.67 mm (95% ic, 0.140.24; p <0.05). Octa angiogram analysis demonstrated a mean of 0.20 0.79 mm (95% ic, 0.150.26; p <0.05). The wilcoxon signed - rank test did not identify a significant difference between fa and octa in patients without dmi (p = 0.9594) (figure 1). The iccs for faz area measurements between 2 observers with respect to fa and octa were 0.96 (ic: 0.360.71) and 0.92 (ic: 0.350.79), respectively, demonstrating the reproducibility and consistency of the methodology . This result highlights the notion that the faz is most often clearly demarcated by a distinct foveal vascular ring and that its abnormalities, in addition to its capillary dropout, can be lucidly obtained from octa images . Etdrs research group connected the severity of macular nonperfusion to the potential for progression in dr . In fact, in dr, the advanced deterioration of macular perfusion is the basis for macular ischemia, and developing a method to perceive perfusion maps may allow correlations between central ischemia and the different stages of dr . However, it is an invasive method requiring venipuncture and contrast infusion; it is a time - consuming test and provides only 2-dimensional images . Therefore, reports of anaphylaxis and death related to contrast injections have been documented, despite being rare . Octa is a noninvasive method, obtains highly detailed 3-dimensional images without requiring injection of a contrast dye, and allows faster acquisition of images (figure 2) [9, 10, 1214]. Octa performed using a split - spectrum amplitude - decorrelation angiography (ssada) algorithm has already been shown to be useful for imaging microvascular changes in dr [811, 14, 20]. Cole et al . Also observed macular nonperfusion in a diabetic patient in a 3 mm 3 mm octa that was centered on the fovea by applying a similar technology . The 3 mm 3 mm octa central sections obtained using sd - oct allowed us to obtain a higher resolution over a small area (figure 3). This area was sufficient for detecting central dmi, but it was not large enough to identify peripheral retinal nonperfusion . High - resolution oct imaging allows measuring thickness of segmented retinal layers in angiographically apparent ischemic dr . Future octa devices improvements may provide clinicians the ability to obtain wider field images with better resolution . In the present study, statistical analysis also did not indicate significant difference between area measurements obtained with fa and octa in patients diagnosed with dmi . The same has occurred among patient without dmi regarding the measurement of normal faz area . The icc for the zaf area between the 2 observers on fa and octa demonstrated the reproducibility and consistency of used methodology . These results highlight the notion that faz is most frequently demarcated by a distinct foveal vascular ring and its abnormalities, in addition to its capillary dropout, can be lucidly obtained from octa imaging device . First, all of the results were obtained during a single appointment, and there was no follow - up . The present study demonstrates that fluorescein angiography and octa provide similar results when used to diagnose macular ischemia in diabetic patients . With further improvements in summary, octa may provide images with higher details regarding macular status, becoming a novel imaging technique for the diagnosis of dmi, and may become an alternative to fa for this purpose . The results also offer improved quantification of faz area in diabetic patients without dmi when compared to diabetic subjects with established macular ischemia.
Geriatric population is on rise globally because of increasing life span . As per the us census, people above 60 years constituted 6.4% of the total population in 1900, which increased to 18.4% in 2010 and predicted to go up to 25.5% by 2050.1 spinal problems and spine surgeries in geriatric population are also showing a similar trend . Lumbar fusion surgeries in people aged 60 years and above have increased by 230% in a decade from 1991 to 2001.2 desire to lead a more active life in advanced age, improved diagnostic techniques, and better operative results are some of the reasons for increasing spine surgeries in the elderly . In general, spine surgery in the elderly in the presence of comorbidities is feared among both patients and surgeon, as it is presumed to have higher perioperative complications and increased cost . Many articles can be found in literature supporting this.345 a study by daubs et al . Involving adult spinal deformity in people over 60 years of age has reported that age and complication rates do not affect the surgical outcome.6 similar studies have reported 91%96% good to excellent results following surgical treatment of lumbar canal stenosis (lcs) by decompression and decompression with fusion on people aged above 6570 years.789 this indicates that in the absence of complications, spinal decompression and fusion surgeries would result in a satisfied patient even in the elderly with comorbidities . Therefore, measures to reduced complications in such patients should be looked at rather than denying surgical management in symptomatic patients due to their old age or comorbidities . This study evaluates the perioperative complications and the contributing factors in patients over 60 years of age undergoing lumbar fusion surgeries . Patients aged 60 years and above with one or more comorbidities undergoing lumbar decompression and instrumented fusion at our institute between january 2012 and december 2013 (2-year period) were included in the study . In all these patients, perioperative complications (intraoperative and complications occurring within 3 weeks postoperative period) and their incidence were recorded . The technique was a standard open technique of pedicle screw instrumentation and fusion, either interbody by transforaminal approach or posterolateral using morcellized bone from the posterior elements or rarely with iliac crest . Age, number of levels instrumented and fused, operative time, blood loss, comorbidities, and the duration of stay were correlated with the incidence of perioperative complications using spss software (ibm, spss statistics v 23.0, new york, united states). Factors contributing to perioperative complications were noted and measures to reduce them were suggested by these results and compared with the available literature in discussion . Analysis of our medical records revealed a total of 52 patients operated by lumbar fusions in the 2-year study period, who were aged 60 years and above and had one or more comorbidities . Most common indication for surgery was spondylolisthesis in 17 (32.7%) followed by lcs in 15 (28.8%) patients . Hypertension (htn) was the most common comorbidity found in 39 patients (75%), followed by diabetes mellitus (type 2) in thirty patients (56.4%). Twenty patients had single comorbidity while 18 patients had two comorbidities and 13 patients were found to have three comorbidities [table 1]. Preoperative mri t2w, midsagittal (a) and axial (b) images showing multilevel listhesis and canal stenosis . Postoperative x - ray lumbosacral spine anteroposterior (c) and lateral (d) views showing implant (pedicle screws) in situ following instrumented fusion diagnosis and comorbidites of patients forty six patients were operated under general anesthesia (ga) while the remaining six patients were operated in regional or spinal anesthesia . 3.8 levels were the average levels instrumented per patient while one patient underwent 9 levels instrumentation . Interbody fusions were performed at single level in 24, 2 levels in 22, and 3 levels in 6 patients . Average operative time and blood loss were 150 min (range 60270 min) and 369 ml (range 901050 ml), respectively . Both operative time and blood loss the levels of instrumentation and fusions were decided on segmental instability observed on dynamic radiographs . The interbody fusions were based on degree of segmental stenosis, disc degeneration, and instability . Clinical details of patients operative time and blood loss with respect to the number of interbody fusion levels a total of 11 complications were noted, 3 systemic and 8 local . Among the systemic complications, 2 were hypostatic pneumonia with secondary infection and one was a psychiatric illness called ganser's syndrome . All the three patients required transfer to icu and one patient with pneumonia expired due to septicemia and shock . The average total duration of stay in the hospital was 6.2 days (range 4 - 14 days). On comparing the complication rates with other variables, we found that the patients with complications had higher blood loss, operative time, number of instrumented levels, and number of interbody fusion levels [tables 4 and 5]. Similarly, the duration of stay was longer in these patients . On analyzing these results statistically by anova, the association of blood loss with complications was found to be statistically significant with p = 0.002 . The duration of stay, operative time, and number of interbody fusion levels were close to significance with p = 0.63, 0.58, and 0.61, respectively, while number instrumented levels and the number of associated comorbidities showed no significance [tables 4 and 5]. The complications and their incidence comparisons of different variables in patients with and without complications on analyzing the correlations between different variables, we found that there was a strong positive correlation of blood loss with operative time, number of instrumented levels, and number of interbody fusions which was statistically significant . Similarly, operative time showed a strong positive correlation with number of interbody fusions and a significant but a weaker positive correlation with number of instrumented levels [table 6]. The perioperative complication rates in the present study occurred in 11 of 52 patients with an incidence of 21% . Increased blood loss strongly correlated with the incidence of complications . Age, operative time, number of levels of fusion, and the duration of stay were also more in patients with complications and were close to statistical significance while number of instrumented levels and number of associated comorbidities were unrelated to the complication rates . Perioperative complication rates in instrumented lumbar fusions in patients above 60 years of age described in literature range from 29% to 62% [table 7]. Reported increased complications in patients with advanced age and surgeries with increased blood loss and number of levels of fusions.1112 carreon et al.11 found increased complication rates with increased operative time while cho et al.12 found no such association . Guigui et al.10 found comorbidities to influence the complication rates and a similar study by acosta et al.4 found ten times higher complication rates in patients with htn while others found no association between comorbidities and the perioperative complications.612 the incidence of complications and factors affecting it described in the literature considering the group of population included, the complication rate in our series was within the acceptable limits compared to literature [table 7].4611121314 our patients were 60 years and above with the average age of 69 years, all of them had one or more comorbidities, and the average number of levels fused was 3.8, making this group more vulnerable for complications . Despite this, the complications in our series were about 21% with most of them being minor reversible complications such as dural tear, transient root deficits, and postoperative persistent radicular pain . Similarly, the operative time and blood loss, in our series, was lesser compared to that described in literature for multilevel fusions [table 7].4611121314 the blood loss in literature ranged from 206 ml in single level to 2056 ml in 9-level fusions and the operative time ranged from 145 min in single level to 415 min in 10.5-level fusions [table 7].4611121314 in comparison in our series with an average of 3.8 levels of fusion, the average operative time and blood loss were 150 min (range 60270 min) and 367.45 ml (range 901050 ml), respectively . This could possibly explain the lesser complication rates in our study as the complications were strongly related to the operative time and blood loss . On reviewing literature and analyzing our surgical technique, we found several strategies that helped in reducing the blood loss and operative time, and hence the complications . Injection tranexamic acid 1 g intravenous was given routinely preoperatively, immediately before skin incision in all cases . Literature describes that a single dose of tranexamic acid 15 mg / kg can effectively reduce blood loss without increasing the risk of deep vein thrombosis.15 the other technique employed in our surgeries was simultaneous exposure and instrumentation on either side by two trained spine surgeons [figure 2]. This reduced the surgery time and also the blood loss as compared to a single surgeon exposing and instrumenting one side after the other . We employed a laminectomy technique described by okuda et al.16 in which lamina was removed as a single fragment using osteotome and making cuts at pars on either side . This further reduced the operative time as compared to the classical technique of removing the lamina piecemeal by rongeurs . (a and b) intraoperative images showing the technique of bilateral exposure and bilateral instrumentation operative time and blood loss were strongly related to the number of levels of vertebra instrumented and fused . Even though operative time and blood loss could be reduced by reducing the number of instrumented and fused levels, not instrumenting or fusing the levels when indicated would compromise the principles of surgery and therefore affect the clinical outcome . Hence, the number of vertebrae fused or instrumented should be restricted to the minimum indicated levels, without compromising on indications . We also found that the blood loss increased steeply with number of interbody fusion levels . The average blood loss in single level interbody fusion was 307 ml which increased to 362 ml in 2 levels and almost doubled in 3-level interbody fusions [table 3]. The reason for this exponential increase being continued bleeding from the bed of prepared interbody levels while performing the next level . Performing interbody fusions at selected levels such as the most stenotic or unstable levels or at the bottom of the construct and posterolateral fusions at other levels also could reduce the blood loss and hence the complications [figures 3 and 4]. Bar diagram showing the relation between the intraoperative blood loss and number of interbody fusion levels postoperative x - rays anteroposterior and lateral views of lumbosacral spine showing interbody fusions at l2l3, l4l5, l5s1 with posterolateral fusion at l3l4 images apart from reduction in operative time and blood loss, a thorough preoperative workup with concerned specialist consultations such as pulmonologist, cardiologist, and optimization of the medical conditions helped in reducing the anesthetic risks during surgery . Six of our patients in the series underwent surgery under spinal or combined spinal epidural anesthesia, due to poor pulmonary or cardiac status . Studies have shown regional anesthesia (ra) in spine surgery to have many advantages over ga in high - risk patients, like lesser anesthetic intraoperative complications, lesser postoperative htn, respiratory and cardiovascular complications, lesser postoperative vomiting, longer postoperative analgesia, and shorter hospital stay.1718 as surgeons we found spinal anesthesia to be satisfactory with reduced blood loss due to stable blood pressure one of these patients underwent surgery in sitting position which has shown in literature to be more convenient for the patient under ra, with the blood draining by gravity, resulting in clearer operative field and also reduce anesthesia complications by creating a hemodynamic status similar to that in othostasis.19 early mobilization postoperatively with optimal control of medical comorbidities also helped in reducing the early postoperative complications . Lumbar fusion surgeries in the elderly with comorbidities have higher complications rates . Increased intraoperative blood loss significantly correlated with the complication rates . Spinal decompression and fusion surgery when indicated should not be denied merely considering the age and comorbidities of the patients, fearing complications . Causes and measures to reduce complications should be considered as the outcome of surgeries in these patients in the absence of complications is good . The authors propose some of the measures to reduce the operative time, blood loss, and hence the complication rates in these patients . Mahesh - research grant by ulrich india and consultancy from medtronic india ltd.upendra - research grant from medtronic india . Mahesh - research grant by ulrich india and consultancy from medtronic india ltd.upendra - research grant from medtronic india.
Management of immature root with a necrotic pulp and apical periodontitis is a challenging task . Obturation of the root canal is difficult because of lack of apical barrier for containing the root filling material . Treatment of choice in such cases is the apexification procedure, i.e., establishing an apical barrier . It has also been shown that the use of calcium hydroxide weakens the resistance of the dentin to fracture . In recent times, a bonded restoration can be placed without any delay, thus reducing the possibility of root fractures . The major disadvantage of mta is its manipulation due to which its placement in the wide apical area is difficult to achieve . A matrix can be used in apexification procedures against which mta can be placed and condensed . Use of an apical matrix allows for the predictable placement and judicious use of this expensive material . Several materials have been recommended to create a matrix including calcium sulfate, hydroxyapatite, resorbable collagen, and platelet - rich fibrin . In this case series, we are presenting two cases of mta apexification using polyglactin - based resorbable suture material as the apical matrix . A 16-year - old male patient reported with a chief complaint of discolored maxillary left central incisor . History revealed that the patient had suffered trauma 8 years back and undergone treatment in a private clinic . Clinical examination revealed grayish discoloration of tooth 21 and attempted access preparation in the same tooth . Periapical radiograph showed well - defined periapical radiolucency and wide open apex in relation to tooth 21 [figure 1a]. The final diagnosis was pulpal necrosis with chronic apical periodontitis in relation to tooth 21 . After discussing different treatment options with the patient's parents, we opted to go for mta apexification with the use of an apical matrix . After rubber dam isolation, the tooth 21 was accessed and working length was established radiographically [figure 1b]. Root canal was chemo - mechanically debrided with circumferential filing using the international organization for standardization (iso) 80 k - file (dentsply maillefer, switzerland) in conjunction with copious amount of 0.5% sodium hypochlorite (shivam industries, india). A volume of 3 ml of 17% ethylenediaminetetraacetic acid (edta) solution (prevest denpro, india) was used for smear layer removal . Apexcal medicament paste (ivoclar vivadent ag schaan, liechtenstein) was placed in the root canal, and access cavity was restored with temp paste (pyrex exports, india). One week later, tooth was again accessed under rubber dam isolation, and copious amount of normal saline was used to remove any remnants of the calcium hydroxide medicament . One end of the suture material was modified to form a mesh, whereas the other end is left as such so that it could be used to adjust the position of apical matrix [figure 1c]. Suture material was saturated with iodine - based radiographic contrast medium iopamidol (bracco, italy) for 10 min to render it radio - opaque [figure 1d]. The matrix was gently placed on working length with the help of preselected hand pluggers [figure 1e], and its position was verified using radiographs [figure 1f]. Any adjustment required was done using the hand pluggers or free end of the suture material . White mta angelus (angelus, londrina, pr, brazil) was mixed according to manufacturer's instructions and using hand pluggers, gently condensed against the matrix to form 4 mm of apical plug [figure 1 g]. After placing a moist cotton pellet, next day, root canal was obturated using lateral condensation of gutta - percha (meta biomed, korea) and ah - plus root canal sealer (dentsply detrey gmbh, germany), and tooth was restored with brilliant ng bonded resin restoration (coltene / whaledent, switzerland) [figure 1h]. The patient was recalled after 3 months for clinical and radiographic evaluation . At the follow - up visit, radiograph demonstrated the resorption of the apical matrix and healing periapical lesion [figure 1i]. (a) preoperative, (b) working length radiograph, (c - f) formation and placement of suture matrix, (g) mineral trioxide aggregate plug, (h) immediate postoperative, and (i) 3 month follow - up a 15-year - old female patient presented with the chief complaint of discomfort while chewing in front region of upper jaw . Patient gave a history of traumatic incident 9 years back for which she had visited a private clinic . Clinical examination revealed slightly grayish discolored tooth 21 and previously attempted treatment in the same tooth . Intraoral periapical radiograph showed a well - defined periapical radiolucency surrounding the wide open apex of tooth 21 [figure 2a]. After rubber dam isolation, tooth was assessed and working length determined using the radiograph [figure 2b]. Root canal debridement was done using iso 80 k - file (dentsply maillefer, switzerland) and 0.5% sodium hypochlorite (shivam industries, india). A volume of 3 ml of 17% edta solution (prevest denpro, india) was used for smear layer removal . Root canal was medicated with apexcal paste (ivoclar vivadent ag schaan, liechtenstein) for 1 week . After 1 week, 4 mm of mta plug was formed by condensing white mta angelus (angelus, londrina, pr, brazil) against vicryl suture (johnson and johnson ltd ., next day, the tooth was obturated using laterally condensed gutta - percha (meta biomed, korea) and ah - plus (dentsply detrey gmbh, germany) [figure 2e]. Bonded composite resin brilliant ng (coltene / whaledent, switzerland) was used for definitive restoration . At the 3-month follow - up visit, periapical radiograph revealed resorbed apical matrix and healing periapical lesion in relation to tooth 21 [figure 2f]. (a) preoperative, (b) working length determination, (c) suture matrix in place, (d) mineral trioxide aggregate plug, (e) immediate postoperative, and (f) 3 month follow - up a 16-year - old male patient reported with a chief complaint of discolored maxillary left central incisor . History revealed that the patient had suffered trauma 8 years back and undergone treatment in a private clinic . Clinical examination revealed grayish discoloration of tooth 21 and attempted access preparation in the same tooth . Periapical radiograph showed well - defined periapical radiolucency and wide open apex in relation to tooth 21 [figure 1a]. The final diagnosis was pulpal necrosis with chronic apical periodontitis in relation to tooth 21 . After discussing different treatment options with the patient's parents, we opted to go for mta apexification with the use of an apical matrix . After rubber dam isolation, the tooth 21 was accessed and working length was established radiographically [figure 1b]. Root canal was chemo - mechanically debrided with circumferential filing using the international organization for standardization (iso) 80 k - file (dentsply maillefer, switzerland) in conjunction with copious amount of 0.5% sodium hypochlorite (shivam industries, india). A volume of 3 ml of 17% ethylenediaminetetraacetic acid (edta) solution (prevest denpro, india) was used for smear layer removal . Apexcal medicament paste (ivoclar vivadent ag schaan, liechtenstein) was placed in the root canal, and access cavity was restored with temp paste (pyrex exports, india). One week later, tooth was again accessed under rubber dam isolation, and copious amount of normal saline was used to remove any remnants of the calcium hydroxide medicament . One end of the suture material was modified to form a mesh, whereas the other end is left as such so that it could be used to adjust the position of apical matrix [figure 1c]. Suture material was saturated with iodine - based radiographic contrast medium iopamidol (bracco, italy) for 10 min to render it radio - opaque [figure 1d]. The matrix was gently placed on working length with the help of preselected hand pluggers [figure 1e], and its position was verified using radiographs [figure 1f]. Any adjustment required was done using the hand pluggers or free end of the suture material . White mta angelus (angelus, londrina, pr, brazil) was mixed according to manufacturer's instructions and using hand pluggers, gently condensed against the matrix to form 4 mm of apical plug [figure 1 g]. After placing a moist cotton pellet, next day, root canal was obturated using lateral condensation of gutta - percha (meta biomed, korea) and ah - plus root canal sealer (dentsply detrey gmbh, germany), and tooth was restored with brilliant ng bonded resin restoration (coltene / whaledent, switzerland) [figure 1h]. The patient was recalled after 3 months for clinical and radiographic evaluation . At the follow - up visit, radiograph demonstrated the resorption of the apical matrix and healing periapical lesion [figure 1i]. (a) preoperative, (b) working length radiograph, (c - f) formation and placement of suture matrix, (g) mineral trioxide aggregate plug, (h) immediate postoperative, and (i) 3 month follow - up a 15-year - old female patient presented with the chief complaint of discomfort while chewing in front region of upper jaw . Patient gave a history of traumatic incident 9 years back for which she had visited a private clinic . Clinical examination revealed slightly grayish discolored tooth 21 and previously attempted treatment in the same tooth . Intraoral periapical radiograph showed a well - defined periapical radiolucency surrounding the wide open apex of tooth 21 [figure 2a]. After rubber dam isolation, tooth was assessed and working length determined using the radiograph [figure 2b]. Root canal debridement was done using iso 80 k - file (dentsply maillefer, switzerland) and 0.5% sodium hypochlorite (shivam industries, india). A volume of 3 ml of 17% edta solution (prevest denpro, india) was used for smear layer removal . Root canal was medicated with apexcal paste (ivoclar vivadent ag schaan, liechtenstein) for 1 week . After 1 week, 4 mm of mta plug was formed by condensing white mta angelus (angelus, londrina, pr, brazil) against vicryl suture (johnson and johnson ltd ., next day, the tooth was obturated using laterally condensed gutta - percha (meta biomed, korea) and ah - plus (dentsply detrey gmbh, germany) [figure 2e]. Bonded composite resin brilliant ng (coltene / whaledent, switzerland) was used for definitive restoration . At the 3-month follow - up visit, periapical radiograph revealed resorbed apical matrix and healing periapical lesion in relation to tooth 21 [figure 2f]. (a) preoperative, (b) working length determination, (c) suture matrix in place, (d) mineral trioxide aggregate plug, (e) immediate postoperative, and (f) 3 month follow - up apexification is defined as a method to induce a calcified barrier in a root with an open apex or the continued apical development of an incomplete root in teeth with necrotic pulp . This chemical has several disadvantages such as difficulty of the patient's recall management and delay in the treatment . Furthermore, there is a risk of tooth fracture after dressing with calcium hydroxide for extended periods . The most promising alternative to calcium hydroxide is mta . The advantages of this material are (i) reduction in treatment time, (ii) immediate restoration of the tooth, (iii) no adverse effect on the mechanical properties of root dentin . In a prospective study, placement of the material in a wide open area is a challenging task and also there is a risk of extruding this expensive material into periapical tissues . Lemon advocated the use of a matrix when the diameter of the perforation is larger than 1 mm to prevent the extrusion of sealing material . Similarly, a matrix can be used for the predictable placement of mta in apexification procedures . Various materials have been advocated to be used as a matrix, for example, calcium sulfate, hydroxyapatite, collagen, platelet - rich fibrin . The common limitation shared by these materials is that once placed their position can't be adjusted as required . Vicryl is a synthetic absorbable suture material composed of 90% polyglycolic acid and 10% polylactic acid . However, the suture material lacks the radio - opacity which makes it difficult to be viewed in the radiographs . Iopamidol was used in our technique to impart radio - opacity to the suture material . It is a water soluble nonionic iodine - based contrast medium which is routinely used for angiography, arteriography, contrast - enhanced computed tomography, and urography . However, a proper history of the patient and sensitivity test should be done before using it . In our technique, one end of the suture material was used for the matrix formation, whereas other end was kept outside the tooth so that the placement of the matrix can be adjusted as required . After the matrix was placed at the desired position, the free end of the suture was cutoff and then mta was condensed against it . The apical matrix mentioned here in our study is cost - effective, easily available, and provides for adjustment of its position . Resorbable suture with a contrast agent may be used as a matrix for mta placement but properly designed clinical trials with long - term follow - up are mandatory to support this novel technique as there is no evidence of such procedure available in the literature presently . Research concentrating on the effect of suture material and contrast media on the healing kinetics can further through some light on this technique.
In interventional cardiology, quantitative coronary arteriography (qca) has been used for on - line vessel sizing for the selection of the interventional devices and the assessment of the efficacy of the individual procedures, for the on - line selection of patients to be included or excluded in clinical trials based on quantitative parameters (e.g. Small vessel disease), and for training purposes . But, in particular, qca has been applied worldwide in core laboratories and clinical research sites to study the efficacy of the procedures and devices in smaller and larger patient populations in off - line situations . Newer developments are directed towards the 3d reconstruction of the coronary arteries and the fusion with ivus or oct [13]. The goal of this paper is to provide a brief overview of the basic principles of a modern qca software package, particularly on the field of coronary bifurcation analysis . This is best illustrated by the qangio xa package (medis medical imaging systems bv, leiden, the netherlands). With the expanding practice of stenting coronary bifurcation lesions worldwide, the need for reliable, standardized and reproducible quantitative bifurcation analyses became apparent . Hence, the qangio xa version7.2 bifurcation application was developed, which contains two bifurcation models: a t - shape bifurcation model (suitable for bifurcations with a standard side branch structure: fig . 1a) and a y - shape bifurcation model (suitable for bifurcations with distal branches of equal size: fig . The particular advantage of these models is that they combine the proximal and two distal vessel segments with the bifurcation core, resulting in a total of two or three sections (depending on the model type), all derived from one analysis procedure, such that each of these sections has its own diameter function and associated parameter data.fig . 1schemes of the at - shape model and by - shape model, explaining the segments, proximal delimiter, interpolated contour and sections terminology . For each model, four segments that represent the building blocks of the models are generated by the software . A using the t - shape model, the arterial and reference diameters of the ostium of the side branch and the whole main section (including the transition within the bifurcation core) can be accurately determined . B using the y - shape model, the arterial and reference diameters up to the carinal point and in the distal 1 and 2 sections can be determined accurately schemes of the at - shape model and by - shape model, explaining the segments, proximal delimiter, interpolated contour and sections terminology . For each model, four segments that represent the building blocks of the models are generated by the software . A using the t - shape model, the arterial and reference diameters of the ostium of the side branch and the whole main section (including the transition within the bifurcation core) can be accurately determined . B using the y - shape model, the arterial and reference diameters up to the carinal point and in the distal 1 and 2 sections can be determined accurately to be able to start a bifurcation analysis, an analysis frame is chosen from the selected image run in which the target vessel is fully contrast - filled (usually second or third cardiac cycle following contrast injection) and in a stable position (preferably end - diastole), i.e. Minimal motion with respect to neighboring frames to prevent motion blur [5, 6]. As a first step, the user selects three pathline points in the image to define the arterial bifurcation segment: a start point in the proximal segment and one end point in each of the two distal segments are required . Subsequently, independent of the model type, two pathlines through the bifurcation segment of interest are computed automatically (fig . 2a) based on the wavefront propagation principle (the wavepath approach) [7, 8], followed by the automated detection of the arterial contours of all three vessel segments at once (fig . C the final analysis contours, plaque filling and the two corresponding diameter functions of the main and side branch sections an example of the t - shape model bifurcation analysis . A the three pathline points with the two detected bifurcation pathlines . C the final analysis contours, plaque filling and the two corresponding diameter functions of the main and side branch sections this arterial contour - detection procedure is carried out in two iterations relative to a model (here explained for a straight vessel, to keep things simple). In the first iteration 3a). To detect the contours, scanlines are defined perpendicular to the model (fig . For each point or pixel along such a scanline, the corresponding edge - strength value (local change in brightness level) is computed as the weighted sum of the corresponding values of the first- and second - derivative functions applied to the brightness values along these scanlines (fig . The resulting edge - strength values are input to the so - called minimum cost contour - detection algorithm (mca) [911], which searches for an optimal contour path along the entire segment (fig . The individual left and right vessel contours, detected in the first iteration, now serve as models for the mca procedure in the second iteration, resulting in the initially detected arterial contours (fig . If the operator does not agree with one or more parts of the initially detected contours, these can be edited / corrected in various ways . In a similar manner, the arterial contours are detected for the bifurcation analysis, resulting in three contours: the left, right and middle contour (fig . 3basic principles of the minimum cost contour - detection algorithm (mca). A initial segment with pathline, b scanlines defined, c straightened for analysis; contours calculated, d contours returned to initial imagefig . Schematic presentation of the brightness profile of an arterial vessel assessed along a scanline perpendicular to the local pathline direction and the computed 1st - derivative, 2nd - derivative, and the combinations of these 1st- and 2nd - derivative function; the maximal values of the last functions determine the edge positions basic principles of the minimum cost contour - detection algorithm (mca). A initial segment with pathline, b scanlines defined, c straightened for analysis; contours calculated, d contours returned to initial image the edge - strength values along a scanline . Schematic presentation of the brightness profile of an arterial vessel assessed along a scanline perpendicular to the local pathline direction and the computed 1st - derivative, 2nd - derivative, and the combinations of these 1st- and 2nd - derivative function; the maximal values of the last functions determine the edge positions the bifurcation core of the t - shape model is defined as the area between the automatically determined proximal delimiter in the proximal main subsection (of which the position is independent of the presence of a lesion) and the carinal point, which is flanked at one side by the first diameter of the distal main subsection (identical to the distal main segment) and at the other side by the interpolated (virtual) contour between the proximal and distal main segments (fig . Two sections are defined: the main section (i.e. The proximal main, distal main and bifurcation core segments merged) and the side branch section (identical to the side branch segment) (fig . 1a). From the left- and right - hand contours of the main section, an arterial diameter function . 5a, yellow graph), while for the side branch section the ostial analysis approach is followed, making sure that the arterial diameters at the ostium of the side branch are measured properly (fig . 5b, yellow graph).fig . 5examples of the a straight analysis and b ostial analysis, each with their own contours and specific diameter function (graph) examples of the a straight analysis and b ostial analysis, each with their own contours and specific diameter function (graph) the most widely used parameter to describe the severity of an obstruction is the percentage of diameter stenosis . Calculation of this parameter requires that a reference diameter value is computed, for which two options are available: (1) a user - defined reference diameter as positioned by the user at a so - called normal portion of the vessel, and (2) the automated or interpolated reference diameter value . In practice, this last approach is preferred because it requires no user interaction and takes care of any tapering of the vessel . For a conventional straight analysis, a reference diameter function is calculated by an iterative regression technique (excluding the influence of any obstructive or ectatic area) and displayed in the diameter function as a (slightly tapering) horizontal straight line (fig . 5a, red line), which represents the best approximation of the vessel size before the occurrence of a focal narrowing . Now that the reference diameter function is known, reference contours can be reconstructed around the actual vessel segment, representing the original size and shape of the vessel before any disease occurred . The value of the reference diameter function at the location of the obstruction diameter equals the reference diameter, so that neither overestimation nor underestimation occurs . Finally, from the reference diameter and the obstruction diameter, the percent diameter stenosis is calculated . However, due to the step down phenomenon caused by the bifurcating vessels, it is not a trivial task to derive a suitable reference diameter function for the entire main section . Therefore, the calculation of the reference diameter function is based on each of the three segments separately . By this approach, it is assured that both the proximal and distal main (interpolated) reference diameter functions are only based on the arterial diameters outside the bifurcation core . Finally, the reference diameter function of the bifurcation core is based on the reconstruction of a smooth transition between the proximal and distal vessel diameters . As a result, the reference diameter function graph of the entire main section will be displayed as one function, which is composed of three different straight reference lines that are linked together (fig . For the side branch section an ostial reference diameter function calculation is used (fig . 5b, red line), which is displayed as one function, which is straight and slightly curved proximally (fig . The bifurcation core of the y - shape model is defined as the area between the automatically determined proximal delimiter in the proximal section and the carinal point (fig . Three sections are defined: the proximal section (i.e. The proximal and bifurcation core segments merged), the distal 1 section and the distal 2 section (fig . For each of these sections the corresponding arterial diameter functions are calculated following the conventional straight analysis approach . This method guarantees that within the bifurcation core the arterial diameters are measured in their fullest extent (e.g. Important for skirt stenting). In order to derive a suitable reference diameter function for each section, again the calculation of the reference diameter function the reference diameter function of the bifurcation core itself is based on reconstructed reference contours between the proximal segment and two distal sections . As a result, the reference diameter function graph of the entire proximal section will be displayed as one function, which is straight for the proximal segment and curved in the bifurcation core . The two reference diameter functions of the distal sections will each be displayed as one function and are straight (fig . 6).fig . The final analysis contours, plaque filling and the three corresponding diameter functions of the proximal, distal 1 and distal 2 sections, respectively an example of the y - shape model bifurcation analysis . The final analysis contours, plaque filling and the three corresponding diameter functions of the proximal, distal 1 and distal 2 sections, respectively the reported bifurcation analysis results of all sections (both models) will be a complete listing of the angiographic parameters similar to the conventional straight qca, including the obstruction, reference, minimum, maximum, and mean diameters and areas, the percent diameter and area stenosis as well as the vessel and lesion lengths . Additionally, an option for edge segment analysis over the bifurcation (both models) will be available for the assessment of (drug eluting) stent segments and the corresponding stent- and ostial edge segments (fig . 7; t - shape). For each of these (sub)segments the complete parameter set including some edge specific parameters (e.g. Mld position relative to the stent boundary or segment start position, etc .) Will be reported, to allow to study the regression and progression of the bifurcation lesion to the fullest.fig . 7a schematic overview of the edge segment analysis for the bifurcation s t - shape analysis model a schematic overview of the edge segment analysis for the bifurcation s t - shape analysis model whichever qca analytical software package is being used, it will always produce numbers describing the morphology of the coronary segment analyzed . However, validation studies must demonstrate the true strengths and weaknesses, as well as the clinical validity of such analytical package . For a qca technique to be acceptable, guidelines for systematic and random error (a.k.a . We have carried out many validation studies, which all have been presented in the international literature [e.g. : 1416]. Considering the bifurcation analysis, the most relevant bifurcation validation studies are of the bifurcation s diameter functions and the intra - observer variability of the t- and y - shape models (see table 2).table 1guidelines for systematic and random errors of a state - of - the - art qca systemtype of studysystemic error (mm)random error (mm)guidelinesresult rangesplexiglass phantom, off patient<0.100.100.130.060.12plexiglass phantom, on patient<0.100.100.130.100.11guidelinesdobsdrefintra - observer variabilities0.100.150.100.13inter - observer variabilities0.100.150.110.13short - term variabilities0.150.250.190.22medium - term variabilities0.200.300.180.34inter - core lab variability0.140.15table 2the intra - observer differences (mean stdev) of the obstruction and reference diameters, for both the t - and y - shape modelsprox main (incl bif core)dist mainside brancht - shaped model (n = 18)dobs (mm)0.01 0.030.01 0.040.01 0.05dref (mm)0.08 0.100.01 0.080.04 0.10y - shaped model (n = 18)dobs (mm)0.00 0.030.02 0.080.01 0.06dref (mm)0.03 0.110.03 0.090.02 0.10all values are very low, demonstrating the high reproducibility of the analyses with both models, including lesions at the bifurcation core guidelines for systematic and random errors of a state - of - the - art qca system the intra - observer differences (mean stdev) of the obstruction and reference diameters, for both the t - and y - shape models all values are very low, demonstrating the high reproducibility of the analyses with both models, including lesions at the bifurcation core semi - automated segmentation techniques are able to detect the luminal boundaries of coronary arteries from two - dimensional digital x - ray arteriograms after minimal user interaction . Qca can be used in an off - line mode for clinical research studies and in an on - line mode during the interventional procedure to support the clinical decision - making process . New approaches have become available for more extensive analyses such as the coronary bifurcation analysis.
It is about 10 km away from osogbo, the state capital of osun state nigeria . There is one public primary school, one secondary school and one primary health center in the community . The study was carried out from may to august 2009, which spanned the rainy season . The study subjects were primary school pupils with age range of 3 to 16 years . The inclusion criteria for the study included: (1) children aged 8 - 16 years; (2) parents or guardians gave written informed consent; and (3) children lived in the study area . Before samples were collected, demographic data such as sex, age, weight, and name of subject were recorded . Ethical clearance for the work was obtained from the osun state ministry of health and ministry of education ethical committee (ins / est/90). Blood samples were collected by finger prick for the determination of p. falciparum para - sitemia . Thick blood films were prepared, airdried, giemsa - stained, and observed under the microscope for identification and quantification of malaria parasites . Malaria parasites were counted against 200 leukocytes, and counts were expressed as the number of parasites per microliter of blood, assuming an average leukocyte count of 8,000 cells/l of blood.26 for packed cell volume (pcv,%), microhematocrit tubes filled with blood were centrifuged in a microhematocrit rotor for 5 min at 10,000 g. pcv values 31% were considered as anemia, which was further classified as mild (2130%), moderate (15 - 20%), or severe (15%).13 clean plastic containers were distributed for stool collection, and instructions were given for proper collection . The formol - ether concentration technique was employed and kato - katz thick smear technique was used for quantitative determination of helminths ova.27 stool samples were processed within 12 hours of collection and examined microscopically within 1 hour of preparation to avoid missing hookworm ova . The intensity of infection was expressed as the number of eggs per gram of feces (epg). Statistical analysis was done using jmp version 5 for windows north carolina, usa . For uni - variate analysis, frequencies were compared using the fisher's exact tests . It is about 10 km away from osogbo, the state capital of osun state nigeria . There is one public primary school, one secondary school and one primary health center in the community . The study was carried out from may to august 2009, which spanned the rainy season . The study subjects were primary school pupils with age range of 3 to 16 years . The inclusion criteria for the study included: (1) children aged 8 - 16 years; (2) parents or guardians gave written informed consent; and (3) children lived in the study area . Before samples were collected, demographic data such as sex, age, weight, and name of subject were recorded . Ethical clearance for the work was obtained from the osun state ministry of health and ministry of education ethical committee (ins / est/90). Blood samples were collected by finger prick for the determination of p. falciparum para - sitemia . Thick blood films were prepared, airdried, giemsa - stained, and observed under the microscope for identification and quantification of malaria parasites . Malaria parasites were counted against 200 leukocytes, and counts were expressed as the number of parasites per microliter of blood, assuming an average leukocyte count of 8,000 cells/l of blood.26 for packed cell volume (pcv,%), microhematocrit tubes filled with blood were centrifuged in a microhematocrit rotor for 5 min at 10,000 g. pcv values 31% were considered as anemia, which was further classified as mild (2130%), moderate (15 - 20%), or severe (15%).13 clean plastic containers were distributed for stool collection, and instructions were given for proper collection . The formol - ether concentration technique was employed and kato - katz thick smear technique was used for quantitative determination of helminths ova.27 stool samples were processed within 12 hours of collection and examined microscopically within 1 hour of preparation to avoid missing hookworm ova . The intensity of infection was expressed as the number of eggs per gram of feces (epg). Statistical analysis was done using jmp version 5 for windows north carolina, usa . For uni - variate analysis, frequencies were compared using the fisher's exact tests . It is about 10 km away from osogbo, the state capital of osun state nigeria . There is one public primary school, one secondary school and one primary health center in the community . The study was carried out from may to august 2009, which spanned the rainy season . The study subjects were primary school pupils with age range of 3 to 16 years . The inclusion criteria for the study included: (1) children aged 8 - 16 years; (2) parents or guardians gave written informed consent; and (3) children lived in the study area . Before samples were collected, demographic data such as sex, age, weight, and name of subject were recorded . Ethical clearance for the work was obtained from the osun state ministry of health and ministry of education ethical committee (ins / est/90). Blood samples were collected by finger prick for the determination of p. falciparum para - sitemia . Thick blood films were prepared, airdried, giemsa - stained, and observed under the microscope for identification and quantification of malaria parasites . Malaria parasites were counted against 200 leukocytes, and counts were expressed as the number of parasites per microliter of blood, assuming an average leukocyte count of 8,000 cells/l of blood.26 for packed cell volume (pcv,%), microhematocrit tubes filled with blood were centrifuged in a microhematocrit rotor for 5 min at 10,000 g. pcv values 31% were considered as anemia, which was further classified as mild (2130%), moderate (15 - 20%), or severe (15%).13 clean plastic containers were distributed for stool collection, and instructions were given for proper collection . The formol - ether concentration technique was employed and kato - katz thick smear technique was used for quantitative determination of helminths ova.27 stool samples were processed within 12 hours of collection and examined microscopically within 1 hour of preparation to avoid missing hookworm ova . The intensity of infection was expressed as the number of eggs per gram of feces (epg). Statistical analysis was done using jmp version 5 for windows north carolina, usa . For uni - variate analysis, frequencies were compared using the fisher's exact tests . One hundred and seventeen stool and blood specimens collected from primary school children aged 4 up to 15 years were microscopically investigated in order to determine the prevalence of intestinal helminths and malaria infection among the children . The mean age of the study population was 9.9 3.1 years while the mean height was 127 14.0 cm and the mean weight was 25.5 6.7 kg . The mean pcv of the study population was 33.6 3.4 g / dl and none of the subjects had severe or moderate anaemia while 15 (12.8%) children had mild anemia . Table 1 shows the prevalence of intestinal helminth and falciparum malaria parasite in the study subjects according to age group . The prevalence of malaria parasite in the study was 25.6% while that of intestinal helminth was 40.2% . Five species of helminths were recovered from the stool samples and these were ascaris lumbricoides (34.2%), hookworm (5.1%), trichuris trichiura (2.6%), diphyllobothrium latum (0.9%) and tri - chostrongylus species (0.9%). The mean malaria parasite density in the study was 173 335 p/l while the mean egg count of a. lumbricoides (1345 3137 ages 4 - 7 years had the highest prevalence (31%) and mean parasite density (109.3 p/l) of malaria . The distribution of mean parasite density of malaria shows a gradual decrease as the age increases . For intestinal helminth, the older age group (12 - 15 years) was generally more infected with a. lumbricoides recording the highest prevalence of 46.5% . The highest intensity (egg output) of 2040 egg / gm of stool was also recorded in a. lumbricoides among age group 8 - 11 years . Age group 12 - 15 years had the highest prevalence of malaria and intestinal helminth co - infection (9.3%). Prevalence of malaria parasite and intensity of intestinal helminth by age the prevalence of malaria parasite and intestinal helminths by sex is shown in table 2 . Females had a higher prevalence (26.5%) of malaria compared to their male counterparts (24.5%) but the difference was not statistically significant . Males tend to have a higher prevalence with intestinal helminths (55.1%) compared to their female counterparts (35.3%) and the difference was statistically significant (p = 0.0390). For a. lumbricoides, males (44.9%) were more infected compared to females (26.5%) and the difference was statistically significant (p = 0.0487). For the co - infection of both malaria and any of the intestinal helminths, females (5.9%) were more infected than males (2.0%) showing no statistically significant difference . Prevalence of malaria parasite and intestinal helminth by sex the association between malaria parasite and intestinal helminths among the study subjects is shown in table 3 . Out of the 30 (25.6%) children that were positive for malaria, 23 (77%) were co - infected with intestinal helminths and the difference was statistically significant (p = 0.0053). Children who were infected with intestinal helminth were almost approximately one time (rr = 0.7295) as likely to be infected with p. falciparum as children with no worm infection . The results of the present study showed the occurrence of asymptomatic falciparum malaria and intestinal helminths parasites of public health importance among schoolchildren in primary schools in kajola, osun state, nigeria . The high prevalence of asymptomatic malaria (25.6%) recorded in the study confirms previous reports that malaria is hyperendemic in osun state nigeria.22 it was previously observed that the majority of malaria infections in individuals living in endemic regions are asymptomatic with the young children bearing the highest burden of disease and carrying asymptomatic infections for most of the time.2829 the decline of p. falciparum parasite density with age, observed in this study is characteristic of both symptomatic and asymptomatic infection in endemic regions.3032 the mean values observed are lower compared to asymp - tomatic values found in a longitudinal survey of malaria infection in individuals living in this area22 and other highly malarious endemic region of papua new.3033 like malaria, soil transmitted helminths are highly prevalent in rural communities as a result of poor sanitary conditions prevailing in most of these areas . This study recorded a prevalence of 43.6% for helminth infections, with a. lumbricoides having the highest prevalence, followed in decreasing order by hookworm and t. trichiura infections . In earlier studies among school children in osun state nigeria, the prevalence of these 3 species were in similar order but these works recorded higher prevalence.3435 the lower prevalence recorded in this study might reflect the increase awareness and improved sanitary conditions brought about by urbanization . While the previous studies were conducted in typical rural communities, the present study was conducted in semi - urban community that shares proximity with osogbo the state capital of osun state nigeria . A significant difference has previously been reported in infection rate between urban and rural school children in this area.36 in the present study, the co - infection of malaria and intestinal helminths is 4.3% and of the children who had malaria, the geo - helminth - positive children tended to be parasitaemic and there was statistically significant association between helminth status and parasitaemia . A similar observation was made in thailand, where, a positive and statistically significant association between geohelminth and malarial infection was also reported.25 in this study, it was observed that children with a. lumbricoides infections were almost two times as likely to have p. falciparum infection as children without a. lumbricoides infection . The mechanism behind this association is not clearly understood, but could be that th2 profile - associated immunoglobulin e production seen in ascaris infection may down - modulate th1 antimalarial immune responses, resulting in increased risk of malaria infection.37 there are, however, conflicting reports on how helminth infection affects malarial infection and vice versa . A similar association was previously observed among cohort of pregnant women in ghana . In uganda, geohelminth positive children tended to be parasitaemic but were not statistically significant.38 in developing countries, although mild anaemia occurs commonly among the general population, moderately severe anaemia is most frequently seen in areas where infections can cause or exacerbate anaemia.39 in this study, the presence of both malaria and helminths parasites among children was not associated with anaemia . This observation could be attributed to the fact that none of the children in this study had a high density of both p. falciparum and helminths infection thus, these parasites are unlikely to have contributed significantly to the presence of anemia in this study population . In individuals with precarious iron balance, however, relatively small hookworm loads may result in anemia . Also, high helminths burden in co - infections involving p. falciparum may in the long run exacerbate anemia caused by the malaria parasites . In conclusion, this study observed co - infection of both p. falciparum and intestinal helminths among school children in osun state nigeria . Given the plausible hypothesis of a biological interaction between helminths and p. falciparum and increasing advocacy for deworming, there is a need for prospective studies of the effects of helminths and the treatment of helminth infection on p. falciparum in this area . This work is undoubtedly the first study to document the prevalence of asymptomatic malaria among children in this area . The success of malaria control will depend on a systematic understanding of the micro - geographic risk of malaria transmission that would enable identification of high risk spots . The identification, quantification, and spatiotemporal mapping of the asymptomatic subpopulation will provide an opportunity to estimate the epidemiological importance of this group to malaria transmission . Oo carried out the design and coordinated the study and also prepared the manuscript, participated in most of the experiments and prepared the manuscript.
Since 1960, the rate of psychiatric beds per 100,000 residents has decreased in oecd countries due to de - institutional policies and the advent of community care support systems . Conversely, in japan, the psychiatric bed rates have increased [1 - 3]. The average length of hospitalisation within a psychiatric ward in 2012 was 298.1 days . While this length is shorter when compared to 1998 (496 days) [1 - 3], it is still too long . A national patient survey in 2005, observed that social hospitalisation, which includes baseless hospital care after a patient has become stable, accounted for 70,000 in - patients, of which about 30% were discharged less than one year after admission and about 70% were discharged after one year . The national patient surveys from 1999 and 2002 revealed that nearly 35,000 patients were discharged within these three years [2 - 4]. Thereafter, about 63,000 patients were newly - admitted and as a result, the number of social hospitalisations virtually did not change . The risk factors associated with long - stay hospitalisation in psychiatric hospitals in japan include diagnoses of dementia, mental retardation, and schizophrenia, and being men, and older [5, 6]. A long stay can directly cause problems regarding a family s acceptance, poor improvement of symptoms, and poor recovery of daily living abilities . The present study was conducted to investigate how the length of a patient s mental health hospitalisation is related to family structure, frequency of visits, and laundry types (self - washing, family washing, supplier washing), which is related to adl . Our hypotheses were that short - stay admission would be related to living with either a partner or family members, to a high frequency of visits, and/or to self - washing . The present study included a re - examination of the medical records of 83 patients discharged between 1 july 2013 and 30 june 2014, and the collection of data about their age, laundry type (self - washing, family washing, and supplier washing), family visits per month, and family structure prior to hospitalisation . Fifty - six patients (men 50.0%, excepting 27 patients discharged due to death) were included in these data . A diagnosis of a mental and/or behavioural disorder was made by mental health specialists according to the icd-10 . All patients received psychotherapy, occupational therapy, and pharmacological interventions as clinically indicated . Patients discharged between 2013 and 2014 were divided into three admission durations (under six months, between six months and 20 years, and over 20 years). The following disorder categories of the icd-10 were used: f00-f09 (organic, including symptomatic, mental disorders), f10-f19 (mental and behavioural disorders due to psychoactive substance use), f20-f29 (schizophrenia, schizotypal and delusional disorders), f30-f39 (mood disorders), and other (mental and behavioural disorders except for f00-f09, f10-f19, f20-f29, and f30-f39). The main independent variables of interest were hospitalisation for medical care and protection, laundry type (self - washing, family washing, and supplier washing), family visits per month, and family structure prior to hospitalisation (living with a parent, with a partner, with a son / daughter / brother, alone, in a care facility, or in a medical hospital). These variables were calculated and compared using tests . We used log - transformed values for admission duration and family visits per month to account for their skewed distribution . A length of stay of more than six months was considered the cut - off point for a long hospital stay . Bivariate logistic regression analyses were conducted to identify factors independently associated with the length of stay . Odds ratios were adjusted for age (continuous), sex, and main mental disorder (f00-f09, f10-f19, f20-f29, f30-f39, and statistical analyses were performed with the statistical package for the social sciences (spss version 22.0; ibm corp, tokyo, japan). Ethics approval was obtained from five ethics committees of the hospital s institutional review board . Due to the non - interventional; retrospective design, no informed consent was required; it was waived by the hospital s institutional review board . The present study included a re - examination of the medical records of 83 patients discharged between 1 july 2013 and 30 june 2014, and the collection of data about their age, laundry type (self - washing, family washing, and supplier washing), family visits per month, and family structure prior to hospitalisation . Fifty - six patients (men 50.0%, excepting 27 patients discharged due to death) were included in these data . A diagnosis of a mental and/or behavioural disorder was made by mental health specialists according to the icd-10 . Patients discharged between 2013 and 2014 were divided into three admission durations (under six months, between six months and 20 years, and over 20 years). The following disorder categories of the icd-10 were used: f00-f09 (organic, including symptomatic, mental disorders), f10-f19 (mental and behavioural disorders due to psychoactive substance use), f20-f29 (schizophrenia, schizotypal and delusional disorders), f30-f39 (mood disorders), and other (mental and behavioural disorders except for f00-f09, f10-f19, f20-f29, and f30-f39). The main independent variables of interest were hospitalisation for medical care and protection, laundry type (self - washing, family washing, and supplier washing), family visits per month, and family structure prior to hospitalisation (living with a parent, with a partner, with a son / daughter / brother, alone, in a care facility, or in a medical hospital). We used log - transformed values for admission duration and family visits per month to account for their skewed distribution . A length of stay of more than six months was considered the cut - off point for a long hospital stay . Bivariate logistic regression analyses were conducted to identify factors independently associated with the length of stay . Odds ratios were adjusted for age (continuous), sex, and main mental disorder (f00-f09, f10-f19, f20-f29, f30-f39, and statistical analyses were performed with the statistical package for the social sciences (spss version 22.0; ibm corp, tokyo, japan). Ethics approval was obtained from five ethics committees of the hospital s institutional review board . Due to the non - interventional; retrospective design, no informed consent was required; it was waived by the hospital s institutional review board . Table 1 shows the characteristics of the patients discharged during the study period, separated by length of stay at discharge . The mean age for patients hospitalised between six months and 20 years was 77.111.7 years, which is nearly 10 years older than those in the other admission groups (p = 0.040). In terms of the main mental disorder, f00-f09, f10-f19, and f20-f29 were the most common categories for those hospitalised between six months and 20 years (10.7%, 5.4%, and 17.9%, respectively). F30-f39 was the most common category for those hospitalised for less than six months (7.1%; p <0.001). Regarding laundry type, self - washing was the most common method for those hospitalised for over 20 years, family washing was most common for those hospitalised for less than six months, and supplier washing was most common for those hospitalised for between six months and 20 years (p <0.001). The bivariate - adjusted odds ratios (95% confidence intervals) for in - patients hospitalised for more than six months was 0.08 (0.01, 0.48) for family washing (p = 0.006) compared with those who used supplier washing . The number of visits per month and family structures before hospitalisation were not significantly associated (table 2). The present study evaluated how length of hospitalisation was related to family variables (family visits per month and living situation prior to hospitalisation) and laundry type, which is related to adl, among in - patients with mental and behavioural disorders in a private psychiatric hospital in japan . Our results revealed that family washing was related to a reduction in the length of the patients hospital stays . In 1961, when japan introduced the universal public insurance system, psychiatric hospitals began to increase in number . Furthermore, psychiatric residents began moving from home care to hospital care [8 - 10]. Although the number of psychiatric beds has decreased in recent years, the number of private hospitals with mental health wards is still high [1 - 3], constituting about 80% of psychiatric hospitals in japan . Therefore, it is difficult to reduce the number of psychiatric beds [8 - 10]. Gigantesco, de girolamo, santone miglio and picard reported that being in a private psychiatric hospital is also a barrier to discharge in italy . Thus, psychiatric medical care has not sufficiently shifted from hospitalisation back to community care [1 - 3]. In terms of the relationship between laundry type and visit frequency per months, family washing was observed to be at its highest peak for more than 60 visits per month, and supplier washing was observed to have two high peaks at 16 - 30 visits and over 46 - 60 visits (fig . 1). In a conventional survey, it was found that patients with dementia, mental retardation, and schizophrenia tended to experience long - stay hospitalisation [5 - 7]. In the present study, diagnostic status did not significantly influence the length of stay . Likewise in italy, no difference of diagnostic status emerged between long - stay and short - stay patients . Kawano et al . Outlined various factors that relate to a long psychiatric hospitalization including being received by the family (51.5%), no symptom improvement (48.8%), and limited life skills (44.0%), whereas for dementia physical diseases (20.8%), and no reliable administrative services (29.2%) account for longer stays . We assumed that the patient who chooses self - washing will have a short - length of stay because they have somewhat higher to adl . The results showed that self - washing did not reduce the length of stay, but family washing was related to a short length of stay . Moriyama, et al . Conducted a follow - up investigation regarding social hospitalisation processes, which included patient, family, and medical worker interviews [13 - 15]. Economy, physical functioning, and the home environment. The factors relevant to the family were health maintaining the health of the caregiver, the patient does not apply for certification of the need for long - term care and has received help from home - visit nursing, a home care worker, or a long - term care health facility, and caregiver burden. The factor relevant to hospitals was the economic condition of the hospital. In other words, if beds are not full, the hospital s profit decreases, affecting the hospitalization - discharge cycle . Thus, if family members intentionally or unintentionally visit when a doctor or chief nurse is absent, the opportunity for discharge and communicative care is diminished . In the present study, if the family was helping with the laundry, the family visited the hospital every two or three days . As such, the opportunity to discuss treatment with hospital staff was more frequent, increasing the probability of earlier discharge . Although one of our hypotheses was that a high frequency of visits would be related to a short length of stay, the frequency of visits was not related to a short length of stay in the present study . Conversely, if a supplier was doing the laundry, there were separate peaks during high visit frequencies (fig . The ratio of patients with a diagnosis of f00-f09 was highest in those hospitalised for between six months and 20 years (table 1). Discharging someone to home care is reasonable for individuals with mild dementia; however, discharge to a care facility is most likely when a patient suffers from severe dementia . This is also the case with other mental disorders (i.e., f10-f19, f20-f29, and f30-f39). In addition, in the present study, illness severity affected the length of stay . Another of our hypotheses was that self - washing would be related to a short length of stay . Self - washing had a ratio high for the f20-f29 category (table 1), and severity of disease affected the length of stay . It has been previously reported that family members with high care efficacy [18, 19] and sufficient economic resources are best able to handle a home discharge for individuals with more severe mental health issues . Another of our hypotheses was that living with a partner or family before hospitalization would be related to a short length of stay . As the present study showed family structure before hospitalisation was not significantly related to reduced hospital stays . Tortnicroft, bebbington, and leff reported that medical staff tend to carefully discharge a patient into the home environment in italy because the severity of psychopathology had influenced the length of stay . They showed that improvements in social networks and a less restrictive social environment promote the discharge to the community from a hospital [22, 23]. Thus, we consider that long - stay patients who are discharged from hospital should be supported to live in the community in oecd countries . In 2015 the ministry of health, labour and welfare reported avenues of reform regarding mental health and medical care welfare . In japan community - based mental health services the main premise is to shift individuals from hospitalisation to community care as a way to recognise and strengthen the medical health care system over the next 10 years . Individuals over the age of 65 years within psychiatric wards accounted for 29% of the patient population in 1996 . Since japan s population of individuals over 65 years of age continues to increase, further increases in disorders (i.e., dementia) associated with this age group will be likely to occur . In the present study, the private hospital has seen an increase in the number of dementia in - patients in recent years (no data). Thus, there will probably be an increase in the number of in - patients requiring long - term hospitalisation . It needs to be determined whether family washing is the result of economic concerns or if it is motivated more by the social and psychological benefits for the patient . Second, we did not assess family care efficacy, it could be the case that care efficacy is directly related to family washing behaviour and/or frequency of family visits . Finally, the present study did not assess a patient s number of a prior social hospitalisations . Further studies are needed to assess how social hospitalisations are related to the variables of interest in the present study . In conclusion, knowing the in - patient family environments helps to encourage discharge and to provide proper care after discharge . The present study indicated that family washing could be an important factor for decreasing the length of stay . In japan, there are still many private psychiatric hospitals . The number of dementia cases is increasing in these psychiatric hospitals, which causes long hospitalisation . In view of this situation further interventional studies should be conducted to investigate the relationship between discharge and communication between in - patients, their families, and hospital staff, and how we are able to help with medical treatment and care.
While the term textiloma is used to describe a mass lesion consisting of surgical sponge, the term gossybipoma is reserved for both the mass of sponge and the foreign body reaction around it . These pathologies can mimic other common spinal mass lesions such as hematoma, abscess, soft tissue tumor, etc . Their presentation is well known but varies with each case due to different kinds of reactions of body . In literature, 46 cases of gossybipoma involving the spine have been reported;[13419] however, it is thought to be more than this number because of medico - legal issues . In this report, we present a case of paravertebral gossybipoma, with a short review of the clinical presentation, radiologic findings and differential diagnosis of these lesions . A 40-year - old woman presented with a history of spinal operation for l4l5 lumbar disk herniation before 8 months and got admitted with non - purulent serous leakage from a small (5 mm) detachment in the surgical wound . The wound was minimal erythematous at the detachment site, but there was no tenderness, swelling or fluctuation . There was no fever, and routine laboratory tests including complete blood count, erythrocyte sedimentation rate, c - reactive protein and blood biochemistry were all normal . Microbiologic investigations of the serous leakage revealed no pathogens . While waiting for the microbiological results, treatment with first - generation cephalosporin was started and continued for 4 days, and the serous leakage stopped immediately with secondary healing of the wound in a 1-week period . However, the patient's same complaints recurred, and thus she got admitted again to our clinic . A computed tomography (ct) of the lumbar vertebrae revealed a hyperdense mass lesion located in the left side of the previous operation site . Magnetic resonance imaging (mri) showed a mass lesion in the left paravertebral area, which was hypointense on t1- and t2-weighted images, with peripheral hyperintense ring in t2-weighted images . Mri post - contrast images showed ring enhancement of the lesion [figure 1]. Lumbar imaging of our case revealed a paravertebral mass lesion located in the left side of the previous operation site (arrows). (a) axial ct scan, (b) axial post - contrast enhanced mri, (c) sagittal t2-weighted mri the patient was operated by lumbar midline reincision, and the exploration of the left paravertebral area revealed a retained sponge . The sponge was found adherent to the surrounding soft tissue by the new formed fibrotic tissue, which required individual dissection of these fibrotic attachments . Histopathologic examination revealed mononuclear cell infiltration and fibrosis formation around the retained sponge [figure 2]. The patient's complaints showed improvement with no neurologic deficits, and the patient was discharged on postoperative day 3 without complications . A photomicrograph shows the mononuclear cell infi ltration and fi brosis formation around the retained sponge (h and e, 200) many different kinds of hemostatic agents absorbable and non - absorbable are used to control intraoperative bleeding in neurosurgical operations . Non - absorbable materials include various forms of cotton pledgets and synthetic hemostats, which should be removed before surgical closure . Retained surgical sponges can be found following abdominal, gynecologic, urologic, thoracic, orthopedic or neurosurgical procedures . They are encountered in abdominal and thoracic cavities more commonly but they are also reported after extremity and spinal surgeries . A retained surgical sponge is thought to be a common entity; however, due to the medico - legal issues, only a few cases in the literature have been reported . While the term textiloma is used to describe a mass lesion consisting of surgical sponge, the term gossybipoma is reserved for both the mass of sponge and the foreign body reaction around it . In the literature, there are 46 reported cases of gossybipoma after spinal surgery since 1965. [13419] in these reports, patients had presented mostly with complaints of back pain, common motor weakness and/or sensory deficits in neurologic examination, with no infectious findings, at which the placement of surgical sponge occurred at least a couple years ago before the admission. [151719] on the other hand, some cases admitted with fluid leakage after only a short time of the first operation . In our case, the patient presented with sterile serous leakage from the operation wound, 8 months after the previous operation . After surgery, the body gives two types of foreign body reactions against retained sponges: (1) the exudative type tissue reaction, which leads to acute abscess formation, with a tendency to form fistulas through the skin and (2) aseptic fibrous tissue reaction, which involves slow adhesion formation, such as encapsulation and granuloma formation . While the time interval to clinical presentation is short with the exudative type tissue reaction, it ranges to even decades after surgery with aseptic fibrous tissue reaction . Surgical sponges with radiopaque markers are used now in most of the medical centers . Due to this imaging characteristic of the gossybipomas, plain radiographs and/or had reported 13 patients with gossybipomas in thorax and abdomen, and remarked that the radiopaque marker inside the gossybipoma was seen in only nine patients (69.2%) and even then did not always lead to diagnosis . In our case, the gossybipoma was revealed in ct scans as a hyperdense mass lesion, although it was not diagnostic for this lesion . Because the differential diagnosis of paraspinal lesions in patients with history of spinal operations include hematomas, abscess or residual / recurrent tumors, mri with intravenous contrast enhancement is known to be the best radiologic investigation modality in these situations [table 1]. Kim et al . Stated that mri usually shows a well - defined mass with a fibrous capsule that exhibits low signal intensity on t1-weighted images compared with the signal intensity of the paravertebral back muscles, high signal intensity in the center with hypointense rim on t2-weighted images, and strong peripheral enhancement in post - contrast images . On the other hand, mri of our case demonstrated a mass lesion, which was hypointense on both t1- and t2-weighted images, with peripheral hyperintense ring in t2-weighted images and peripheral enhancement in post - contrast images . Accordingly, we believe that despite the importance of the mri in the diagnosis of gossybipoma lesions, the definitive diagnosis must be mainly aided by the high suspicion profile of the physician and the intraoperative findings . In patients with the history of spinal operation, gossybipomas should always have a place in the differential diagnosis of newly found lesions, as it is believed that they are much more common than they are reported . However, no pathognomic radiologic characteristics are defined for these lesions . For this reason, the definitive diagnosis must be mainly aided by the high suspicion profile of the physician and the intraoperative findings . Thus, it must be remembered that careful inspection of the surgical field before closure is still an important basic rule in surgery.
Colonic lipoma is the second most common, but very rare, tumor after colon polyps.1 although most cases are found incidentally in asymptomatic patients, some cases may present with symptoms such as abdominal pain, diarrhea, intestinal obstruction and bleeding . The reported size of lipoma varies from 2 mm to 30 cm, with increased likelihood of having symptoms as the size is bigger.1 - 3 patients with a larger lesion or symptomatic patients require treatments . Surgical resection has been considered better suited for giant (2 cm) lesions than endoscopic management, which is highly likely to cause complications such as perforation or bleeding in a giant lesion . With recent breakthrough in endoscopic excision technology, however, reports of successful endoscopic management is increasing even for giant lesions.4 - 6 two endoscopic methods to manage lipoma, complete resection and partial resection (including unroofing technique), have been reported.7 - 9 the difference of effectiveness between these two methods is not proven,9 but partial resection is thought to be much safer than complete resection due to lower chances of complication . Now, we report our experiences of three cases of giant colon lipoma that were cleared up after partial resection, together with a literature review . A 65-year - old female was referred to our center due to a giant colon lipoma detected on a colonoscopy . A wide - based 33-cm size yellowish protruding mass without mucosal change was detected at the ileocecal valve during colonoscopy (fig . We planned to perform an unroofing technique with a snare catheter (asm-1-s; wilson - cook medical inc ., winston - salem, nc, usa) at the center of the lesion so that the remaining lipoma could flow out of the exposed surface . It was deemed as appropriate to remove the roof of the center because the wide base of the lipoma could impede the conduction of electric current . Erbe icc 200 (elektromedizin, tubingen, germany) was used as the electrosurgical unit; at the endo - cut mode (120 w, effect 3), partial resection was performed twice, each time lasting for less than 5 seconds, to resect about half of the mass (fig . The patient was relieved of the abdominal discomfort afterwards . At 3-year follow - up colonoscopy, a 46-year - old female was referred to the center with a colonoscopic finding of 33 cm giant lipoma at the sigmoid colon during a health examination performed 3 years ago . Being asymptomatic, the patient had been followed on an outpatient basis but was referred to our center due to continued abdominal discomfort in the left lower quadrant . A wide - based 33-cm yellowish globular mass without mucosal change was detected 30 cm above the anal verge during colonoscopy (fig . Partial resection technique was performed with a snare catheter at the center of the lesion so that the remaining lipoma could flow out of the exposed surface (fig . Since the mass was attached in a globular form, snare catheter was placed slightly apart from the base for optimal constriction . At the endo - cut mode (120 w, effect 3), partial resection was performed for less than 10 seconds to resect most of the mass . The colonoscopic finding at the next day confirmed flowing out of the remaining lipoma tissue at the exposed lesion (fig . The patient was relieved of the abdominal discomfort afterwards . At 1-year follow - up colonoscopy, a 48-year - old male was referred to the center with diagnosis of a benign lesion in the ascending colon detected by abdominal - pelvic computed tomography and abdominal discomfort in the right lower quadrant . At colonoscopy, a 47-cm cylindrical protruding mass without mucosal change was detected originating from the proximal ascending colon (fig . 3a). As the size of the mass was much bigger than the other cases, we first tried partial resection with a snare catheter at the apical end of the mass unlike the other cases . At the endo - cut mode (120 w, effect 3), partial resection was performed for 20 seconds unsuccessfully due to the impeded conduction of the electric current by the submucosal lipoma tissue (fig . It was found that the diameter of the mass became decreased toward the base of the cylindrical mass, which was why we retried partial resection slightly apart from the origin . Simulations were performed several times near the base of the mass, before performing the resection, in order to find the proper placement of the snare catheter with optimal constriction while avoiding possible thermal injury (fig . Partial resection was performed for less than 5 seconds to reset most of the mass . As observed from the resected specimen of the mass, the apical end of the mass was too thick to apply electric current without inflicting severe thermal injury on the adjacent tissues, while the actual resected part was relatively thinner and the electric current was well - applied with rare thermal injury on the adjacent tissues (fig . At 1-year follow - up colonoscopy, a surgical scar was observed but not any remaining lipoma . A 65-year - old female was referred to our center due to a giant colon lipoma detected on a colonoscopy . A wide - based 33-cm size yellowish protruding mass without mucosal change was detected at the ileocecal valve during colonoscopy (fig . We planned to perform an unroofing technique with a snare catheter (asm-1-s; wilson - cook medical inc ., winston - salem, nc, usa) at the center of the lesion so that the remaining lipoma could flow out of the exposed surface . It was deemed as appropriate to remove the roof of the center because the wide base of the lipoma could impede the conduction of electric current . Erbe icc 200 (elektromedizin, tubingen, germany) was used as the electrosurgical unit; at the endo - cut mode (120 w, effect 3), partial resection was performed twice, each time lasting for less than 5 seconds, to resect about half of the mass (fig . The patient was relieved of the abdominal discomfort afterwards . At 3-year follow - up colonoscopy, a 46-year - old female was referred to the center with a colonoscopic finding of 33 cm giant lipoma at the sigmoid colon during a health examination performed 3 years ago . Being asymptomatic, the patient had been followed on an outpatient basis but was referred to our center due to continued abdominal discomfort in the left lower quadrant . A wide - based 33-cm yellowish globular mass without mucosal change was detected 30 cm above the anal verge during colonoscopy (fig . Partial resection technique was performed with a snare catheter at the center of the lesion so that the remaining lipoma could flow out of the exposed surface (fig . Since the mass was attached in a globular form, snare catheter was placed slightly apart from the base for optimal constriction . At the endo - cut mode (120 w, effect 3), partial resection was performed for less than 10 seconds to resect most of the mass . Typical yellow tissue indicating lipoma the colonoscopic finding at the next day confirmed flowing out of the remaining lipoma tissue at the exposed lesion (fig . The patient was relieved of the abdominal discomfort afterwards . At 1-year follow - up colonoscopy, a surgical scar was observed but not any remaining lipoma (fig . 2f). A 48-year - old male was referred to the center with diagnosis of a benign lesion in the ascending colon detected by abdominal - pelvic computed tomography and abdominal discomfort in the right lower quadrant . At colonoscopy, a 47-cm cylindrical protruding mass without mucosal change was detected originating from the proximal ascending colon (fig . 3a). As the size of the mass was much bigger than the other cases, we first tried partial resection with a snare catheter at the apical end of the mass unlike the other cases . At the endo - cut mode (120 w, effect 3), partial resection was performed for 20 seconds unsuccessfully due to the impeded conduction of the electric current by the submucosal lipoma tissue (fig . It was found that the diameter of the mass became decreased toward the base of the cylindrical mass, which was why we retried partial resection slightly apart from the origin . Simulations were performed several times near the base of the mass, before performing the resection, in order to find the proper placement of the snare catheter with optimal constriction while avoiding possible thermal injury (fig . Partial resection was performed for less than 5 seconds to reset most of the mass . As observed from the resected specimen of the mass, the apical end of the mass was too thick to apply electric current without inflicting severe thermal injury on the adjacent tissues, while the actual resected part was relatively thinner and the electric current was well - applied with rare thermal injury on the adjacent tissues (fig . The patient was relieved of the abdominal discomfort afterwards . At 1-year follow - up colonoscopy, generally, simple follow - up is sufficient for asymptomatic colon lipomas since they are highly unlikely to develop to malignancy.10 excision becomes a necessity, however, when the size of the lipoma gets as big as to require histologic examinations for differential diagnosis from other malignancies and the patient accompanies symptoms such as abdominal pain, constipation, bleeding, or intestinal obstruction.4,10,11 furthermore, ileocecal valve hypertrophy or lipomas occurring at the ileocecal valve, as presented in the case 1, is believed to cause small bowel obstruction.12 recent development of endoscopic instruments and their accessories has enabled endoscopic excision of huge lipomas, but the complication rate is still higher than polypectomy.6 pfeil et al.11 performed endoscopic removal of colon lipoma in seven patients and reported perforation in three of them . Adipose tissue contains not enough water to facilitate conduction of electric current, which is why endoscopists apply higher electrical output for snare during procedure, causing thermal injury on the colon wall adjacent to the mass and increasing the likelihood of perforation.6,13 it seems that, in case of a pedunculatd type with a long stalk, it is relatively safe to perform a procedure, as if performing polypectomy, bearing the low conduction in mind . However, in case of a wide - based lipoma, surgical resection is inevitable since forced endoscopic resection of such cases may not only cause acute complications such as bleeding or perforation, it may also lead to a severe stricture due to a large iatrogenic ulcer occurring after the resection.6 two endoscopic methods to manage lipoma, complete resection and partial resection (including unroofing technique), have been reported.7 - 9 the unroofing technique, first reported by mimura et al.,7 is a new method of endoscopic resection for colorectal lymphangioma, a benign tumor composed of several interconnected cavities . The unroofing technique only cuts off the upper half of the submucosal tumor, thus reducing the risk of complications . The difference of effectiveness between the two methods is not proven,9 but partial resection is thought to be much safer than complete resection due to lower chances of complication . However, in case of lipoma of big size (like case 3), remnant lipoma after cutting of the upper half could be possible . Therefore, in order to complete removal, more resection is more effective . In this report, we performed endoscopic partial resection for wide - based giant lipomas to facilitate spontaneous flowing out of the remaining tissues with time . Practical methods to apply this strategy in three patients with distinct shapes of giant lipomas were described in detail . In the case 1, the patient had a wide - based lipoma . In order to expose the lesion by opening the cover, the partial resection of the central tissues was safely performed twice, and the remaining tissues were left to flow out spontaneously (unroofing technique). In the case 2, the lipoma was globular but wide - based, suggesting high risk of deep thermal injury on the adjacent tissues when resected in the form of polypectomy . Snare catheter was placed slightly apart from the base ensuring optimal constriction and the resection was performed for less than 10 seconds . The examination on the next day revealed the lipoma tissues flowing out of the exposed surface . In the case 3, we could personally witness the fact that snare resection of a giant lipoma could only lead to serious thermal injury on the adjacent tissues, suggesting high chance of complication after lipoma resection close to the base . We prefer and recommend using higher power cutting mode with shorter coagulation time, which can minimize cutting time and reduce heat - induced damage of adjacent tissues . But, in cases of polypectomy, polyps larger than 1 cm in the right colon or larger than 2 cm in the left colon and multiple polyps carried an increased risk of bleeding and perforation.14 therefore, we think that lipoma with a broad base or a large diameter has the risk for complication after endoscopic resection . Although surgery is not necessarily the first - line treatment option any more, the following circumstances are good candidates for operation; in case with wide - base sessile lipoma, unclear diagnosis, intussusception or obstruction, involvement of muscular and serosal layers.15 in conclusion, endoscopic resection could be preferentially recommended for giant lipomas, provided that the lipoma was well - characterized and the procedure was applied safely . The lesions could be cleared up spontaneously by unroofing technique and more aggressive resection will be needed in case of big size.
The clinical outcome of total knee arthroplasty (tka) remains suboptimal in some patients . Recently, a level ii study suggested that although there is a substantial improvement in functionality, the effect size of that improvement is greater for the physician - derived measures than it is for the patient - derived measures . This suggests that there is some discord between end points for physicians when compared with patients . Many factors may be involved in the differences observed in the clinical and functional outcome following tka . One possible underlying variable that may be affecting this discrepancy is postoperative limb length discrepancy (lld). Change in limb length is not commonly measured after tka, yet lld has been shown to increase the incidences of back pain and sciatica, gait disorders, and general dissatisfaction . It has been demonstrated that mechanical load and isometric torque placed on the long limb are significantly greater than that of the shorter limb . These biomechanical differences in gait have been implicated in many clinical presentations, including low back pain, arthritic changes of the hip, running associated injuries, and others . The amount of lld necessary to generate such pathologies is widely debated with sources suggesting 20 to 50 mm as being clinically relevant . A wide range of treatments are available for lld . For mild lld (up to 60 mm), shoe lifts and inserts are suggested . For severe cases, limb shortening by bone resection the lld is common in the general population in as much as 40% to 70% of the population . The extent of lld after tka has been correlated to the degree of the preexisting hip knee ankle angle (hka) of the lower extremity . Severe preoperative offset (varus or valgus) from neutral alignment there tended to be a greater increase in postoperative leg length . Severity of osteoarthritis in the knee may also play a role in lld after tka . It is debated whether or not body mass index (bmi) affects the outcome of total joint arthroplasty, and its role in lld has not been well - delineated . Lange et al found that a kellgren and lawrence (kl) grade above 2 does not produce a larger difference in postoperative leg length between the operative and nonoperative limbs . The purpose of this study is to examine the extent to which limb length change occurs after tka and to compare the change in limb length to the degree of valgus or varus joint position preoperatively . The role of bmi and kl grade in limb length change will also be assessed . It is expected that overall there will be an increase in limb length after tka and that the limb length change postoperatively will be greater in those with severe preoperative malalignment . It is also suspected that the limb length change will be greater in those with a higher kl grade but will not be dependent on bmi . This study received institutional review board s approval and was compliant with the health insurance portability and accountability act . A cross - sectional prospective study was conducted using a joint replacement database from 203 patients at a university medical center, who received a primary tka from november 2012 to july 2014 . Both preoperative and postoperative standing full - length radiographs were used to assess the limb length and hka for the analysis . Thus, those individuals who had not received adequate imaging were excluded from the study . Other exclusion criteria included those with gross bony deformities or poor quality radiographs, which prevented accurate measurement . Of the 203 patients originally identified, 57 did not have the adequate imaging required, 2 had significant bony deformity, and 7 had additional hardware that would have impacted the measurement . After excluding these patients, 137 tkas were included . For the purpose of this study, 0 was considered neutral, negative values were assigned to the valgus category, and positive values were assigned to varus . A standard approach was used, and access to the joint space was obtained in a medial parapatellar fashion . Minimal amount of bone resections was attempted in order to be able to use the thinnest polyethylene insert available . Standing anteroposterior full - length digital images of the lower extremities were obtained using fujifilm computed radiography system (fujifilm, valhalla, new york) before and after tka using standard departmental protocol . The digital images were viewed on agfa impax pacs software (agfa morsel, belgium), and measurements were obtained using a digital cursor . Measurement of the hka was done using 2 intersecting lines on the film following the protocol outlined by cooke et al . Briefly, the mechanical axis was obtained using a line intersecting the center of the femoral head extending to the intercondylar notch of the distal femur . Next, the tibial mechanical axis was obtained by drawing a line from the center of the tibial plateau extending distally through the center of the tibial plafond . The hka is defined as the angle between the 2 axes as shown in figure 1 . As outlined by lang et al, the leg length was measured using the full - limb radiograph by measuring the distance from the top of the femoral head to the base of the tibial plafond as shown in figure 2 . Each of these measurements was obtained on full - length radiographs in both a pre- and postoperative setting . Figure 1.the image demonstrates the measurement of the hip knee ankle angle (hka). The tibial and femoral mechanical axes are measured on the film, and the axis between them is the hka . The line is extended from the top of the femoral head to the base of the tibial plafond . The image demonstrates the measurement of the hip knee ankle angle (hka). The tibial and femoral mechanical axes are measured on the film, and the axis between them is the hka . The line is extended from the top of the femoral head to the base of the tibial plafond . To ensure accuracy of the individual who performed the measurements, a second individual measured each of the end points on a randomly selected group of patients in the study . Neither of the 2 individuals who measured the said parameters was the primary surgeon . To determine the variability in the measurement, this coefficient was calculated for each of the end points in this study and demonstrates that the variability in measurements was insignificant . The change in limb length was evaluated after organizing the patients into several different groups . First, the overall average change in limb length after tka was determined and compared using a 2 sample t test . Next, the patients were separated into preoperative limb alignment (valgus / varus), and the average limb length differences for each group were determined and compared . Next, the sample was separated by the severity of hka, with those in either alignment greater than 10 placed into a separate group . Again the patients were then separated based on bmi (> 35 placed in a separate group), and the same calculations were obtained . Finally, the patients were separated on degree of osteoarthritis as classified by the kl scale . Those with a grade of 3 or greater were compared with those with a lesser grade . All statistical analyses were performed using microsoft office 2010 . Using the data analysis tool, the calculations of averages and standard deviations (sds) were obtained, and a 2 sample t test was executed giving the associated p value and sd for each of the different categories . Varus alignment is much more common in the general population and was more common in this study as well . Females are more likely to receive tka, and this is consistent with the population included in our study . Table 1.patient demographics.parameterwhole samplevalgus, <0varus,> 0number of tkas1324587age68 (10)67 (11.7)68 (9.7)bmi30 (5.0)29 (4.8)31 (4.8)female823151abbreviations: sd, standard deviation; tkas, total knee arthroplasties; bmi, body mass index . Values are expressed as mean (sd). Abbreviations: sd, standard deviation; tkas, total knee arthroplasties; bmi, body mass index . For this study, 59.1% of the patients experienced an increase in limb length with an average increase of 0.438 cm . Overall, there was no statistically significant change, as the p value from the 2 sample t test of the total sample was 0.598 . In an effort to delineate potential confounding factors, the patients were separated into different groups, and their change in limb length was compared . As seen in table 2, the patients were separated into valgus and varus groups based on their preoperative alignment . The average increase in limb length was greatest in those who were preoperatively valgus (0.556 cm), despite the fact that the original average alignment (6.67) in this group was closer to neutral than the preoperative varus group (9.34). The preoperative valgus group did, however, have a greater average change in limb alignment (6.08) when compared to the preoperative varus group (4.99). Parameterwhole samplevalgus, <0varus,> 0preoperative hka4.16 (8.71)6.67 (5.79)9.34 (4.66)postoperative hka2.76 (4.29) 0.468 (3.57)4.86 (3.03)change in alignment1.40 (7.49)6.08 (5.72)4.99 (4.77)lengthening, cm0.438 (1.12)0.556 (1.13)0.377 (1.11) p value0.5980.7350.686percentage lengthening,% 0.500 (1.40)0.702 (1.46)0.457 (1.33)abbreviations: hka, hip knee ankle angle; sd, standard deviation . Pre- and postoperative hka, change in alignment, lengthening, and percentage lengthening given as average . Measurement results grouped by preoperative alignment . Abbreviations: hka, hip knee ankle angle; sd, standard deviation . Pre- and postoperative hka, change in alignment, lengthening, and percentage lengthening given as average . Those within 10 of neutral alignment were placed into 1 group, and those with severe valgus or severe varus, classified as greater than 10 from neutral, were placed into 2 other groups . Table 3 shows the change in limb length was greatest in the severe valgus group (1.51 cm). The change in alignment was also greatest for this group at 13.8, as it was 0.07 and 8.67 for the moderate and severe varus groups, respectively . The p value for the change in limb length after a 2 sample t test for each of the groups was 0.870, 0.730, and 0.567 for the moderate, severe valgus, and severe varus groups, respectively . The pre- versus postoperative alignment and percentages lengthened are also shown in table 3 . Table 3.measurement results stratified by preoperative degree of deformity.parametermoderate, 10 to 10severe valgus, <10severe varus,> 10number of tkas93935preoperative hka2.18 (5.38)15.0 (2.86)14.1 (2.91)postoperative hka2.11 (4.21)1.18 (4.74)5.41 (2.62)change in hka0.07 (5.22)13.8 (5.81)8.67 (4.14)change in limb length, cm0.164 (1.03)1.51 (1.00)0.851 (1.07) p value0.8700.7300.567percentage lengthened,% 0.20 (1.24)1.95 (1.50)1.02 (1.30)abbreviations: hka, hip knee ankle angle; sd, standard deviation; tkas, total knee arthroplasties . Pre- and postoperative hka, change in alignment, lengthening, and percentage lengthening given as average . Abbreviations: hka, hip knee ankle angle; sd, standard deviation; tkas, total knee arthroplasties . Pre- and postoperative hka, change in alignment, lengthening, and percentage lengthening given as average . The preoperative kl grade was determined, and the patients were separated into 2 groups as seen in table 4 . Those with severe osteoarthritic deformity (kl grade> 2) demonstrated an average lengthening of 0.594 cm, while those with less severe deformity had an average shortening of 0.2 cm . The limb length of the 2 groups was compared pre- and postoperatively and did not demonstrate a significant difference, as the p value was 0.916 and 0.521 for the less severe and severe osteoarthritic deformity groups, respectively . Preoperative kl grade 1 and 23 and 4preoperative hka2.87 (4.75)4.48 (9.41)change in alignment0.704 (4.52)1.57 (8.04)lengthening, cm0.200 (.887)0.594 (1.11) p value0.9160.521percentage lengthening,% 0.206 (1.07)0.723 (1.40)abbreviations: hka, hip knee ankle angle; sd, standard deviation . Preoperative hka, change in alignment, lengthening, and percentage lengthening given as average . Abbreviations: hka, hip knee ankle angle; sd, standard deviation . Preoperative hka, change in alignment, lengthening, and percentage lengthening given as average . Values in parentheses indicate sd . Those with a bmi of greater than 35 were isolated, and the pre- versus postoperative leg lengths were compared . An analysis was also done with the group of individuals with a bmi of less than 35 . The average change in limb length and associated p value was 0.86 cm and 0.617 for the group with bmi of less than 35 and 0.31 cm and 0.869 for the group with bmi greater than 35 . This was done by comparing change in limb length after tka based on hka, bmi, and kl grade . The results of this study though were inconsistent with those done previously that showed limb lengthening after tka . Those with a valgus alignment tended to have a greater increase in limb length . For this group, the average change in alignment was also greater, and this could be the reason for the increased length . Lange et al showed that in patients with severe malalignment, the limb lengthening is greater after tka when compared to patients with less severe malalignment . In this study, the trend found is consistent with their findings, but was not found to be statistically significant . Notably, there were only 9 tkas with a severe preoperative valgus deformity (10) and 35 with a severe preoperative varus deformity (> 10). A larger number of patients within these groups would strengthen the power of this study . Lange et al did not find a difference in limb length based on kl grade . In this study, there was a reduction in limb length for the patients with minimal radiographic osteoarthritis (kl grades 1 and 2), while the group with severe osteoarthritis (kl grade> 2) did have an average increase in limb length . Patients with minimal osteoarthritis have less joint space narrowing, and thus, the tibial insert does not serve as a source of increased joint length because that space was still partially intact preoperatively . There are several other possible explanations for why there was no significant limb lengthening in this study . One possibility is the surgical technique in selecting the tibial insert prosthetic size . A further investigation of the protocol followed by the senior author could help identify a surgical reason for the lack of limb lengthening . Standing ap full - length radiographs were used for the measurement of both limb length and alignment . Thus, it is possible that deformity other than that in the coronal plane may have had a role and gone unrecognized . One study suggests, however, that the measurement of limb length is not affected by flexion contracture if it is less than 15. third, when measuring the angles, identifying the pertinent landmarks occasionally proved difficult . Digitally manipulating the contrast of the film to compensate for over penetration of the lower extremities was done to assist in identifying these landmarks . Finally, the clinical significance of lld after tka is not well known . Correction of lld during tka is difficult due to the high priorities of ligament and gap balancing and the correction of preoperative deformity . Fang et al demonstrated that maintaining a postoperative hka between 2.4 and 7.2 valgus produces improved outcomes . Their results indicated that failure rates are significantly higher when the postoperative hka alignment is not within this range . This study suggests that it may be possible to minimize limb lengthening in tka while still achieving adequate alignment and stability . A deeper analysis of the technique used by the senior author may be beneficial in determining what was done to prevent postoperative limb lengthening . In conclusion, the lld is associated with increased incidences of pathologies that reduce patient satisfaction and quality of life . Further research is necessary to better characterize the incidence of lld after tka and to determine whether a need exists to adjust surgical techniques to prioritize maintaining limb length during the procedure.
Multiple sclerosis (ms, omim 126200) is the most common cause of nontraumatic chronic neurological disability of the central nervous system (cns), characterized by demyelination, axonal degeneration, and inflammation (1 - 5). The disease onset usually occurs in young adults and it is more common in females, with prevalence rates varying across ethnic groups and depending on geographic latitude (6 - 8). Most ms patients (85%) present a relapsing - remitting ms (rrms) clinical course at the onset, while the remaining groups of patients present primary - progressive ms (9). Epidemiological analysis shows that ms results from unknown environmental factors, acting on genetically susceptible individuals (10). Susceptibility to ms is held to have a strong genetic component in a large degree, as shown by the increased risk of the disease seen among relatives of affected individuals (11). Genetic contributions are clearly included at least in 20% of affected individuals, indicating the family background of the disease . Concurrence rates comprise 26% monozygotic twins compared to 3% in dizygotic twins (12). Although the etiology of ms is unknown, epidemiological data recommend that predisposition to the disease is approximately genetically found out and linked to a number of genetic loci . An association with human leukocyte antigen (hla) class ii is well recorded (13). Tumor necrosis factor (tnf-) gene is located between the hla - b (class i) locus and the class region in tandem in the major histocompatibility complex (mhc) region . The location of the tnf locus within the mhc region has generated notion about the emphasis of tnf in the etiology of mhc - associated diseases, especially those with an inflammatory or autoimmune history . Ms is not figured as a hereditary disease; although, a lot of genetic variations have been exhibited to increase the risk of developing the disease (8, 13). The role of tnf- in a demyelinating disease such as experimental allergic encephalomyelitis (eae) is well understood and increasing evidence exists to recommend a role for tnf in the pathogenesis of ms (13). Considering the significant differences between the tnf- microsatellite polymorphism gene in patients with ms and healthy controls in europeans, we studied the role of tnf- genotype in ms by determining the correlation between the tnf- microsatellite located in the hla region and ms in patients of two southern provinces of iran (hormozgan and fars). From february to november 2012, all individuals involved in this comparative case - control study gave written informed consents for the genetic analysis according to the iran medical committee . The 81 unrelated patients with ms (26 males and 55 females) considered in this study lived in these two southern areas of iran (hormozgan and fars provinces) and had iranian origins dating back at least three generations from both maternal and parental sides . They were classified according to the poser criteria (14) and diagnosed with either clinically definite (90%), laboratory supported definite (7%) or clinically probable (3%) ms . All the controls were free from acute or chronic internal and neurological diseases, determined by physical examinations . Hla typing had been previously performed for hla class ii alleles: increase of drb1 * 15 allele (p <0.005) in the patients was the most important point . Genomic dna was extracted from peripheral blood using dna extraction kit (dnptm, cinnagen co., iran) according to the manufactures protocol and stored at -20c until used . Using spectrophotometry, the dna quantity was evaluated in each sample . The microsatellite marker used in this study contained ac / gt repeats and had 13 alleles . The amplification was carried out in a pcr reaction using 5'-gcctctagatttcatccagccaca-3' and 5'-cctctctcccctgcaacacaca-3' primers . Of the genomic dna, a 100 ng sample was amplified in a total volume of 10 l containing 10 mm tris - hcl, 50 mm kcl, 1.5 mm mgcl2, 250 m of each dntp, 1 m of each primer and 0.5 u of taq dna polymerase (fermentas, germany). The following thermal - cycling conditions were used: initial denaturation at 94c for two minutes, 30 cycles of 94c for 45 seconds, 65c for 30 seconds and 72c for 45 seconds, and final extension at 72c for 10 minutes . The amplified products were loaded into a 9% polyacrylamide gel with a standard x174/hinf1 marker (15). Chicago, il, usa) in which all the averages were reported as mean sd and frequencies as percentage . Allele, genotype and haplotype the frequencies were analyzed using gene pop program (http://genepop.curtin.edu.au) and the comparison of intergroup differences were conducted by the chi - square and t tests . Test of hardy - weinberg (hw) equilibrium was carried out for tnf- microsatellite gene in the case and control populations to check any significant deviation . Our study was performed in a case - control design consisting of 163 people (81 patients with ms and 82 hcs). Basic demographic details of the patients were unremarkable with mean age [mean sd] of 31.20 9.11 years, mean expanded disability statue score (edss) of 4, mean disease duration of 8.55 years, mean age at diagnosis of 27.49 9.07 years, and female: male ratio of 2.12 . In total, 83.95% of the patients had rrms, 11.11% had secondary progressive disease (spms), and 4.94% had primary progressive multiple sclerosis (ppms) at the time of assessment . A total of 82 unrelated healthy controls (27 males and 55 females; mean age 34 4.7 years) with almost the same ages, sexes and geographical origins as cases were selected for this study . Most of the case group had a family history of ms (89.9%, p <0.005). All the 13 alleles were identified for tnf- microsatellite and genotypic distribution of tnf- microsatellite differed significantly between the two population (p <10 - 4) (table 2). Tnf - a11 and tnf-10 alleles increased significantly in patients with ms (0.25, p <0.005 and 0.19, p <0.005, compare to 0.06 and 0.08, p <0.005, respectively). In contrast, tnf-1 and tnf-2 allele frequencies decreased in patients (0.01 and 0.02 respectively, p <0.005), compared to hcs (0.15 and 0.21, p <0.005). The heterozygosity for the tnf- microsatellite gene was particularly high in this study (0.8); the tnf- genotypes in studied population were in hardy - weinberg equilibrium (p = 0.002 for cases and p = 0.05 for controls). Linkage analysis is a main method for recognition of genetic factors which can cause diseases . However, this procedure is not enough for finding the etiology of complex diseases such as ms . To date, crohn's disease is the only one that can be diagnosed successfully using linkage analysis (16). We screened tnf- microsatellite polymorphism in patients with ms to find their association in an iranian population . To our knowledge, this is the first study on tnf- microsatellite performed in a tropical area of iran, showing association with pathogenesis of ms . The position of tnf- gene within the hla region has led to paying more attention to the role of tnf- alleles in etiology of ms (15). The tnf- gene is located tandemly and maps within the mhc region centromeric to hla - b and telomeric to the class region on chromosome 6p2l.3 . Its chromosomal region and the immunomodulatory influence of this gene have caused much more speculation about the role of the tnf locus in mhc - linked diseases (17). It has been repeatedly suggested that genetic predisposition to ms might be influenced by tnf gene polymorphism (3, 18). The investigation of polymorphic markers (including microsatellites) within the tnf locus has resulted in a lot of studies which have shown the relationship between tnf haplotypes and pathogenesis of the disease (17). To find the association of tnf- polymorphism and ms, goertsches et al . Performed a research on 200 patients with ms (67 males and 133 females) and 200 hcs in a spanish caucasian population . All the patients had clinically definite ms according to poser and mcdonald criteria (14, 19, 20). Performing pcr according to the genetic analysis of ms in europeans (games) consortium (http://www-gene.cimr.cam.ac.uk/msgenetics/games), tnf- polymorphism showed a significant correlation, which confirmed the association of this microsatellite marker and the etiology of this disease (20). As a part of the games collaborative project, godde et al . Screened microsatellite markers in 198 germans with ms and 198 hcs to identify any susceptible region to ms . Performing pcr and statically analysis, tnf- marker residing in the hla region on chromosome 6p21 showed the most significant relationship (12). Comparing gene frequencies of tnf- alleles in a french population of 74 patients with ms and 75 hcs, lucotte et al . Showed a strongly significant positive association between tnf-11 allele and ms (15). Our results were consistence with those of a study on europeans patients with ms in the huge games project using 6000 microsatellite markers . The games collaborative screened 19 case - control cohorts and 10 trio family - based cohorts (8, 21), as described in the original publications: australia (22), belgium (23), finland (24), france (25), germany 1 (6), germany 2 (26), hungary (27), iceland (28), ireland (29), italy (30), poland (31), portugal 1 (32), portugal 2 (33), sardinia (34), scandinavia (35), spain (20), turkey (36), and uk (37). It was found that some special alleles of these markers were noticed significantly more usual in ms groups, as compared with the hc groups . In the games project altogether involving 13896 individuals, meta - analysis determined by the fisher's method for the six markers was validated . Three non - mhc markers and three mhc markers (including tnf- marker) were identified to be potentially associated with ms (10). Our findings also demonstrated that the tnf-11 allele was more frequent in rrms, ppms and spms groups than the hcs, suggesting a possible genetic predisposition to ms in iranians patients with ms . A large number of microsatellites from the human mhc region presented the polymorphism information content (pic) of around 0.75 . For the tnf- microsatellite locus, pic values around 0.85 have been observed (38). In the iranian population (current study), the findings indicated the association between tnf- microsatellite polymorphism in the hla region and the risk of developing ms in the native iranian population . Our findings also confirmed the relationship between tnf- microsatellite and predisposition to ms, as reported previously by the games project . However, achieving the exact results need similar studies in other parts of iran as well as in other parts of asia.
Renal cell carcinoma (rcc) is defined by the national cancer institute (nci), as the most common type of kidney cancer, which develops in the lining of the renal tubules of the kidney . The highest incidence of rcc between 1998 and 2006 was observed in north america and the czech republic . Cytokine therapy is beneficial only to a small number of patients (without multiple unfavorable prognostic factors) and is associated with the occurrence of severe adverse effects . Even though substantial progress in general cancer diagnosis techniques has been achieved, as much as around 2030% of patients are diagnosed with metastatic rcc (mrcc). What is more, 20% of patients will relapse after nephrectomy and will develop mrcc in the first 12 months after surgery . Molecular therapies such as targeting the mammalian target of rapamycin (mtor) and vascular endothelial growth factor (vegf) are the main achievements in modern rcc treatment . Subtypes of rcc are as follows: clear cell, 83%.papillary (chromophil), 11%.chromophobe, about 4%.collecting duct carcinoma, about 1% . Rcc is one of the most vascularized solid cancers and angiogenesis plays a pivotal role in growth of renal tumors (especially ccrcc), because of upregulation of proangiogenic vegf and platelet - derived growth factor (pdgf). The upregulation is caused by mutation in the von hippel - lindau (vhl) gene, which induces overexpression of hypoxia - inducible factor (hif) [3, 5, 6]. The main function of tyrosine kinase is to transfer a phosphate group from atp to a protein in a cell . Tyrosine kinases function plays a pivotal role in many signal transduction cascades in which extracellular signals are transmitted through the cell membrane to the cytoplasm or nucleus, where gene expression is modulated . Tyrosine kinase receptors (tkrs) are cell surface receptors for many polypeptide growth factors, hormones, and cytokines . Tkrs are the key regulators of normal cellular processes and have a critical role in the development and progression of many types of cancer . Vegf and pdgf receptors play crucial roles in angiogenesis, mainly via induction of cell survival, invasion, and proliferation, as well as exhibition of tyrosine kinase activity . To block the physiological function of some of the tkr class receptors, tyrosine kinase inhibitors (tkis) currently the food and drug administration (fda) approved the following tkis: sunitinib, sorafenib, axitinib, and pazopanib [5, 9] for use in therapy . Bevacizumab, which is an anti - vegf monoclonal antibody, is commonly used, with clinical benefit . Sunitinib (sigma - aldrich) is a tyrosine kinase receptor inhibitor, which targets vegf - r1, vegf - r2, vegf - r3, pdgf - r, pdgf - r, kit, flt3, csf-1r, and ret . Although novel treatment such as using sunitinib significantly prolongs time to progression, the overall survival of patients diagnosed with rcc is still not satisfactory, which justifies the search for new methods and therapy targets, for which it is necessary to understand the mechanisms responsible for the development and progression of this cancer . The thyroid hormones (ths), thyroxine (t4) and its active form triiodothyronine (t3), are produced by the thyroid gland . In order for the thyroid gland to produce t3 and t4, the most common form of th in blood is t4 (with the ratio t4/t3 = 20/1); however, t4 is converted to four times more potent t3 by 5-iodinase within the cell . Production of t3 and t4 is regulated via a closed loop feedback high levels of t3 and t4 in blood plasma inhibit the production of thyroid - stimulating hormone in the pituitary gland . Despite the fact that t3 and t4 are lipophilic, they are not able to diffuse passively through the phospholipid membrane of the cell . Instead, the t3 and t4 hormones are dependent on transmembrane iodothyronine transporters [10, 11]. Thyroid hormones are known for their impact on metabolism, development, and normal growth mostly during fetal development . In adults, deregulation of thyroid hormone levels can cause hypo- and hyperthyroidism, which is connected with a vast number of clinical symptoms [1215]. What is more, ths may play an important role in tumor promotion or suppression . Ths act mainly by their nuclear receptors, which serve as hormone - modulated transcriptional regulators . Thyroid hormone receptors (trs) are encoded by two genes, and, which are located on chromosome 17 and chromosome 3, respectively [18, 19]. The thr gene resides in 3p21 - 25 chromosomal region, which is known to be a hot spot for mutations in genes involved in rcc pathogenesis . The transcripts of thr and thr are alternatively spliced to produce three major isoforms of the receptor: tr1, tr1, and tr2 . Due to their ability to recruit coactivators and corepressors, those receptors are bipolar in their transcriptional properties, which means that they can activate or repress target gene expression . The chromatin template is modified by auxiliary proteins, which interact with general transcriptional machinery to produce appropriate transcriptional output [21, 22]. Mutations in thr are usually the cause of resistance to thyroid hormone (rth) syndrome . Ccrcc is one of the cancers in which aberrances in tr are frequently observed, due to localization of the gene in the hot spot for mutations . In addition to its metabolic role, tr1 has been known for its tumor - suppressive function . Disrupted expression and activation of tr1 have been previously described in tumors of the thyroid, lung, breasts, or insulinomas . Based on research conducted on the cell lines of breast cancer and hepatocellular carcinoma it was proposed that hypothyroidism accelerates the development of metastases and increases the invasiveness of tumor cells; however, tumor growth is slower . Studies on those cell lines in a mouse cancer model also showed that changes in stromal cells of tumor microenvironment generated in response to low thyroid hormone levels significantly alter the development of cancer . Activation of the kinase - signaling pathway3-phosphatidyl - inositol (pi3k), serine - threonine protein kinase akt, and protein kinase b is associated with a mutation in the tr and the development of cancer . Moreover, the function of tr1 as tumor suppressor indicates that the low expression of the receptor can affect the proliferation of cancer cells . For example, in prostate cancer it was shown that the induction of cells by t3 can increase the proliferation of tumor cells by reducing the b - cell translocation gene 2 (btg2) gene expression which is responsible for the regulation of the g1/s transition in the cell cycle . Research on the role of thyroid hormone and its receptor has proven over the years to be extremely complicated . The problem is the fact that the impact of thyroid hormone differs in different types of cancer . Tr1 is known for its tumor - suppressive role, so it is understandable that expression of this receptor in cancer cell lines is reduced . Reports of the possibility of existence of an alternative thyroid hormone - signaling pathway can be found in literature; shibusawa et al . It is known that thyroid hormones are essential for inner ear development; therefore they checked if the same effect occurs in tr mutant mice . They concluded that although not completely normal, inner ear development could occur in the absence of thyroid hormone receptor; thus it is possible that an alternative and novel thyroid hormone - signaling pathway may exist . Moreover, analysis of the kegg database showed that thyroid hormones could influence the cell through mapk and pi3k - akt signaling pathways, bypassing thyroid hormone receptors as it is shown in figure 1 . Finally, nongenomic (therefore not - thr mediated) actions of thyroid hormones include the regulation of ion channels, mitochondrial gene transcription, and oxidative phosphorylation . T4 through its own mechanism of action can promote angiogenesis and cell proliferation extracellular signal - regulated kinase (erk) 1/2for transduction of the signal initiated by t4 . What is more, lin et al . Proposed a model of action for t3 and t4 at the receptor domain of v3 . In this two - site model, site one (s1) is exclusively reserved for t3 and activates pi3k and src kinase . Activation of those kinases leads to the transcription of hif-1. Site two (s2) however can be activated by both t3 and t4 and act through erk1/2 activation to cause tumor cell proliferation . Since signaling pathway activated by t4 via v3 can lead to gene expression, thus, both nongenomic effects of t3 and t4 as well as genomic effects of t3 may overlap in the nucleus . What is more, tetrac which is a deaminated t4 analog has been shown to block hormone binding at s1 and s2 of integrin v3 . Tetrac blocks nongenomic action of t3 at s1 and both t3 and t4 action at s2; therefore tetrac suppresses the proliferative nongenomic activity of thyroid hormone on cancer cells . This data is supported by finding of yalcin et al . Who show that tetrac and tetrac nanoparticles reduced tumor volume in human rcc xenografts . The effect of th depends on the cell developmental state (stem / progenitor / differentiated) and type and physiological state (cancerous / healthy). Since in this case healthy and cancer cell lines are compared, it is understandable that the effects of t3 hormone are different . Effect of t3 on the cell cycle in rcc cell lines has never been described previously . According to the literature, effects of t3 can promote or decrease the proliferation rate in different cell lines [17, 39, 40]. T3 is produced by (extrathyroidal) conversion of t4 by di highly expressed in kidney . The expression of di has also been reported as downregulated in rcc (in comparison to normal kidney). It had been previously shown that the thr gene is frequently mutated in rcc [4244]; however it was only shown for tissue samples but not established cell lines . In first report kamiya et al . Found that 40.9% of tested ccrcc samples had at least one tr mutation; however, there is no information about the reference sequence or the percentage of mutations in samples derived from the healthy kidney . Nevertheless, mutations were reported to be found in seven of twenty - two investigated cancer samples . Finally, if the ccrcc samples were compared with samples from the healthy kidney, there is no information if sequences obtained from control samples were identical to each other . Future sequencing research with a broader panel of rcc cell lines and with different primers is needed; however, it is possible that commercially available cell lines are an inadequate model to investigate mutations in rcc . Thr mutations were further studied functionally and it was shown that most of these rcc mutants result in expression of a receptor that is impaired in t3-mediated transcriptional activity . Moreover, receptors that are not fully functional are dominant - negative inhibitors of wild type thr1 expressed from second allele . Mutations in the t3-binding domains of the receptor s380f, e299k, h412r, y321h, and l456s reduce t3 binding activity at different rate between 35 and 60% . In specific cases including q252r w219l, f451s, f451i, f417l, and mutations in dna binding domain like k155e may result in the 100% loss of dna binding activity of thyroid hormone receptor . In selected mutants loss of transcriptional activity results from lower avidity and defective t3 binding . In other cases, transcriptional inefficiency reported for thr1 mutants results from altered corepressors binding and release . These mutants may require significantly higher hormone concentration to release the corepressor and activate transcription . In selected cases increase in corepressor affinity is accompanied with modified corepressor and corepressor splice variants specificity and this may render thr1 mutants toward resistance repression inhibition . Finally, thr1 mutants were shown to harbor greatly expanded target gene specificity that is not homologous with that of the wild type thr1 . Interestingly upregulation of the von hippel - lindau (vhl) tumor suppressor was found in rc15-tr1 mutant cells . In functional study t3 had no direct effect on transcription of hif-1, but activation of thr1/rxr- (retinoid x receptor alpha) heterodimer by t3 stimulated expression of hlf (hepatic leukemia factor), which finally promoted hif-1 expression . Disrupted tr1 activity may be the result of reduced tr1 expression, loss of heterozygosity at the thrb locus, decreased intracellular concentrations of t3, mutation in dna at the thrb locus, altered interaction with coregulators, aberrant splicing and sequence of tr1 utrs, or increased expression of mirnas that interact with the thr1 3 utr . In first reports thr1 at the transcriptome level (mrna) was reported as overexpressed in 30% and downregulated in 70% of tumor samples . In the same set of tissues, at the proteome level, expression of thr1 was decreased 1.7 times in tumors tissues when compared to healthy kidney from the same patients . In this report it was revealed that decreased thr1 protein level was found in 87% (20/23) cancer samples when compared with normal adjacent tissue from the same kidney . Another dissonance between the relative amount of thr mrna and protein in the cancer cell line when compared to healthy (control) cells was observed by master et al . . Expression of thr was correlated with thr in cancer ccrcc tissues, but not in normal kidney samples . In more recent study tr1 mrna and protein levels were reduced by 70% and 91% . This 70% reduction was reported on thr mrna coding sequence, but reduced expression of 5utr variants a and f has been reported in 75% and 62% tumors, respectively . In rcc cell culture model it was shown that weakly folded variant a (ay286465.1) of 5utr promotes the highest level of thr expression, while strongly folded variants f (ay286470.1) and f1 (gq456950) result in low transcription and translation rates and low thr expression and in rcc the delicate balance between a and f / f1 variants is disturbed . Reported discordance in tr1 mrna level and protein along with mutations in thr 5utrs deregulating mrna stability and transcription rate suggests that thr expression is regulated mostly by posttranscriptional events along with epigenetic modifications and specific transcription initiation control [31, 45, 49]. Tyrosine kinase inhibitor rcc therapy is associated with development of hypothyroidism in up to 25% of patients as per registered trials (average value for subclinical and clinical hyperthyroidism) [5052] and up to 100% in selected it is important to notice that subnormal levels of serum tsh do not always reflect the presence of subclinical hyperthyroidism and in the major part of available literature the definition of hypothyroidism is unclear . Among patients treated with sorafenib, sunitinib, pazopanib, and axitinib, those treated with axitinib develop thyroid dysfunction faster and more pronounced compared to the others . The mechanism through which this tyrosine kinase inhibitor (tki) causes changes in function of the thyroid is not yet fully understood; however, emerging evidence suggests that sunitinib induces hypothyroidism by altering t4/t3 metabolism and thyroid capillary regression . Sunitinib was found to have antithyroperoxidase activity with potency equivalent to 25% of propylthiouracil (6-propyl-2-sulfanylpyrimidin-4-one). It was found that on sunitinib treatment oxidation of iodide ions to iodine atoms required for the synthesis of t4 is reduced . In cell culture model, sunitinib was shown to induce thyroid cells proliferation inhibition (ic50, 14.6 m) and dose - related increase of (125) i - iodide uptake . Computed tomography volumetry of thyroid gland on sunitinib treatment revealed that more than 50% reduction in volume may develop in half of the patients . Histological study revealed that thyroid atrophy of thyroid follicles with degeneration of follicular epithelial cells is found on autopsy of patients treated with sunitinib for rcc . Surprisingly no decease in vascular volume in the thyroid gland was found in these patients . In clinical research it was also shown that iodine uptake is impaired in rcc patients treated with sunitinib and the lowest uptake is reported at the end of on - drug period of sunitinib treatment (end of 4th week of treatment). Most recently it was investigated if autoimmunity is coresponsible for thyroid dysfunction in rcc patients treated with sunitinib . It was found that up to 25% of patients develop anti - thyroid peroxidase (tpoab) autoantibodies within 1218 month of therapy with sunitinib . Moreover it was found that progression - free survival (pfs) is significantly longer in patients developing these tpoab in comparison to those who do not develop autoimmune response (10.8 months versus 5.8 months). There are also a few other possible explanations: tki - induced inhibition of vegf receptor tyrosine kinases on thyroid cells that result in capillary regression [59, 60], inhibition of iodine uptake, and inhibition of the protein product of the ret protooncogene . Moreover, hypothyroidism induced by tkis such as sunitinib may not be an unwanted toxicity because t3 promotes proliferation of cancer stem cells and metastatic and primary cell lines . . Showed that patients who developed subclinical hypothyroidism during treatment with sunitinib had a significantly greater probability of responding to treatment . Although correlations between hypothyroidism and treatment outcome have been noticed and many researchers confirmed an association between progression - free survival (pfs) and hypothyroidism, the rate of objective remission (orr) (hypothyroid patients versus euthyroid patients: 46.7 versus 13.7%, resp . ; p = 0.001), and the median overall survival (hypothyroid patients versus euthyroid patients: 39 versus 20 months, resp . ; p = 0.019), some researchers argue that there is no certainty that hypothyroidism can be considered as an independent prognostic marker, because it is still unclear if the induction of hypothyroidism is the mode of sunitinib action or this phenomenon is dependent on pharmacokinetics, height, and weight of the patient or affinity to tyrosine kinase receptor . It was suggested that although pfs is longer in these patients, there might be no difference in overall survival of hypothyroid patients . Finally, in their study schmidinger et al . Reported that there is a statistically significant correlation between hypothyroidism during the treatment of kidney cancer using tyrosine kinase inhibitors and the percentage remission (patients with hypothyroidism versus euthyroid patients: 28.3% versus 3.3%, resp . ; p <0.001) and the mean survival time (above and below 13.9 months, resp . ; p = 0.016). In another study hypothyroidism was associated with a longer pfs in patients treated with sunitinib or sorafenib (16.0 months versus 6.0 months) and replacement with l - thyroxine did not show influence on pfs . Recent meta - analysis that covered 250 patients has shown that there is no significant statistical difference in pfs between patients with hypothyroidism on sunitinib and patients without acquired hypothyroidism (hr = 0.82 and p = 0.22). Similar result was calculated for os (hr = 0.52 and p = 0.01). This leads the authors to the conclusion that development of hypothyroidism during tyrosine kinase inhibitor therapy may not be an independent predictive factor in patients with metastatic renal cell cancer . It is known for a long time that t3 has high impact on cancer development and maintenance; however, there is no fundamental and comprehensive analysis of the role of thyroid hormone in different populations of cancer . Of course, many research groups study t3 influence on cancer; however those findings are not structured and cannot be compared to each other, because thyroid hormone can act differently in different types of cancer . Many research groups provided very important data about t3 and cancer; for example, it has been shown by poplawski and nauman that t3 promotes cell proliferation in rcc in vitro but does not have the same effect on a cell line derived from healthy kidney . What is more, puzianowska - kuznicka et al . And master et al . Observed that cells derived from rcc have disrupted tr expression on both the mrna and protein level; therefore disrupted posttranscriptional regulation has been proven for rcc . Research by kamiya et al . Showed numerous mutations in the thr transcript in the samples derived from rcc patients . However, it is important to mention that studies cited in this paper used supraphysiologic concentrations of t3 in in vitro experiments . Concentration of t3 in human sera was described in numerus studies by many groups as 3.07 nm, 1.92 nm, 1.69 nm, and 5.07 nm . What is more, based on these and many other publications it is possible to design and perform functional analysis of the tr1 in different populations of cancer, representing primary tumor, metastatic tumor, and cancer stem cell populations of rcc . It is extremely important to understand the mechanisms responsible for the disrupted influence of t3 hormone on cells of renal cell carcinoma because of its role as a cancer regulator and its link to therapy with sunitinib and other multitargeted receptor tyrosine kinase inhibitors.
Subjects for the gokind collection were recruited through two centers with different methods of ascertainment and recruitment (14). The george washington university (gwu) biostatistics center coordinated the recruitment of volunteers (through mass media advertisement) living throughout the u.s . (excluding new england) and canada to 1 of 27 clinical centers located across the u.s . And the section of genetics and epidemiology at the joslin diabetes center (jdc) recruited and examined patients of the joslin clinic from new england who were already enrolled in the joslin kidney study on the genetics of diabetic nephropathy, a clinic - based cohort study in which case subjects with diabetic nephropathy and a random sampling of eligible control subjects were identified and recruited (16). Briefly, subjects enrolled in gokind had type 1 diabetes diagnosed before age 31, began insulin treatment within 1 year of their diagnosis, and were between 18 and 59 years of age at the time of enrollment . Participation in the dcct / edic study was an exclusion criterion so that the two study populations would be independent . Case subjects with diabetic nephropathy had either persistent proteinuria, defined by a urinary albumin - to - creatinine ratio 300 g / mg in two of the last three measurements taken at least 1 month apart, or esrd (dialysis or renal transplant). Control subjects had type 1 diabetes for at least 15 years and normoalbuminuria, defined by an albumin - to - creatinine ratio <20 g / mg in two of the last three measurements taken at least 1 month apart (if a third measurement was required, a value <40 g / mg was necessary for inclusion), without ever having been treated with ace inhibitors or angiotensin receptor blockers, and they were not being treated with antihypertensive medication at the time of recruitment into the study . For additional information regarding the definition of case and control subjects used in this analysis, (14). In total, 1,879 subjects (935 case and 944 control subjects) were recruited into gokind . The gwu panel included 437 case subjects with diabetic nephropathy (58 with proteinuria and 379 with esrd) and 446 control subjects; the jdc panel included 498 case subjects with diabetic nephropathy (268 with proteinuria and 230 with esrd) and 498 control subjects . Further details are also provided in the supplementary information, which is available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db08-1514/dc1 . Confirmation of our findings in the gokind collection was sought in genome - wide association data from the dcct / edic study, a long - term, prospective investigation of the development of diabetes - associated complications (17,18). Of the original dcct cohort recruited between 1983 and 1989, participants in edic underwent baseline examinations between 1994 and 1995 and have since participated in annual follow - up examinations to assess the development or progression of complications . As of edic year 12 (2005), this cohort had 1622 years of follow - up, and 132 cases of severe nephropathy (proteinuria or esrd) had been documented in 1,304 caucasian dcct / edic participants . Detailed clinical characteristics of the dcct / edic study have been published (13,17,18). The gokind collection was genotyped on an affymetrix 5.0 500k single nucleotide polymorphism (snp) array by the gain genotyping laboratory at the eli and edythe l. broad institute (cambridge, ma). Additionally, two snps, rs39075 and rs1888747, were genotyped in the gokind collection using taqman (applied biosystems, foster city, ca) technology by the genetics core of the diabetes and endocrinology research center at the jdc in accordance with the manufacturer's protocols . Dna samples used for the genotyping of these snps in the gokind collection were obtained through the national institute of diabetes and digestive and kidney diseases central repository (www.niddkrepository.org). Several quality control metrics, including filters for minor allele frequency <0.01, rejection of hardy - weinberg assumptions (p 10), and differential rates of missing data (by case / control status) were applied to these data . After reconciliation of snps eliminated by these analyses, the resulting data contained 359,193 autosomal snps . The application of quality control criteria reduced the gokind population from 1,879 to 1,705 individuals of european ancestry . Samples from the two gokind panels that constitute this sample are 379 gwu case subjects (49 with proteinuria and 330 with esrd), 413 gwu control subjects, 441 jdc case subjects (235 with proteinuria and 206 with esrd), and 472 jdc control subjects . The measured genotypes for these individuals were augmented by imputation of ungenotyped snps across two mega base pair regions flanking each of the lead snps in the gokind collection (where linkage disequilibrium, as measured by r, decayed to <0.20 for all lead snps). A total of 8,245 imputed hapmap snps across these four loci were included in our association analysis . Further details of sample quality control and imputation procedures are available in the supplementary information and supplementary table 2 . Gokind samples were collected under two separate ascertainment protocols (jdc and gwu panels) so that tests for latent residual stratification were performed . Cochran - armitage tests of trend for the jdc versus gwu control subjects, and jdc versus gwu case subjects, revealed an overdispersion in the test statistics for both control and case subjects compared with the complete null . The median genomic control parameters were estimated at = 1.13 for control subjects and = 1.097 for case subjects . Permutation analysis within the control subjects and within the case subjects resulted in a stratification significance of p <10 for both case and control subjects . Therefore, the primary association analysis used in the study was a stratified test of association combining case - control tests of allele frequencies in jdc and gwu strata . Combined p values and odds ratios (ors) were calculated using a cochran - mantel - haenszel procedure . All genome - wide statistical association analyses were performed using plink and r (19). Further details of quality control procedures, software, statistical analysis and adjustments, and cluster plots are available in the supplementary information . Data from our analysis of the gokind collection are available for specific snps and/or genes upon request . Genotypes of the dcct / edic study participants were generated with the illumina human1 m beadchip . Multivariate cox proportional hazard analyses were performed on data from 1,304 caucasian subjects using time to onset of severe nephropathy, defined by an albumin excretion rate (aer)> 300 mg/24 h on at least two consecutive examinations or dialysis / renal transplant with prior persistent microalbuminuria (two consecutive aers> 30 mg/24 h) as the outcome phenotype (n = 132). Among those with severe nephropathy, 116 subjects developed only proteinuria (aer> the dcct cohort (primary prevention versus secondary intervention), treatment (intensive versus conventional), and interaction between cohort and treatment were used as covariates in the analysis of the effect of an independent additive snp genetic factor . This model was examined for all associated loci in gokind and subsequently tested for both statistical significance and the same direction of effect for associated alleles (2022). The expression of candidate genes was examined in four primary human cell lines derived from cells that have been implicated in the pathogenesis of kidney complications (endothelial cells from the iliac artery, adult dermal fibroblasts, mesangial cells, and epithelial cells from proximal tubules) by quantitative real - time pcr . Sources of these cells, cell culture conditions, and protocols used in these experiments are available in the supplementary information . The gokind collection was genotyped on an affymetrix 5.0 500k single nucleotide polymorphism (snp) array by the gain genotyping laboratory at the eli and edythe l. broad institute (cambridge, ma). Additionally, two snps, rs39075 and rs1888747, were genotyped in the gokind collection using taqman (applied biosystems, foster city, ca) technology by the genetics core of the diabetes and endocrinology research center at the jdc in accordance with the manufacturer's protocols . Dna samples used for the genotyping of these snps in the gokind collection were obtained through the national institute of diabetes and digestive and kidney diseases central repository (www.niddkrepository.org). Several quality control metrics, including filters for minor allele frequency <0.01, rejection of hardy - weinberg assumptions (p 10), and differential rates of missing data (by case / control status) were applied to these data . After reconciliation of snps eliminated by these analyses, the resulting data contained 359,193 autosomal snps . The application of quality control criteria reduced the gokind population from 1,879 to 1,705 individuals of european ancestry . Samples from the two gokind panels that constitute this sample are 379 gwu case subjects (49 with proteinuria and 330 with esrd), 413 gwu control subjects, 441 jdc case subjects (235 with proteinuria and 206 with esrd), and 472 jdc control subjects . The measured genotypes for these individuals were augmented by imputation of ungenotyped snps across two mega base pair regions flanking each of the lead snps in the gokind collection (where linkage disequilibrium, as measured by r, decayed to <0.20 for all lead snps). A total of 8,245 imputed hapmap snps across these four loci were included in our association analysis . Further details of sample quality control and imputation procedures are available in the supplementary information and supplementary table 2 . Gokind samples were collected under two separate ascertainment protocols (jdc and gwu panels) so that tests for latent residual stratification were performed . Cochran - armitage tests of trend for the jdc versus gwu control subjects, and jdc versus gwu case subjects, revealed an overdispersion in the test statistics for both control and case subjects compared with the complete null . The median genomic control parameters were estimated at = 1.13 for control subjects and = 1.097 for case subjects . Permutation analysis within the control subjects and within the case subjects resulted in a stratification significance of p <10 for both case and control subjects . Therefore, the primary association analysis used in the study was a stratified test of association combining case - control tests of allele frequencies in jdc and gwu strata . Combined p values and odds ratios (ors) were calculated using a cochran - mantel - haenszel procedure . All genome - wide statistical association analyses were performed using plink and r (19). Further details of quality control procedures, software, statistical analysis and adjustments, and cluster plots are available in the supplementary information . Data from our analysis of the gokind collection are available for specific snps and/or genes upon request . Genotypes of the dcct / edic study participants were generated with the illumina human1 m beadchip . Multivariate cox proportional hazard analyses were performed on data from 1,304 caucasian subjects using time to onset of severe nephropathy, defined by an albumin excretion rate (aer)> 300 mg/24 h on at least two consecutive examinations or dialysis / renal transplant with prior persistent microalbuminuria (two consecutive aers> 30 mg/24 h) as the outcome phenotype (n = 132). Among those with severe nephropathy, 116 subjects developed only proteinuria (aer> 300 mg/24 h), whereas 16 progressed to esrd . The dcct cohort (primary prevention versus secondary intervention), treatment (intensive versus conventional), and interaction between cohort and treatment were used as covariates in the analysis of the effect of an independent additive snp genetic factor . This model was examined for all associated loci in gokind and subsequently tested for both statistical significance and the same direction of effect for associated alleles (2022). The expression of candidate genes was examined in four primary human cell lines derived from cells that have been implicated in the pathogenesis of kidney complications (endothelial cells from the iliac artery, adult dermal fibroblasts, mesangial cells, and epithelial cells from proximal tubules) by quantitative real - time pcr . Sources of these cells, cell culture conditions, and protocols used in these experiments are available in the supplementary information . The application of metrics for snp and sample quality resulted in the analysis of 359,193 autosomal snps and 1,705 gokind samples of european ancestry (885 control subjects and 820 case subjects) (see research design and methods and the supplementary information). Because different ascertainment protocols were used by the jdc and gwu, the resulting data were found to exhibit significant stratification . As a result baseline clinical characteristics of the gokind collection clinical characteristics are the mean values sd for all caucasian patients (n = 1,705) included in the current analysis after the application of quality control metrics (see supplemental information regarding sample quality control analysis and population substructure and ancestry analysis). * the duration of type 1 diabetes in control subjects and in subjects with proteinuria is based on the duration at examination . Among esrd case subjects, this is based on the duration of type 1 diabetes at the onset of esrd . Mean a1c values do not include data from case subjects that have undergone pancreas transplantation (11% of jdc case subjects and 58% of gwu case subjects); percentages are of esrd group . Although no snp achieved genome - wide significance (0.05/359,193 = 1.4 10), the primary association analysis identified 11 snps representing four distinct chromosomal regions with p <1 10 (fig . 1 and table 2), which were considered for replication . The strongest association with diabetic nephropathy occurred on chromosome 9q with rs10868025 (or = 1.45, p = 5.0 10). This snp is located near the 5 end of the 4.1 protein ezrin, radixin, moesin (ferm) domain containing 3 (frmd3) gene . The log10 p values calculated using the cochran - mantel - haenszel method (adjusting for sex and gokind subcollection [jdc / gwu]) across the entire genome are shown for the combined gokind collection . The horizontal dashed line corresponds to a log10 p value = 5.0 (p = 1 10). Snps shown in green (n = 11) exceed this threshold (because of the resolution of this image, some of the snps located on chromosome 13 [n = 7] appear indistinguishable). Summary of snps associated with diabetic nephropathy in the gokind collection the most strongly associated snps from the combined analysis of the gwu and jdc gokind panels are presented along with the risk allele frequencies and p values (calculated using the cochran - mantel - haenszel method, adjusting for sex, between case and control subjects within each collection) for each separate collection . Combined p values and ors were calculated using the cochran - mantel - haenszel method . Chromosomal locations, snp positions, and gene annotations are in reference to ncbi build 36.1 . A summary of the genotype frequencies for the most strongly associated snps in the gokind collection are presented in supplementary table 3 . Rs39075 and rs1888747 were identified through imputation and genotyped using taqman assays in the gokind collection; rs1411766 and rs17412858 were both genotyped on the affymetrix array and are in complete linkage disequilibrium (r = 1.0); rs6492208 and rs2391777 were both genotyped on the affymetrix array and are in complete linkage disequilibrium (r = 1.0). Three additional genomic regions located on chromosomes 7p, 11p, and 13q were also associated with diabetic nephropathy . The rs39059 snp (or = 1.39, p = 5.0 10) localizes to the first intron of chn2 (-chimerin) isoform 2 and upstream of an alternatively spliced cpvl (serine carboxypeptidase vitellogenic - like) transcript on chromosome 7p . The rs451041 snp (or = 1.36, p = 3.1 10) is located on chromosome 11p in an intronic region of the cars (cysteinyl - trna synthetase) gene . And, finally, the region bounded by rs1411766/rs1742858 (or = 1.41, p = 1.8 10) is located in a 42 kb intergenic region on chromosome 13q . Analyses of the imputed snps in our lead loci identified 11 additional snps that were highly correlated withthe original associations (p <1 10). Of these, two were more strongly associated with diabetic nephropathy than our lead genotyped snps . Imputed snp rs1888747 (chromosome 9q), which is in partial linkage disequilibrium (r = 0.81) with rs10868025, was more strongly associated with diabetic nephropathy than the original snp (p = 4.7 10) (fig . Similarly, two imputed snps in the 7p region (rs39075 and rs39076) were also more strongly associated than the original snp in that region (rs39059) (fig . Both imputed snps were genotyped in the gokind samples, and the associations with the imputed data were confirmed (rs39075, p = 6.5 10; and rs1888747, p = 6.3 10) (table 2). Summary of genome - wide association results for the chromosome 7p, 9q, 11p, and 13q loci . A: genome - wide association scan and imputed data for the chromosome 7p locus . Rs39059 (solid red triangle) is located at position ivs1 + 21350 relative to exon 1 of chn2 isoform 2 and is in tight linkage disequilibrium with rs39075 (r = 0.96), located at position ivs1 + 42572 . Rs39059 and rs39075 reside 69,318 and 90,540 kb, respectively, upstream of cpvl isoforms 1 and 2 . A third alternate transcript (isoform 3) is predicted for cpvl and contains an exon that extends to intron 1 of chn2 . Rs39059 and rs39075 are located at positions 20579 and 41801, respectively, relative to this transcript ., snps genotyped on the affymetrix array (n = 163);, imputed snps (n = 694). * imputed snp rs39075 was genotyped in the gokind samples to confirm the imputation . B: genome - wide association scan and imputed data for the chromosome 9q locus . A total of 100 genotyped snps from the affymetrix array data and 450 imputed snps are shown . Rs10868025 (solid red triangle) is located at position 10829 relative to frmd3's transcription start site . Rs10868025 is in complete linkage disequilibrium (r = 1.0) and only 253 bp from imputed snp rs13289150 (superimposed on rs10868025). Rs1888747, located at position 2204, is in partial linkage disequilibrium (r = 0.81) with rs10868025 . * c: genome - wide association scan and imputed data for the chromosome 11p locus . A total of 33 genotyped snps from the affymetrix array data and 190 imputed snps are shown . Rs739401 and rs451041 (solid red triangles) are in strong linkage disequilibrium (r = 0.97). Rs739401 is located in intron 16 (isoforms a and c)/17 (isoforms b and d) of the cars gene (position ivs16 + 687/ivs17 + 687). Rs451041 is located in intron 4 (isoforms a and c)/5 (isoforms b and d), position ivs4 d: genome - wide association scan and imputed data for the chromosome 13q locus . A total of 68 genotyped snps from the affymetrix array data and 268 imputed snps are shown . Seven lead snps (rs1041466, rs1411766, rs17412858, rs6492208, rs2391777, rs7989848, and rs9521445) from this region are indicated in red . Rs1411766 and rs17412858 are in complete linkage disequilibrium (r = 1.0). Similarly, rs6492208 and rs2391777 are in complete linkage disequilibrium (r = 1.0). Rs7989848 and rs9521445 are in strong linkage disequilibrium (r = 0.87), whereas only modest linkage disequilibrium exists between all other snp pairs (r = 0.300.65). The two nearest genes are myo16 and irs2, located 384 kb centromeric and 120 kb telomeric of this region, respectively . If the etiology of diabetic nephropathy involves the interaction of a locus with the cumulative effect of hyperglycemia, the association of the locus with diabetic nephropathy can vary according to diabetes duration at diabetic nephropathy onset, such that it is strongest in early - appearing case subjects and diminishes in later ones, even reversing in direction in very late - appearing case subjects (23). We examined the snps in table 2 according to diabetes duration by stratifying case and control subjects across tertiles of diabetes duration (at the onset of esrd or at enrollment into gokind for proteinuria patients and control subjects). The strength of the associations was consistent across these strata (data not shown). Additionally, if a locus influences mortality risk, the high mortality experienced by patients with esrd would alter its association with diabetic nephropathy according to the duration of survival with esrd and may mask the effect of a diabetic nephropathy risk allele or produce a false association . For this reason, we also analyzed the lead snps in table 2 according to duration of esrd . For each of these snps, the ors were consistent across tertiles of esrd duration (supplementary table 4), a pattern consistent with the absence of survival bias . However, the current study is underpowered to formally exclude the presence of such effects . Data from a genome - wide association scan of the dcct / edic study were used to assess whether genome regions identified in the gokind collection were associated with advanced diabetic nephropathy in an independent collection . Among the 11 snps identified in gokind, eight were included on the illumina array used in the dcct / edic study (table 3). The three snps not included on this platform, rs39059, rs739401, and rs9521445, were in strong linkage disequilibrium (r 0.87) with rs39075, rs451041, and rs7989848, respectively . Analysis of time to onset of severe nephropathy confirmed the significant associations with diabetic nephropathy in gokind for rs1888746 (frmd3, p = 0.02), rs13289150 (frmd3, p = 0.05), and rs451041 (cars, p = 0.01). Hrs for the development of severe nephropathy during 1622 years of follow - up in the dcct / edic study for snps associated with diabetic nephropathy in gokind data are from multivariate cox proportional hazard analysis of time to onset of severe nephropathy . As of 2005 chromosomal locations, snp positions, and gene annotations are in reference to ncbi build 36.1 . * the risk alleles that are presented are in reference to those identified in the gokind collection; one - sided p values (consistent with the current best practices for replication in gwa scans) (2022) are used to test for the same direction of effect as in the gokind collection; rs1888746 was genotyped on an illumina array in dcct / edic and is in complete linkage disequilibrium (r = 1.0) with rs1888747 (genotyped using a taqman assay in gokind); rs13289150 was genotyped on an illumina array in dcct / edic and is in complete linkage disequilibrium (r = 1.0) with rs10868025 (genotyped on an affymetrix array in gokind). Previous studies, as well as publicly available gene expression data (www.ncbi.nlm.nih.gov/geo), have shown that genes closest to the lead snps identified in gokind are expressed in a variety of human tissues, including kidney (2426). To further test whether these candidate genes may be involved in the development of diabetic nephropathy the expression of chn2, cpvl, frmd3, and cars was examined in four primary human cell lines: iliac artery endothelial cells, adult dermal fibroblasts, mesangial cells, and renal proximal tubule cells . Our data show that cars expression was high in all four of the cell lines that we examined (table 4). Frmd3 expression was also detected in each cell type, with its highest expression being observed in renal proximal tubule cells . Of the two candidate diabetic nephropathy genes located in chromosome 7p region, neither was detected in mesangial cells, whereas cpvl expression was greatest in proximal tubule cells . Relative gene expression of novel candidate diabetic nephropathy susceptibility genes in primary human cell lines the relative gene expression of cpvl, chn2, frmd3, and cars was determined in four primary human cell lines using real - time pcr . Relative gene expression of each gene was calculated in reference to a normalization control (-actin) and is presented as the mean ct (ctvalue from each gene minus ct value from the normalization control) and sd from three separate experiments . Mean ct = 0 equals high relative gene expression (i.e., expression similar to -actin). The application of metrics for snp and sample quality resulted in the analysis of 359,193 autosomal snps and 1,705 gokind samples of european ancestry (885 control subjects and 820 case subjects) (see research design and methods and the supplementary information). Because different ascertainment protocols were used by the jdc and gwu, the resulting data were found to exhibit significant stratification . As a result baseline clinical characteristics of the gokind collection clinical characteristics are the mean values sd for all caucasian patients (n = 1,705) included in the current analysis after the application of quality control metrics (see supplemental information regarding sample quality control analysis and population substructure and ancestry analysis). * the duration of type 1 diabetes in control subjects and in subjects with proteinuria is based on the duration at examination . Among esrd case subjects, this is based on the duration of type 1 diabetes at the onset of esrd . Mean a1c values do not include data from case subjects that have undergone pancreas transplantation (11% of jdc case subjects and 58% of gwu case subjects); percentages are of esrd group . Although no snp achieved genome - wide significance (0.05/359,193 = 1.4 10), the primary association analysis identified 11 snps representing four distinct chromosomal regions with p <1 10 (fig . 1 and table 2), which were considered for replication . The strongest association with diabetic nephropathy occurred on chromosome 9q with rs10868025 (or = 1.45, p = 5.0 10). This snp is located near the 5 end of the 4.1 protein ezrin, radixin, moesin (ferm) domain containing 3 (frmd3) gene . The log10 p values calculated using the cochran - mantel - haenszel method (adjusting for sex and gokind subcollection [jdc / gwu]) across the entire genome are shown for the combined gokind collection . The horizontal dashed line corresponds to a log10 p value = 5.0 (p = 1 10). Snps shown in green (n = 11) exceed this threshold (because of the resolution of this image, some of the snps located on chromosome 13 [n = 7] appear indistinguishable). Summary of snps associated with diabetic nephropathy in the gokind collection the most strongly associated snps from the combined analysis of the gwu and jdc gokind panels are presented along with the risk allele frequencies and p values (calculated using the cochran - mantel - haenszel method, adjusting for sex, between case and control subjects within each collection) for each separate collection . Combined p values and ors were calculated using the cochran - mantel - haenszel method . Chromosomal locations, snp positions, and gene annotations are in reference to ncbi build 36.1 . A summary of the genotype frequencies for the most strongly associated snps in the gokind collection are presented in supplementary table 3 . Rs39075 and rs1888747 were identified through imputation and genotyped using taqman assays in the gokind collection; rs1411766 and rs17412858 were both genotyped on the affymetrix array and are in complete linkage disequilibrium (r = 1.0); rs6492208 and rs2391777 were both genotyped on the affymetrix array and are in complete linkage disequilibrium (r = 1.0). Three additional genomic regions located on chromosomes 7p, 11p, and 13q were also associated with diabetic nephropathy . The rs39059 snp (or = 1.39, p = 5.0 10) localizes to the first intron of chn2 (-chimerin) isoform 2 and upstream of an alternatively spliced cpvl (serine carboxypeptidase vitellogenic - like) transcript on chromosome 7p . The rs451041 snp (or = 1.36, p = 3.1 10) is located on chromosome 11p in an intronic region of the cars (cysteinyl - trna synthetase) gene . And, finally, the region bounded by rs1411766/rs1742858 (or = 1.41, p = 1.8 10) is located in a 42 kb intergenic region on chromosome 13q . Analyses of the imputed snps in our lead loci identified 11 additional snps that were highly correlated withthe original associations (p <1 10). Of these, two were more strongly associated with diabetic nephropathy than our lead genotyped snps . Imputed snp rs1888747 (chromosome 9q), which is in partial linkage disequilibrium (r = 0.81) with rs10868025, was more strongly associated with diabetic nephropathy than the original snp (p = 4.7 10) (fig . Similarly, two imputed snps in the 7p region (rs39075 and rs39076) were also more strongly associated than the original snp in that region (rs39059) (fig . Both imputed snps were genotyped in the gokind samples, and the associations with the imputed data were confirmed (rs39075, p = 6.5 10; and rs1888747, p = 6.3 10) (table 2). Summary of genome - wide association results for the chromosome 7p, 9q, 11p, and 13q loci . A: genome - wide association scan and imputed data for the chromosome 7p locus . Rs39059 (solid red triangle) is located at position ivs1 + 21350 relative to exon 1 of chn2 isoform 2 and is in tight linkage disequilibrium with rs39075 (r = 0.96), located at position ivs1 + 42572 . Rs39059 and rs39075 reside 69,318 and 90,540 kb, respectively, upstream of cpvl isoforms 1 and 2 . A third alternate transcript (isoform 3) is predicted for cpvl and contains an exon that extends to intron 1 of chn2 . Rs39059 and rs39075 are located at positions 20579 and 41801, respectively, relative to this transcript ., snps genotyped on the affymetrix array (n = 163);, imputed snps (n = 694). * imputed snp rs39075 was genotyped in the gokind samples to confirm the imputation . B: genome - wide association scan and imputed data for the chromosome 9q locus . A total of 100 genotyped snps from the affymetrix array data and 450 imputed snps are shown . Rs10868025 (solid red triangle) is located at position 10829 relative to frmd3's transcription start site . Rs10868025 is in complete linkage disequilibrium (r = 1.0) and only 253 bp from imputed snp rs13289150 (superimposed on rs10868025). Rs1888747, located at position 2204, is in partial linkage disequilibrium (r = 0.81) with rs10868025 . * c: genome - wide association scan and imputed data for the chromosome 11p locus . A total of 33 genotyped snps from the affymetrix array data and 190 imputed snps are shown . Rs739401 and rs451041 (solid red triangles) are in strong linkage disequilibrium (r = 0.97). Rs739401 is located in intron 16 (isoforms a and c)/17 (isoforms b and d) of the cars gene (position ivs16 + 687/ivs17 + 687). Rs451041 is located in intron 4 (isoforms a and c)/5 (isoforms b and d), position ivs4 d: genome - wide association scan and imputed data for the chromosome 13q locus . A total of 68 genotyped snps from the affymetrix array data and 268 imputed snps are shown . Seven lead snps (rs1041466, rs1411766, rs17412858, rs6492208, rs2391777, rs7989848, and rs9521445) from this region are indicated in red . Rs1411766 and rs17412858 are in complete linkage disequilibrium (r = 1.0). Similarly, rs6492208 and rs2391777 are in complete linkage disequilibrium (r = 1.0). Rs7989848 and rs9521445 are in strong linkage disequilibrium (r = 0.87), whereas only modest linkage disequilibrium exists between all other snp pairs (r = 0.300.65). The two nearest genes are myo16 and irs2, located 384 kb centromeric and 120 kb telomeric of this region, respectively . If the etiology of diabetic nephropathy involves the interaction of a locus with the cumulative effect of hyperglycemia, the association of the locus with diabetic nephropathy can vary according to diabetes duration at diabetic nephropathy onset, such that it is strongest in early - appearing case subjects and diminishes in later ones, even reversing in direction in very late - appearing case subjects (23). We examined the snps in table 2 according to diabetes duration by stratifying case and control subjects across tertiles of diabetes duration (at the onset of esrd or at enrollment into gokind for proteinuria patients and control subjects). The strength of the associations was consistent across these strata (data not shown). Additionally, if a locus influences mortality risk, the high mortality experienced by patients with esrd would alter its association with diabetic nephropathy according to the duration of survival with esrd and may mask the effect of a diabetic nephropathy risk allele or produce a false association . For this reason, we also analyzed the lead snps in table 2 according to duration of esrd . For each of these snps, the ors were consistent across tertiles of esrd duration (supplementary table 4), a pattern consistent with the absence of survival bias . However, the current study is underpowered to formally exclude the presence of such effects . Data from a genome - wide association scan of the dcct / edic study were used to assess whether genome regions identified in the gokind collection were associated with advanced diabetic nephropathy in an independent collection . Among the 11 snps identified in gokind, eight were included on the illumina array used in the dcct / edic study (table 3). The three snps not included on this platform, rs39059, rs739401, and rs9521445, were in strong linkage disequilibrium (r 0.87) with rs39075, rs451041, and rs7989848, respectively . Analysis of time to onset of severe nephropathy confirmed the significant associations with diabetic nephropathy in gokind for rs1888746 (frmd3, p = 0.02), rs13289150 (frmd3, p = 0.05), and rs451041 (cars, p = 0.01). Hrs for the development of severe nephropathy during 1622 years of follow - up in the dcct / edic study for snps associated with diabetic nephropathy in gokind data are from multivariate cox proportional hazard analysis of time to onset of severe nephropathy . As of 2005 chromosomal locations, snp positions, and gene annotations are in reference to ncbi build 36.1 . * the risk alleles that are presented are in reference to those identified in the gokind collection; one - sided p values (consistent with the current best practices for replication in gwa scans) (2022) are used to test for the same direction of effect as in the gokind collection; rs1888746 was genotyped on an illumina array in dcct / edic and is in complete linkage disequilibrium (r = 1.0) with rs1888747 (genotyped using a taqman assay in gokind); rs13289150 was genotyped on an illumina array in dcct / edic and is in complete linkage disequilibrium (r = 1.0) with rs10868025 (genotyped on an affymetrix array in gokind). Previous studies, as well as publicly available gene expression data (www.ncbi.nlm.nih.gov/geo), have shown that genes closest to the lead snps identified in gokind are expressed in a variety of human tissues, including kidney (2426). To further test whether these candidate genes may be involved in the development of diabetic nephropathy the expression of chn2, cpvl, frmd3, and cars was examined in four primary human cell lines: iliac artery endothelial cells, adult dermal fibroblasts, mesangial cells, and renal proximal tubule cells . Our data show that cars expression was high in all four of the cell lines that we examined (table 4). Frmd3 expression was also detected in each cell type, with its highest expression being observed in renal proximal tubule cells . Of the two candidate diabetic nephropathy genes located in chromosome 7p region, neither was detected in mesangial cells, whereas cpvl expression was greatest in proximal tubule cells . Relative gene expression of novel candidate diabetic nephropathy susceptibility genes in primary human cell lines the relative gene expression of cpvl, chn2, frmd3, and cars was determined in four primary human cell lines using real - time pcr . Relative gene expression of each gene was calculated in reference to a normalization control (-actin) and is presented as the mean ct (ctvalue from each gene minus ct value from the normalization control) and sd from three separate experiments . Mean ct = 0 equals high relative gene expression (i.e., expression similar to -actin). In this report, we describe the results of a genome - wide association scan in the gokind collection to identify loci associated with risk of diabetic nephropathy in type 1 diabetes . Although the biology underlying these associations remains to be elucidated, they implicate chn2/cpvl, frmd3, cars, and an intergenic region on chromosome 13q as novel genes / genetic regions involved in the pathogenesis of diabetic nephropathy . None of these loci overlap with previously reported associations between candidate genes and the development of any stage of diabetic nephropathy (10,11). Importantly, replication in a cox proportional hazard analysis of the associations at the frmd3 and cars loci with time to the onset of severe nephropathy in the dcct / edic study bolsters the significance of these two findings; that two studies having such different designs (one a case - control study and the other a prospective cohort study) yielded similar ors strengthens confidence in this conclusion . Frmd3 encodes the 4.1o protein, a structural protein with unknown function and a member of the 4.1 family of proteins (26). Members of the 4.1 protein family have well - characterized roles as cytoskeletal proteins, maintaining both cellular shape and form, in a variety of cell types, including mouse nephron (27,28). Although membership of the 4.1o protein in this family has recently been questioned, it does contain a ferm domain, which is a module that is integral in maintaining cell integrity through its interactions with transmembrane proteins and actin filaments (29,30). Frmd3 is detectable in adult ovaries as well as in fetal skeletal muscle, brain, and thymus (26). Our data extend the expression profile of frmd3 to specifically include mesangial and proximal tubular cells . Interestingly, among 18 genes that contain ferm domains, including several members of the 4.1 protein family, we identified nominally significant associations with diabetic nephropathy for snps located in eight of these genes (supplementary table 5), including farp2 (ferm, rhogef and pleckstrin domain protein 2; p = 3.0 10) and epb41l2 (erythrocyte membrane protein band 4.1-like 2; p = 2.3 10). Although these findings require further study, including replication in additional collections, it is interesting to speculate that these data may point to the involvement of new, previously unsuspected pathways in the pathogenesis of diabetic nephropathy . The cars gene encodes cysteinyl - trna synthetase, one of several aminoacyl - trna synthetases (arss) that have been identified in humans (31,32). Arss are important regulators of intracellular amino acid concentrations and protein biosynthesis in both the cytoplasm and mitochondria (a process facilitated by specialized mitochondria - specific and bifunctional arss). In the initial steps of protein translation, the function of these enzymes is to attach amino acids to their cognate trna molecules . To date, both autosomal dominant and recessive mutations in ars - encoding genes have been identified only in neurodegenerative disease, including missense changes in glycyl - trna synthetase (gars) and both missense mutations and in - frame deletions in tyrosyl - trna synthetase (yars) in charcot - marie - tooth disease (32). Cars has been implicated in cystinosis, an autosomal recessive renal tubule disorder caused by the accumulation of free cystine in cellular lysosomes (33,34). A recent study identified defects in lysosomal cystine transport as the primary cause of the disease (35). However, esrd is prominent in this disorder, and such an outcome may be due to vulnerability of specific renal cells to damage by excess cystine . Interesting, in this light, is the observation that of all the associated snps, only those in the cars locus were associated primarily with esrd (supplementary table 4). Further work is needed to characterize the role of cars in the pathway that is involved in the development of esrd in diabetes . Similar to the set of genes containing ferm domains, analysis of 21 ars genes identified nominally significant associations with diabetic nephropathy for snps located in four members of this class of genes (supplementary table 6), with the most significant association (p = 9.1 10) occurring at the tars (threonyl - trna synthetase) locus . Two additional loci were strongly associated with diabetic nephropathy in both panels of the gokind collection . Of the two genes located on chromosome 7p, cpvl, a carboxypeptidase that is highly expressed in the kidney and, more specifically, in proximal tubules, is a particularly interesting candidate gene . Other carboxypeptidases, such as ace and bradykinin, are important regulators of renal hemodynamics and have previously been implicated in the pathogenesis of diabetic nephropathy (36,37). Previously, genomic deletions of this locus have been linked to congenital renal abnormalities (38). The two genes closest to the associated snps, myo16 (myosin heavy - chain myr 8) and irs2 (insulin receptor substrate 2), are located 384 kb centromeric and 120 kb telomeric of this region, respectively . Although there is little linkage disequilibrium between the variants within this block and those in the vicinity of either myo16 or irs2, the multiple signals identified in this region give credence to the association detected in our analysis . The findings presented in our study contribute to understanding the genetic susceptibility of diabetic nephropathy in type 1 diabetes . As has been reported for other complex genetic disorders, no single major gene that contributes to an increased risk of disease emerged (20,39). However, given the incomplete coverage of the genome by the genotyping platform and the suboptimal study design (prevalent rather than incident cases of esrd), detection of any existing major gene effect was not guaranteed . For example, because most of the case subjects with esrd had survived many years on dialysis or with a kidney transplant, a disease allele that not only increased susceptibility to diabetic nephropathy but also increased mortality in patients with esrd could go undetected . Appreciably, the snps that we identified in the gokind collection were mortality neutral (supplementary table 4). The optimal study design for detecting all disease loci, regardless of their effect on mortality, would be a large cohort of incident esrd case subjects . The gokind collection is heavily weighted with case subjects with esrd; thus, the small number of case subjects with proteinuria limited our ability to detect variants primarily associated with the risk of proteinuria . Second, because of the limited power of the dcct / edic study and the need to contain inflation of the -error in seeking replication for multiple snps in this dataset, our replication efforts refrained from considering snps less significant than p = 1 10 . It is certainly possible that additional variants among those not meeting this threshold may truly be associated with diabetic nephropathy; however, given these limitations, these variants remain to be identified . Similarly, despite replication in the dcct / edic cohort, we acknowledge that positive associations at both the frmd3 and cars loci require additional study to be certain of these findings . Third, although the locations of the variants confirmed in this study implicate both frmd3 and cars as novel genes involved in the pathogenesis of diabetic nephropathy, the underlying mechanisms of disease of these associations need to be elucidated . And, finally, although confirmation in dcct / edic has been achieved for variations near frmd3 and cars, additional cohorts, particularly non - caucasian, would be useful to further characterize the pathogenic role of these, and other, candidate genes identified in the gokind collection.
Twin studies have proved that neurodevelopmental disorders, such as attention deficit hyperactivity disorder (adhd), autism spectrum disorders (asd), as well as psychiatric conditions like schizophrenia (scz) and bipolar disorder (bd) have an important genetic background . Nevertheless, until very recently, causal genes have only been found in the context of intellectual disability (i d). Classical genetic studies have failed to identify genes with high penetrance in pndd, thus indicating that the genetic background of these disorders is highly heterogeneous . Recent developments in dna analysis and sequencing, such as next - generation sequencing, snp arrays, exome sequencing or analysis of copy number variations (cnvs), allow to study the whole genome of large cohorts of affected individuals, enabling the analysis of cns disorders with highly heterogeneous genetic etiology . Several of these studies have focused on pndds, uncovering new genes with potential roles in these disorders . Interestingly, many of the genes identified are involved in synaptic physiology, pointing towards synaptic dysfunction as an important contributing factor in many of these disorders . Although numerous psychiatric conditions have traditionally been ascribed to unbalances in monoaminergic systems, it is also accepted that alterations in the glutamatergic system are involved in these disorders . In particular, an important group of genes expressed at the synapse identified in the context of pndds code for glutamate receptor subunits . Glurs comprise three families of ionotropic receptors: ampa (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), nmda (n - methyl - d - aspartate) and kainate; as well as three groups of metabotropic receptors (mglurs i - iii). Ionotropic receptors are found as tetramers of various subunits: 4 gria genes code for ampa subunits, 7 grin genes code for nmda subunits and 5 grik genes code for kainate subunits . Finally, metabotropic receptors, which are g - protein coupled receptors, are coded by 8 grm genes . Functionally, ionotropic glurs are specialized on different aspects of synaptic transmission . While nmda receptors act as coincident detectors of postsynaptic membrane depolarization and glutamate release, ampa receptors mediate fast transmission in excitatory synapses . We discuss recently identified mutations in glur subunits in the context of pndds, including large genomic rearrangements directly affecting these genes or point mutations predicted to be deleterious . Linkage and association studies of natural variation, such as snps or microsatellites, have not been included in this work as these have a less direct implication in disease . Of all four genes coding for ampa receptor subunits, only mutations in gria2 and gria3 have been related with pndds (table 1). Alterations in these two genes have been associated with some cases of asd, but have mainly been found concomitant with i d . Although chromosomal deletions encompassing gria2 had been described for individuals with mental and developmental retardation (see ref . 12 for review), only recent studies have identified specific mutations in gria2 in the context of i d, suggesting that gria2 haploinsufficiency might cause i d . Gria3 was first identified as a candidate gene for x - linked i d in 1999, in a female with a balanced translocation directly involving this gene . Since then, several other gria3 mutations have been associated with i d, including complete or partial duplications, mutations on its 5utr and a whole gene deletion . Interestingly, both duplications and deletions of gria3, translate into a diminished or absent synthesis of glua3 protein . Partial duplications would cause either reduced gria3 transcripts or aberrant protein levels ultimately contributing to i d . Missense gria3 variants have also been found linked to i d and, with the exception of the g833r mutation (see table 1) these individuals express gria3 at normal levels . Nevertheless, when functionally tested glua3 variants displayed altered channel function either in homomeric combination or in heteromers with normal glua2 . Glua3 is normally present at synapses together with glua2 contributing to the normal cycling of ampa receptors . From the studied individuals with i d, it can be inferred that the lack of glua3 is not crucial for neuronal viability . In fact, synaptic targeting and function of these receptors are not significantly altered in glua3 ko mice . Remarkably, long - term potentiation (ltp), widely thought to be the cellular basis of learning processes, is abnormal in these animals . Nevertheless, in humans, the lack of glua3 could impair normal neuronal wiring or stabilization of activated synapses during development . Ampa receptor auxiliary subunits, tarps and cnihs, control receptor function by modulating channel trafficking and kinetics . It is interesting to note that a mutation affecting cacng2 (tarp -2) has been described in an individual with moderate i d . This mutation caused a decreased association with ampa subunits, altering the receptor trafficking and reducing mepscs in hippocampal neurons . Finally, cnih2 deletion has also been found in a boy with mild i d . Thus, it is noteworthy that malfunctions of ampa receptor auxiliary can also be associated with i d . These receptors are composed of two obligatory subunits (glun1) and two variable ones, which consist of either glun2(a - d) or glun3(a, b). Of the variable subunits, glun2b expression starts very early in development and is critical for synaptogenesis and neuronal survival in cortical brain areas, thus making it a candidate factor in neurodevelopmental disorders . Indeed, grin2b is the most frequently mutated grin gene (see table) in pndds, being mainly related with i d . Specific grin2b gain - of - function mutations have also been associated with asd, supporting the hypothesis of an imbalance between excitatory and inhibitory neurotransmission in asd etiology, as well as in west syndrome with severe developmental delay . Grin2a de novo mutations and microdeletions have also been associated to i d, indicating the viability of glun2a haploinsufficiency . Likewise, a rare grin2a de novo mutation was recently associated with schizophrenia, although the role of glun subunits with scz is under debate . Although less frequently, mutations in grin1 gene, the obligatory nmda receptor subunit, have also been identified . A mutation in grin1 has been found to cause non - syndromic intellectual disability (nsid), an observation functionally validated using cellular models . Interestingly, regarding grin2c, grin3a and grin3b, only rare truncating mutations affecting both healthy individuals and asd / sz patients have been reported . In contrast, no truncating mutations were found in grin1, grin2a, grin2b and grin2d genes, suggesting a more critical function of these genes during neurodevelopment and the lethality of the putative loss - of - function . Taken together, these recent reports suggest that de novo mutations of nmdar subunits are frequently associated with i d, although some specific mutations are also associated with psychiatric diseases . Previous classic genetic association studies suggested linkages to mood disorders for some of the kainate receptor - encoding genes, mainly grik2 and grik3 (see ref . More recently, cnvs in grik2 were found enriched in, but not exclusive of, children with i d, indicating limited pathogenic burden . Interestingly, a complex loss - of - function mutation in grik2 was found to co - segregate with nsid . This grik2 mutation involves various deletions and inversions spanning exons 7 to 11, resulting in loss of the first ligand - binding domain, the adjacent transmembrane domain, and the putative pore loop of gluk2 . This study strongly indicates that loss of gluk2 protein can cause severe - to - moderate cognitive impairment in humans . A grik4 variant with an insertion - deletion in the 3utr region (which results in increased gluk4 levels) was found to confer protection against bipolar disorder . Moreover, this grik4 variant increased hippocampal activation during face processing, suggesting a link between kainate receptor - mediated excitation in the hippocampus and bipolar disorder . So far, the subfamily of kainate glutamate receptors is the one for which less mutations have been identified in the context of pndds . However, collectively taken, these results support the notion that mutations leading to up- or down - regulation of kainate subunits can cause learning disabilities and modulate mood disorders . Currently, a limited number of papers report deleterious mutations related to pndds in grms (see table 1). Of these, two performed grm1 exon sequencing in scz and bd, another sequences the grm3 gene in a cohort of individuals with bd and one performed a genome - wide copy number variation (cnv) association study on attention deficit hyperactivity disorder (adhd). Finally, a mutation in the kozak s sequence of grm3 assocaited with scz has also been reported . I d, intellectual disability; nsid, non - syndromic i d; asd: autism spectrum disorder; bd, bipolar disorder; scz, schizophrenia; adhd, attention deficit hyperactivity disorder; * copy number variation study with average cnv size of 62 kb it is important to highlight that, as it happens with genes giving susceptibility to psychiatric diseases, none of the reported mutations supports for a causal role in disease . In most cases, the small number of mutations identified for grms make it still difficult to conjecture on their relevance to disease . Nevertheless, a striking observation can be made: there is no report implicating grm mutations in neurodevelopmental disorders such as i d or asd . Despite the extensive literature on the role of these receptors, especially grm1 and grm5, in fragile x - syndrome and asd, deleterious mutations on grms have so far been found only in the context of psychiatric disorders, such as scz, bp or adhd . Recently developed dna analysis tools are allowing for the rapid uncovering of glurs mutations in the context of pndds . This can be seen by the exponential increase in the number of papers reporting glur mutations in most recent years . Based on this, we expect that new glur mutations will be identified in the future, hopefully allowing for a better understanding of glur etiological contribution to pndds . Although the number of studies reporting glur mutations in pndd is so far restricted, some initial conclusions can be drawn . These will need to be examined in the light of future studies . In the first place, mutations of subunits of some receptor subtypes are related to certain disease types but not to others . In this regard, mutations in ampa subunits have only been found in the context of i d and asd, both neurodevelopmental disorders . Similarly, mutations in genes coding for ampar auxiliary proteins are also related with i d . Along these lines, mutations in nmda subunits are mostly linked to i d and asd . In stark contrast, mutations in metabotropic receptors are only related to psychiatric disorders . Accordingly, the data available would suggest that mutations in ionotropic glutamate receptors predispose towards neurodevelopmental disorders, while mutations in metabotropic receptors would predispose towards psychiatric disorders . One can also draw a parallel between the extent of mental disability and the contribution to neurotransmission of the affected receptor type . Thus, loss - of - function mutations in ampa and nmda subunits are frequently found in patients with i d . This is consistent with their important role in neuronal development, fast transmission, and synaptic plasticity . On the other hand, the abundance of grm mutations in individuals with psychiatric disorders is consistent with the more modulatory role of mglurs . The occurrence of i d in carriers of a mutant gluk2 suggests that tuning of neuronal network activity by kainate receptors can have profound effects on cognitive abilities . While some genes accumulate many potentially deleterious mutations, no mutations have been found in others . Amongst ampa subunits, for instance, gria1 and gria4 have not been found mutated in the context of pndd, while twelve different mutations have been described for gria3 . Remarkably, the spectrum of mutations in nmda subunits concentrates in particular coding regions, namely, the extracellular and pore - forming domains . This observation suggests that impaired ion selectivity and conductance of nmda receptors is closely linked to developmental defects, while the role of its intracellular tail might have a less critical role in disease . There are several potential explanations why some glur genes do not appear mutated in relation to disease; they might play indispensable biological functions, thus leading to lethality even in heterozygosity, or other molecules could compensate for their dysfunction . Interestingly, we do not see an increased number of mutations in glur subunits expressed early in development, which a priori, should be more relevant to neurodevelopmental disorders . For instance, a similar number of mutations has been found for grin2b, which starts to be expressed early in development, and for grin2a, that is expressed post - natally . In contrast, no mutations have been described for gria4, also highly expressed during development . Nevertheless, this observation should be taken cautiously as mutations in developmental genes could cause lethality and also because gene expression data, mostly obtained from rodent species, might not be completely valid for humans . Although the etiopathology of pndds is complex and multigenic, a growing set of genetic and functional evidences indicate the contribution of glutamate receptors in these damaging disorders . The following years will be crucial to understand whether the different receptor subunits are associated with certain pndds or not, as well as their interaction with genetic background and environmental factors.
Maintenance of strict glycemic control prevents or delays the risk of microangiopathy and, to a lesser degree, of macroangiopathy in patients with type 2 diabetes.1,2 the primary aim of therapy in diabetes is, therefore, to achieve target glycemic control, with a rate of hypoglycemic episodes or adverse events as reduced as possible.3,4 besides diet and physical exercise, both metformin and -cell secretagogues can be administered by an add on modality.5 in case of therapeutic failure, a further opportunity is given by glucagon - like peptide-1 (glp-1) receptor agonists, such as exenatide.68 glp-1 receptor agonists exert their hypoglycemic effect through stimulation of glucose - dependent insulin release coupled with a reduction of glucagon release from pancreatic -cells, cumulatively leading to protective effects on -cell mass.9,10 in addition, glp-1 receptor agonists elicit satiety sensation and slow gastric empting.1113 in conclusion, glp-1 receptor agonists such as exenatide obtain their therapeutic effects by reducing the risk of hypoglycemic events and, at the same time, by leading to significant weight loss, especially in obese patients.1418 many randomized controlled trials have, over the past years, confirmed the efficacy of exenatide either in monotherapy, or in an add on therapeutic strategy,5,19 while only few studies have been published about exenatide therapy in real world clinical settings;20,21 scarce data exist about gender differences concerning the use of exenatide in routine practice . An additional interest also lies in the observation that in normal weight individuals, the release of endogenous glp-1 elicited by fiber - rich nutrients is sexually dimorphic, being selectively potentiated in women.22 exenatide, which has been available in italy since february 2008, is prescribed in our country as an adjunctive therapy to metformin, sulphonylureas, their combination, or thiazolidinediones and until 2010 its use, as well as its reimbursement, have been approved, only after recording patients into a specific web - assisted - computerized database . We have, therefore, utilized this database to investigate whether and possibly how the therapeutic response by exenatide in type 2 diabetes is different between genders . This multicentric, observational, retrospective study involved type 2 diabetic patients enrolled in five diabetes outpatient clinics in tuscany, italy (livorno, lucca, massa, pistoia, and versilia) between february 2008 and august 2010 . Patients were recruited at each center if they had experienced therapeutic failure despite maximum - tolerated oral treatment (metformin, or combination metformin + sulphonylureas), or had adverse events after conventional oral therapy . There was also a very exiguous number of patients who were in therapy with sulphonylureas (n = 8) or thiazolidinediones (n = 3) only and, consequently, for the purpose of this study, we considered only two therapeutic groups (those on metformin / thiazolidinediones and those on combination therapy / sulphonylureas). All patients began treatment with exenatide 5 g twice daily (bid), injected subcutaneously 3040 minutes before principal meals, for 4 weeks; if treatment was well tolerated, the dosing was increased to 10 g twice daily . Background therapy was generally kept at prestudy dosage unless patients had experienced hypoglycemic episodes . According to scheduled follow - ups, each patient was reevaluated after 4 months, 8 months, and 12 months . At each visit, body weight, waist circumference, fasting plasma glucose, hemoglobin a1c (hba1c) and adverse events fasting plasma c - peptide was measured and homeostasis model assessment - b (homa - b) was calculated as:23 with these premises, the purpose of this study was to retrospectively identify predictors of target glycemic response (hba1c 7%), and of weight loss in males as compared to females, adding further information about the rates of drop - outs and of side effects in our population . Plasma glucose, hba1c, and plasma c - peptide were measured in all centers by means of standardized methods, with periodical quality control checks . In each center, use of information contained in the database was approved by the local ethics committee . Differences between means were evaluated according to both parametric (student s t - test) and nonparametric (wilcoxon rank - sum test) methods, when appropriate . Predictors of main outcomes at 1 year (hba1c or body weight loss at target) were identified in both genders, calculating the odds ratio from multiple logistic models including sets of continuous or categorical independent variables . For continuously distributed independent variables, odds ratios measured variations in risk according to either the increase or decrease is proportional to the consensual change of the unity of measure of the independent variable . Data are expressed as means standard deviation, or as median values (interquartile range), and a p - value 0.05 was considered statistically significant . All analyses were performed with sas software, version 8.2 for windows (sas institute, cary, nc, usa). This multicentric, observational, retrospective study involved type 2 diabetic patients enrolled in five diabetes outpatient clinics in tuscany, italy (livorno, lucca, massa, pistoia, and versilia) between february 2008 and august 2010 . Patients were recruited at each center if they had experienced therapeutic failure despite maximum - tolerated oral treatment (metformin, or combination metformin + sulphonylureas), or had adverse events after conventional oral therapy . There was also a very exiguous number of patients who were in therapy with sulphonylureas (n = 8) or thiazolidinediones (n = 3) only and, consequently, for the purpose of this study, we considered only two therapeutic groups (those on metformin / thiazolidinediones and those on combination therapy / sulphonylureas). All patients began treatment with exenatide 5 g twice daily (bid), injected subcutaneously 3040 minutes before principal meals, for 4 weeks; if treatment was well tolerated, the dosing was increased to 10 g twice daily . Background therapy was generally kept at prestudy dosage unless patients had experienced hypoglycemic episodes . According to scheduled follow - ups, each patient was reevaluated after 4 months, 8 months, and 12 months . At each visit, body weight, waist circumference, fasting plasma glucose, hemoglobin a1c (hba1c) and adverse events fasting plasma c - peptide was measured and homeostasis model assessment - b (homa - b) was calculated as:23 with these premises, the purpose of this study was to retrospectively identify predictors of target glycemic response (hba1c 7%), and of weight loss in males as compared to females, adding further information about the rates of drop - outs and of side effects in our population . Plasma glucose, hba1c, and plasma c - peptide were measured in all centers by means of standardized methods, with periodical quality control checks . In each center, use of information contained in the database was approved by the local ethics committee . Differences between means were evaluated according to both parametric (student s t - test) and nonparametric (wilcoxon rank - sum test) methods, when appropriate . Predictors of main outcomes at 1 year (hba1c or body weight loss at target) were identified in both genders, calculating the odds ratio from multiple logistic models including sets of continuous or categorical independent variables . For continuously distributed independent variables, odds ratios measured variations in risk according to either the increase or decrease is proportional to the consensual change of the unity of measure of the independent variable . Data are expressed as means standard deviation, or as median values (interquartile range), and a p - value 0.05 was considered statistically significant . All analyses were performed with sas software, version 8.2 for windows (sas institute, cary, nc, usa). The group of patients under study was composed of 154 males and 161 females, who were well matched for age, baseline body mass index (bmi), and hba1c (table 1). Among females, baseline mean fasting plasma glucose was higher among males, while females were characterized by a longer duration of diabetes as well as by a higher prevalence of patients treated with combination therapy (sulphonylureas + metformin) (table 1). No differences in mean values of mean plasma glucose, hba1c, or bmi at baseline, at 4 months, 8 months, and 12 months were observed between genders (table 1 and figure 1). The percentage of patients who achieved the target glycemic value (hba1c 7%) was significantly higher among males than females after 4 months (41% versus 28%; = 6.21; p = 0.01), as well as after 12 months (38% versus 27%; = 4.66; p = 0.03; figure 1b). Bmi was, on average, greater (even if not significantly) in females than in males throughout the whole period (table 1, and figure 1a). Target weight loss, arbitrarily expressed as 1-year percent loss 75th percentile in the whole population (8.5%), was more often achieved among females at 8 months (28% versus 15%; = 8.04; p = 0.004) and 12 months (33% versus 17%; = 10.98; p = 0.0009) (figure 1b). After substituting body weight with bmi, predictors of target metabolic response at 1 year, (hba1c 7%), was evaluated by using a multiple logistic model where the dependent variable (y) was the positive target outcome attained (yes = 1; no = 0), and age, duration of diabetes, baseline hba1c, bmi, and background antidiabetic therapy (metformin / thiazolidinediones or combination therapy / sulphonylureas, respectively categorized as 0 and 1), were independent variables . Duration of the disease was inversely related with the probability of achieving 1-year target glycemic outcomes, and in the whole population, each increase in 1-year diabetes duration reduced the probability of achieving the glycemic target by about the 6% . Each 1% increase of hba1c reduced the probability of achieving the glycemic target at 12 months by about 35% (table 2 and figure 2). Longer disease duration and higher basal hba1c levels were significantly negative predictors of target metabolic response in men, while in women negative achievement of the 1-year glycemic target was associated with baseline treatment with sulphonylureas or a combination oral therapy, with baseline therapy with metformin being related to a positive outcome at 1 year (or: 0.321 [95% ci: 0.1480.696]) (table 2). Increase in baseline homa - b was significantly related to 1-year positive metabolic response only in females (table 2). A longer duration of diabetes was a significant predictor of a target weight loss response in the overall population; also, in this case there was a difference between genders since lower hba1c levels at baseline was a significant 1-year response predictor in females, while longer diabetes duration was a significant 1-year response predictor in males (table 3). Table 4 shows the types of side effects and reasons for drop - outs; there was no clear gender difference with regards to the prevalence of the most common gastrointestinal side effects . In this observational study, exenatide was used at the standard dose as an add - on strategy in diabetic patients experiencing therapeutic failure . As expected, the duration of diabetes and the baseline hba1c levels were among the main predictors of therapeutic glycemic response . In fact, a longer duration of diabetes and increasingly elevated hba1c levels are both significant predictors of -cell dysfunction,2427 and are thus responsible for impairing the therapeutic effect of antidiabetic drugs including glp-1 receptor agonists . The therapeutic glycemic effect was similar to what has been reported by previous intervention trials,16,18,20 and the new data of the present study illustrates the observed gender difference in the attainment of metabolic targets achieved by a significantly higher proportion of males (38%) than females (27%). Currently, to the best of our knowledge, there are no reports about the influence of gender on hypoglycemic responses in diabetic patients treated with exenatide, since previously published studies refer to randomized controlled trials, which are often of short duration and they usually compare exenatide with other active drugs.18 furthermore, in many trials, gender distribution was unbalanced with the percentage of males (in most cases) being above 50%,18 suggesting that the final results could have been influenced by gender mismatching . Very recently, a retrospective post hoc meta - analysis of pencek et al28 regarding 16 studies and 2067 diabetic patients treated with exenatide bid did not reveal any gender difference as to the glycemic outcome . This may be due to the fact that the main characteristics of patients analyzed by pencek et al28 were quite different from those of our patients with regards to age, matching of sex, background therapy, duration of trial, and race . It is interesting, however, to note that in pencek et als28 analysis, the final weight loss observed in patients was significantly higher in females than in males, which is in agreement with our data . Why glycemic response was better in men is not clear from our data; one possible explanation could be that women had a much longer duration of diabetes and were more frequently on therapy with metformin + sulphonylureas, suggesting a more preserved baseline -cell function among men, which is also indirectly suggested by their higher homa - b median value . This hypothesis is validated by two findings: the first is the prediction that there is a negative effect on achievement of the glycemic target exerted by background baseline combination therapy; and the second is the positive relation linking basal homa - b and 1-year metabolic response, both of which were observed uniquely among females . In agreement with previous experiences, weight loss after 1-year of exenatide therapy was about 5 kg, on average, in both sexes;18,20 however, females achieved weight loss targets more frequently than males after 8 months and 12 months of exenatide therapy . The reason for this divergent response between genders which was interestingly also reported in a previous real world study,20 as well as in pencek et als28 meta - analysis is not clear . The slightly higher mean bmi observed in women, even if nonsignificant, may have contributed to a greater weight loss after exenatide . Additional results of this study were the relationship between the duration of diabetes and weight loss, which was significant only in men, as well as and the inverse association between higher basal hba1c levels and the achievement of significant weight loss only in women . This latter finding among the females in our study was confirmed by the evidence that there was a lower mean baseline hba1c concentration in (8.49% 0.88% versus 8.88% 1.06%; p = 0.02). Since previous studies reported that weight is an important risk factor for diabetes in women,29 the idea that plasma glucose is selectively related to body weight in obese women,30 and that women are more obese than men at the time of diagnosis of diabetes,31 it is possible to speculate that all these gender - specific traits may suggest that females carry an overall greater sensitivity to losing weight after exenatide treatment, especially in the presence of poor baseline metabolic control . Why there was a significant link between longer duration of diabetes and significant weight loss in males, as well as in the overall population, is unknown . The prevalence of adverse reactions and drop - outs was quite similar between genders, which was in agreement with previously published trials.28 no relation was found between weight loss and gastrointestinal side effects in both males and females . Post hoc design; but, nevertheless, we believe that the study holds the advantage of being a real world experience . To our knowledge, there are few studies that have been performed in clinical settings that have been especially aimed at investigating gender difference with regards to therapy with glp-1 receptor agonists . In summary, this study confirms that therapy with exenatide is efficacious in improving metabolic control, which is associated with a significant weight loss in patients with type 2 diabetes, irrespective of sex . Some baseline variables predict 1-year outcomes; specifically, lower baseline hba1c values and a shorter duration of disease predict a better glycemic response in the overall population . Therefore, the first conclusion that can be drawn from our results is that therapy with exenatide should be initiated not too late, seemingly before patients present with treatment failure while on oral therapy . Interestingly, the duration of disease acts biphasically a higher duration of diabetes was associated with a worse glycemic response, while exactly the opposite was observed for a reduction in body weight . A second aspect coming from the data of the present study is that males seem to have a more efficacious response after exenatide therapy, probably due to their reduced duration of disease as well as to a less exhausted -cell mass at baseline, as indirectly suggested by the lower rate of males on combination therapy . In addition, gender seems to have a different impact on weight - lowering effects exerted by exenatide, with women being seemingly more sensitive than men . The findings of this study support the hypothesis that dissimilar baseline diabetes - related characteristics between genders are mostly responsible for the different therapeutic outcomes after exenatide therapy . However, due to the observational retrospective feature of this study, all suggested gender differences need further confirmation by trials specifically designed to assess these factors.
Each year there are nearly 795,000 individuals who suffer from a new or recurrent stroke; 10% of these are cases of intracerebral hemorrhage . Stroke is the 4th leading cause of mortality according to the latest cdc statistics . Among all strokes, the case fatality rate for hemorrhagic strokes (37 - 38% mortality) is the highest, and most survivors have poor functional outcomes . Female gender has been recognized as an important risk factor for stroke, with nhanes reporting that women between 45 and 54 years of age were almost twice as likely to suffer from a stroke than males . A greater decline was also seen in stroke - related deaths among males as compared to females between 1980 and 2005 . A recent review concluded that although the incidence of stroke was higher in males, females were more severely ill . Internationally, it has been reported that the stroke burden is higher in females, because of a higher prestroke and poststroke disability [58]. This difference in disability after stroke between men and women is seen not only physically but also psychologically . No published literature, however, answers the question of gender differences specifically in acute hemorrhagic stroke . Since acute nontraumatic intracerebral hemorrhage (ich) contributes greatly to the poststroke morbidity and mortality burden on the health system, we sought to determine gender differences in an exclusive cohort of first time hemorrhagic stroke patients presenting to our emergency department . This was an institutional review board approved, consecutive cohort study, conducted in the emergency department (ed) of our academic institute with an annual census of 75,000 visits . All adult patients who presented with a diagnosis of spontaneous nontraumatic intracerebral hemorrhage (ich), from january 2006 to december 2008, were eligible for inclusion in the study cohort . All pediatric patients, patients with subdural, extradural, or subarachnoid hemorrhage, and patients with a recurrence of intracerebral hemorrhage were excluded from the final cohort . Written consent was obtained for use of medical records from patients at admission to the hospital . The medical records of these patients were reviewed by two independent abstractors for demographics, arrival characteristics, vitals, symptoms, signs, laboratory parameters on presentation, past history of risk factors, treatment, and discharge status . The stroke severity was obtained in the form of nihss scores, calculated on the basis of the examination notes of the neurologist on call [10, 11]. The clinical interpretation program of the ge / marquette mac-8 digital ecg chart determined the intervals on the ecg . The functional outcome at discharge was noted as the modified rankin score (mrs) at discharge . This was stratified into two categories good outcome (mrs <3) and bad outcome (mrs 3). The ct scans of these patients were studied to calculate the volume of hemorrhage using the abc/2 method . The topographic area affected by the hemorrhage and the intraventricular extension of the hemorrhage were also noted . A close network of primary care and a single unified hospital system, in our county, enabled us to collect data on mortality outcomes on all patients in the study . Abstractors met periodically to discuss possible ambivalent data and to ensure uniformity in data collection . All associations between nominal variables were analyzed using the pearson chi - square test or fisher's test wherever applicable . Multivariate logistic regression was performed to adjust for potential confounding factors to evaluate the association between gender and stroke outcome . Survival analysis based on time to death for each of the genders was also performed . Of the 261 adult patients with ich who presented to the ed, 13 declined to consent for research; for 3 patients, this was a recurrence of ich, and hence these patients were excluded from the cohort . In the final cohort of 245 patients, there were 125 (51.0%) females (f) and 120 (49.0%) males (m). The median age for the females was 77 years (interquartile range iqr 65 to 83 years), which was significantly higher than the median age for males (median age 69 years, iqr 59 to 80 years; p = 0.007). Only 12.6% of the patients were <50 years of age (12.5% females, 12.8% males). From the cohort, 57.6% females and 56.7% males were referred to our academic ed, from elsewhere (p = 0.883). There was also no difference (p = 0.46) in the proportion of females and males who were brought in by ambulances (59.2% f, 51.7% m), helicopters (28.8% f, 32.5% m), or private vehicles (12% f, 15.8% m). On analyzing symptoms on presentation to the ed 37.6% females presented with headache versus 29.2% males, 28.8% females presented with vomiting / nausea versus 12.5% males, 7.2% females presented with syncope versus 5.8% males, and 46.8% females presented with weakness versus 51.3% males . Further analysis revealed that females had 2.8 times higher odds of presenting with vomiting / nausea than males (95% ci 1.45 to 5.51; p = 0.002). About 58.9% females and 34.4% males presented with left - sided weakness, and 37.5% females and 62.5% males presented with right - sided weakness (p = 0.002), implying that there might be a significant difference in the area of affection, which we analyzed on ct scans . We also looked at the glasgow coma scale (gcs) evaluation of patients in our cohort . There was no difference between the proportion of females (26.4%) and males (25%) who arrived comatose (p = 0.802). There was also no difference in the stroke severity on arrival as defined by nihss between females (median nihss 7, iqr 2 to 23) and males (median nihss 8, iqr 2 to 19; p = 0.76). On assessing the ct scans on arrival of these patients, we found that there was no significant difference between the volumes of hemorrhage between females (median volume 30 cc, iqr 5.2 to 135.7 cc) and males (median volume 22.9 cc, iqr 6.5 to 85.8 cc; p = 0.415). There was also no difference (p = 0.886) in the prevalence of intraventricular extension of hemorrhage between females (47.6%) and males (46.7%). On analyzing the topographic distribution of the hemorrhage in the brain, we found that 60% of the females had a right - sided hemorrhage as compared to 41% males . Males were more likely to have a left - sided hemorrhage (56%) as compared to females (38%). The odds of having a cerebellar hemorrhage were 4.2 times higher in females than males (95% ci 1.63 to 10.75; p = 0.002). This association remained significant even after controlling for nausea / vomiting as the latter has been reported to be associated with higher incidence of cerebellar hemorrhage (odds ratio 3.42, 95% ci 1.16 to 10.04, p = 0.02). We compared history of ischemic stroke, coronary artery disease, hypertension, diabetes mellitus, dyslipidemia, atrial fibrillation, cancer, coagulation abnormalities, seizures, head trauma, smoking, and cessation of smoking between the genders . Females had 0.51 times odds for having coronary artery disease (p = 0.024) as compared to males . Also, only 30.4% females from the cohort had a past history of smoking as compared to 61.7% males (odds 0.27, 95% ci 0.16 to 0.46; p <0.0001). There was, however, no difference between the proportions of females (25.6%) and males (36.5%) who stopped smoking (p = 0.243). Associations between past history of other diseases and gender did not reach statistical significance . On analyzing past medication use, there was also no statistically significant difference between the class of antihypertensive medication, anticoagulants, antiplatelets, steroids, and statins (all of which are known to influence ich) used by males and females . There was no difference in the skin temperature (p = 0.467), heart rate (p = 0.725), and systolic blood pressure (p = 0.271) recorded on arrival to the ed between males and females . The women, however, did have a lower diastolic blood pressure on arrival (median 81 mmhg, iqr 71 to 95 mmhg) when compared to the men (median 87 mmhg, iqr 75 to 102 mmhg; p = 0.011). On analyzing the ecg intervals (pr, qrs, and qtc), we found a significantly lower qrs interval in females (median 90 ms, iqr 82 to 98 ms) when compared to males (median 94 ms, iqr 88 to 106 ms; p = 0.0125). On analyzing hemogram characteristics, we noted that females had lower hemoglobin (median 13.2 mg / dl, iqr 12.3 to 14.1) as compared to males (median 14.1 mg / dl, iqr 12.7 to 15.3 mg / dl; p <0.0001), higher leukocyte counts (median 10.7 10/cc for females versus 9.4 10/cc for males; p = 0.039), and higher platelet counts (median 244 10/cc for females versus 223 10/cc; p = 0.0031), but all these were within normal limits . There were no differences between the genders with regard to the coagulation parameters inr (p = 0.532) and aptt (p = 0.096). Females also had higher total cholesterol levels (median 199 mg / dl iqr 174 to 211 mg / dl) when compared to males (median 168 mg / dl, iqr 146 to 208 mg / dl; p = 0.033). Women also had higher measured blood glucose on arrival to ed (median 146 mg / dl iqr 115 to 191 mg / dl) when compared to men (median 129 mg / dl, iqr 106 to 157 mg / dl; p = 0.0096). There were also statistically significant differences that were noted between serum sodium (p = 0.0386), serum potassium (p = 0.0045), and serum creatinine (p <0.0001) levels between females and males, with females having lesser values than males . A total of 12.8% females had a craniotomy / surgery as compared to 10.8% of the male subjects, and nearly 10.4% females had an extraventricular drainage (evd) as compared to 8.3% males . However, there was no difference in proportion of the females undergoing surgery / craniotomy and evd as compared to the males (craniotomy / surgery p = 0.62, ivd, p = 0.58). There was no difference in the median length of hospital stay between females (median 4 days, iqr 2 to 10 days) and males (median 5 days, iqr 3 to 13 days; p = 0.179). Females had a worse functional outcome on discharge (median mrs 4, iqr 2 to 6) when compared to males (median mrs 4, iqr 0 to 5; p = 0.0062). Females had 1.94 times the odds of having a poor outcome as compared to males (95% ci 1.12 to 3.3). There were also more deaths within 7 days in the female subset (n = 38, 30.4% patients) as compared to the males (n = 23, 19.2% patients). The odds of dying within 7 days were 1.84 times higher in females than in males (95% ci 1.02 to 3.33; p = 0.0421). New dnr orders were instituted over a period of 113 days from the date of admission . Overall, 62 (49.6%) females had dnr orders versus 36 (30%) of males . Despite having no differences between the stroke severity, volume of hemorrhage, and past history of diseases, females were also more likely to have dnr (do not resuscitate) orders instituted after admission to the hospital with ich (odds ratio 2.33, 95% ci 1.37 to 2.97; p = 0.0016)., we first made a subcohort of all patients who had dnr orders instituted within 24 hours (n = 44). In this subcohort, we excluded any patients who received any operative or interventional procedures after arrival to the ed (n = 2), because it was assumed that these patients were only made dnr after significant interventional albeit futile, efforts . The final subcohort (n = 42; females 28, males 14) was then omitted from our initial cohort of 245 patients . This final subset of nonearly dnr patients (n = 203) consisted of 97 females (47.8%) and 106 males (52.2%). On analysis of association of gender with early mortality, we still found that females were 2.6 times (or) more likely to die within 7 days of presentation with ich than males (95% ci 1.3 to 5.4; p = 0.006). It is a known fact that increasing age, stroke severity, cerebellar hemorrhage, and intraventricular extension of hemorrhage are associated with poor outcome and death after ich . We ran a logistic fit incorporating all these variables in a multivariate analysis and found that female gender remained an independent predictor of poor outcome at discharge (adjusted or = 2.30, 95% ci 1.04 to 5.25; p = 0.0398) and death within 7 days of ich (adjusted or 3.03, 95% 1.24 to 7.79; p = 0.0144). The adjusted or for mrs for males was 0.44 (95% ci 0.19 to 0.96) and for death within 7 days was 0.33 (95% ci 0.13 to 0.81). We also ran a survival analysis based on time to death for each of the genders . The curves were near similar and the statistical analysis of difference did not reach significance on the wilcoxon test (p = 0.4403). A closer look at the curves did show a clear separation between the mortality trends among females and males between 10 and 150 days after ich (figure 1). In this research, we have reported important differences between males and females who presented with first time intracerebral hemorrhage (ich). Our demographic finding of women being older than men at the time of ich concurs with the findings of roquer et al . Who reported that women were on an average 6 years older than men at the time of stroke . Although there were no differences between the modes of arrival of patients for both genders, or their state of consciousness and orientation (measured by gcs), we did find that women were more likely to have nausea / vomiting as a symptom on presentation . Investigators have previously reported some differences in the presentation symptoms between males and females with stroke, with regard to ability to walk, aphasia, visual field disturbances, dysphagia, and coma . The most important difference that we noted however, was that women were more likely to be affected in the right hemisphere, causing left - sided weakness . We also noted that though the volume of hemorrhage was similar in both genders, females had nearly 4 times the odds to have a cerebellarhemorrhage when compared to males . This actually steps away from previous reports on ischemic stroke, which state that infra - tentorial strokes are less prevalent in females [7, 14]. Previous experience with subarachnoid hemorrhage (sah) patients has revealed that females with sah may be more prone to hypokalemia and qt prolongation . Females also had lower hemoglobin, higher leukocyte, and platelet counts as compared to males, but again these were all within normal limits . Women did have higher glucose levels on arrival to the emergency department, although there was no difference in the prevalence of diabetes mellitus between males and females . It has been reported in the past that hyperglycemia on presentation can lead to worse early outcome in patients with spontaneous ich . We concentrated on studying the early mortality outcomes for our cohort, as we wanted to negate influences of complications that arise due to the morbidity and disability after stroke . We found that, despite having similar premorbid status, similar prior medication use, similar hemorrhage volumes and frequency of intraventricular extension, women still experienced higher 7-day mortality post ich . The higher odds of cerebellar ich and hyperglycemia at presentation, could be contributory factors to this phenomenon . It is known that hyperglycemia early on in the course of spontaneous ich does influence the early outcome in these patients . The long - term mortality experience for males and females was, however, similar, as we saw on the survival curves . At discharge from the hospital, females were almost twice as likely as males to have bad functional outcome . This association held true even after adjusting for predictors of worse outcome in a multivariate model . Higher inflammatory response in hemorrhagic stroke has been associated with poor outcomes [17, 18]; studies have reported that females tend to have more robust inflammatory response in some diseases . This could have led to poor outcome in females, although we were not able to capture data regarding inflammatory measures such as midline shift, perihematomal edema, or herniation . For example, it has been reported that progesterone and estrogen may be important neuroprotective agents in ischemic stroke [2023]. Research has suggested that estrogens may serve this purpose by a way of stimulating growth factor supply, attenuating inflammatory processes, free radical scavenging, stimulation of intrinsic antiapoptotic pathways, and other interactions with intrinsic cell - cell pathways [2426]. Some researchers have also suggested the mitochondria as a possible site of estrogen - mediated, neuronal survival . Other studies have reported that women experience worse functional outcome and early mortality after ischemic stroke [8, 28]. Still others, however have credited the female gender for conferring a survival advantage after ischemic stroke, consistent with the notion that female sex hormones are candidates for neuroprotective therapy . Some researchers have reported that gender differences in outcome are not seen in hemorrhagic stroke . Our study reports the first comprehensive gender differences in a cohort of exclusively first time hemorrhagic stroke patients . The significant differences outcomes and early mortality, with female sex driving bad outcome, may indicate that unlike in ischemic stroke, female sex hormones might not present a survival advantage in ich . This may be due to intrinsic differences in the neuronal damage mechanisms in ich and ischemic strokes, which need to be studied extensively in the near future . We noted that female patients in our cohort were more likely to have dnr orders instituted after admission for ich, despite the similarities to males on premorbid conditions, stroke severity and hemorrhage volumes . The variation in the use of dnr orders has been studied extensively including differences by gender, race, insurance status, patient preference, hospital, and specialty [31, 32]. It might be suggested that dnr orders could have influenced the outcomes and could have been an important confounder in our study . By doing the subset analysis, wherein we excluded patients who had early dnr orders, a surrogate for no interventions, we believe that we tried to get an understanding of this influence . If anything, the female gender became more strongly associated with early mortality in this subcohort of nonearly dnr patients (odds ratio increased from 1.84 to 2.33). For convenience, we looked at functional outcome at the hospital discharge, rather than at a fixed time the point from point of onset of ich itself . This could have influenced our assessment of mrs, though we did not notice any statistically significant difference in hospital length of stay between the two genders . In addition to this, we did not report long - term outcome for our patients . Although prospective large - scale stroke trials typically assess outcome at 36 months, due to the unavailability of data and the retrospective nature of our study, we were unable to do so . We also could not assess the effects of hematoma expansion in our cohort, as the initial ct scan was done at different time points from onset of ich, and results of repeated ct scan were not looked at . It is known that hematoma expansion can continue for up to 48 hours after stroke; hence, we may have overestimated or underestimated the volume of hemorrhage in our patients . Referral of a large subset of patients from other hospitals and initial treatment given at other sites could have influenced our assessment of initial gcs . Since only 12.5% of our female subset was below the age of 50 years (the median age for menopause), we might not be seeing the protective effects of estrogen on our cohort, which may truly hold true . We also did not look at specific causes of mortality in our cohort of patients . But since we are reporting early mortality after ich, we have fair reason to believe that death within 7 days was primarily related to the ich itself . We have described important gender differences in patients with first episode of ich . We have concluded that unlike in ischemic stroke females are more likely to experience cerebellar hemorrhagic strokes, than males . Also, we have reported a greater inclination for females to experience a right hemispheric ich than males, which could influence functional recovery and mortality outcomes . We found that females have nearly twice the odds to die within 7 days after an ich as compared to males . Female gender is an independent predictor of early mortality and bad functional outcome at hospital discharge, after adjusting for age, stroke severity, volume of hemorrhage, and intraventricular extension of hemorrhage, all of which are known to predict worse outcome.
Bladder cancer is the fourth most common malignancy in men and the ninth most common in women, with an estimated incidence of 67,160 and 13,750 estimated deaths (2007) in the united states . Transitional cell carcinoma (tcc) refers to those bladder cancer tumors derived from urothelial tissue, more than 90% of which originate in the urinary bladder and present as nonmuscle - invasive disease in the majority of patients at diagnosis.1 of those patients who respond to current standard of care, approximately two - thirds will have recurrence, and 10%20% of recurrent tumors will have progressed to muscle - invasive disease.2 typically, treatment for high - risk, nonmuscle - invasive bladder cancer (nmibc) is transurethral resection of the bladder tumor and adjuvant therapy with bacillus calmette guerin (bcg). Although bcg treatment can reduce the risk of recurrence and progression, its use is limited by the adverse effect profile and intolerance that occurs in 20% of patients.36 epithelial cell adhesion molecule (epcam) is overexpressed in many carcinomas relative to their normal tissue counterparts, as is the case in tcc.7,8 in addition, epcam expression increases as these cancers progress from lower to higher grades.811 together, these features make epcam a clinically relevant antigen for targeted therapy in bladder cancer . Vb4 - 845 is a recombinant fusion protein that targets epcam - positive cancer cells . It consists of an anti - epcam humanized single - chain variable fragment (scfv) linked to a truncated form of pseudomonas exotoxin a (eta252608) that lacks the cell - binding domain.12 once bound to epcam on the surface of carcinoma cells, vb4 - 845 is internalized, whereupon the exotoxin portion of the fusion protein induces apoptosis.13,14 one concern of targeted therapies has been the toxicity associated with systemic administration of this class of drug.15 moreover, repeated use of therapeutics comprising foreign proteins is limited by their immunogenicity . Therefore, it is desirable to develop therapies designed for local administration, thereby increasing the clinical benefit of these treatments while minimizing any drug - related effects . Accordingly, locoregional delivery of eta - conjugated antibodies has been demonstrated to be well tolerated and clinically effective in patients with glioblastoma multiforme, erbb2-expressing breast tumors, and squamous cell carcinoma of the head and neck.1618 nonclinical studies showed a significant reduction in toxicity with locally administered vb4 - 845 . Similarly, local injections of vb4 - 845 were well tolerated in cynomolgus monkeys with adverse events (aes) being mild and easily managed.19 in addition to the strong nonclinical safety profile, vb4 - 845 exhibits highly potent activity against epcam - expressing tumor cell lines and has been shown to localize to epcam - positive tumor xenografts.12 based on these preclinical results, a phase i dose - escalation trial was performed using vb4 - 845 as an intravesical therapy in bcg - refractory and bcg - intolerant patients with grade 2 or 3 nmibc . Only patients 18 years of age or older with immunohistochemically confirmed epcam - positive grade 2 or 3 nmibc (ta, t1, in situ carcinoma [tis]), either refractory to (recurrence within 2 years following at least one complete cycle of bcg therapy) or intolerant of bcg therapy, were eligible for this study . Other key inclusion criteria were adequate renal (serum creatinine 1.5 the upper limit of normal (uln) or creatinine clearance 60 mls / min), hepatic (alanine aminotransferase and aspartate aminotransferase 2.5 uln and bilirubin levels 1.5 uln), and hematological (granulocytes 1500/l, platelets 100,000/l, and hemoglobin> 8 g / dl) function . Women of child - bearing potential, and all men, must have agreed to use adequate contraception prior to and for the duration of the study . Key exclusion criteria included patients with muscle - invasive tumors, nodal involvement, or distant metastases; patients with a history of upper tract tcc, adenocarcinoma, or squamous cell carcinoma of the bladder; and patients with disease involving the prostatic ducts or stroma . Moreover, excluded were patients with a history of pelvic malignancy, hydronephrosis, or clinically significant abnormalities of the upper urinary tract and those who had undergone bcg therapy within 6 weeks prior to the start of vb4 - 845 dosing . Written informed consent was obtained from each participant before any study - related activity was performed . This study was conducted according to section c.05.010 of division 5 of the food and drug regulations of the government of canada, ich harmonized tripartite guidelines for good clinical practice, the declaration of helsinki (2002), and all local laws and regulations concerning clinical studies and the protection of study patients . Regulatory clearance for the study was obtained from the biologics and genetic therapies directorate of health canada in canada . The study was an open - label, multicenter, dose - escalating trial of intravesically administered vb4 - 845 . Eight dose levels were initially evaluated, starting at 0.1 mg once weekly for 6 consecutive weeks and escalating through 0.2, 0.33, 0.66, 1.32, 2.64, 5.28, and 10.56 mg / dose . The maximum tolerated dose (mtd) was not reached; therefore, an additional escalation through 13.73, 17.85, 23.20, and 30.16 mg was undertaken . Each dose was administered to the bladder through a catheter and held for 2 h prior to voiding . Safety data from each dose cohort was evaluated after 3 weeks of treatment before proceeding to the next dose cohort . Dose - limiting toxicity (dlt) was defined as the occurrence of treatment - related aes, including intractable cystitis persisting for more than 1 week associated with severe pain, urgency, and/or frequency not relieved by measures considered to be routine standard of care according to each clinical site; significant hematuria leading to clot obstruction; grade 4 flu - like symptoms; grade 3 or higher hematological toxicity (if it is a two grade increase from baseline); or any other grade 3 nonhematological toxicity (with the exception of alopecia). If two patients experienced a dlt at any dose, that dose would be defined as the mtd . Specific additional evaluations were performed depending on the timing of the visit, as described below . Pretreatment screening evaluations were conducted within 4 weeks of the baseline visit and included medical history, pregnancy test, 12-lead electrocardiogram, and assessment of karnofsky performance status . Immunohistochemistry on snap - frozen tissue samples was performed at a single facility to determine whether tumors were epcam positive . In addition to the evaluations performed at every study visit, patients were monitored for aes for 3 h following drug administration on days 1, 8, 15, 22, 29, and 36 . The toxicity grade of any ae was classified according to the national cancer institute common toxicity criteria, version 3 . Aes were considered to be treatment related by the investigators if they were possibly, probably, or definitely related to vb4 - 845 administration . On day 2 additional assessments performed included pregnancy test, 12-lead electrocardiogram, and karnofsky performance status . To evaluate tumor response, a biopsy was required if the cystoscopy revealed lesions suspicious for malignancy, or cytology results were suspicious or positive for malignancy . Blood samples for pharmacokinetic analysis were taken on day 1 prior to and 1, 2, and 3 h postdosing . Predose samples were also taken on days 8, 15, 22, 29, and 36 . For each sample, 3 ml of venous blood was collected into a tube containing lithium heparinate and placed on ice . Vb4 - 845 plasma levels were measured using a glp - validated, mts - based, potency assay.18 the assay detects intact vb4 - 845 by its ability to kill the epcam - positive cell line cal-27, where the measured ic50 is directly proportional to the concentration of intact drug . The assay was shown to have a lower limit of detection of 14 pg / ml . Blood samples for assessing humoral immune reactivity to vb4 - 845 were taken prior to dosing on days 1, 8, 15, 22, 29, 36, and at the final study visit . Samples were collected as for the pharmacokinetic analysis, and antibody titers to the scfv and eta252608 portions of the fusion protein (human antihuman antibodies (haha) and human anti - pseudomonas antibodies (hapa)) were measured using an enzyme - linked immunosorbent assay.18 responses were expressed as the geometric mean of measurable titers at each time point . Tumor response was assessed on evaluable patients 46 weeks following the last dose of vb4 - 845 . The evaluation was based on cytology, cystoscopy, and, if required, biopsy . Complete response was defined as nonpositive urinary cytology and either normal cystoscopy or abnormal cystoscopy with negative biopsy . Only patients 18 years of age or older with immunohistochemically confirmed epcam - positive grade 2 or 3 nmibc (ta, t1, in situ carcinoma [tis]), either refractory to (recurrence within 2 years following at least one complete cycle of bcg therapy) or intolerant of bcg therapy, were eligible for this study . Other key inclusion criteria were adequate renal (serum creatinine 1.5 the upper limit of normal (uln) or creatinine clearance 60 mls / min), hepatic (alanine aminotransferase and aspartate aminotransferase 2.5 uln and bilirubin levels 1.5 uln), and hematological (granulocytes 1500/l, platelets 100,000/l, and hemoglobin> 8 g / dl) function . Women of child - bearing potential, and all men, must have agreed to use adequate contraception prior to and for the duration of the study . Key exclusion criteria included patients with muscle - invasive tumors, nodal involvement, or distant metastases; patients with a history of upper tract tcc, adenocarcinoma, or squamous cell carcinoma of the bladder; and patients with disease involving the prostatic ducts or stroma . Moreover, excluded were patients with a history of pelvic malignancy, hydronephrosis, or clinically significant abnormalities of the upper urinary tract and those who had undergone bcg therapy within 6 weeks prior to the start of vb4 - 845 dosing . Written informed consent was obtained from each participant before any study - related activity was performed . This study was conducted according to section c.05.010 of division 5 of the food and drug regulations of the government of canada, ich harmonized tripartite guidelines for good clinical practice, the declaration of helsinki (2002), and all local laws and regulations concerning clinical studies and the protection of study patients . Regulatory clearance for the study was obtained from the biologics and genetic therapies directorate of health canada in canada . The study was an open - label, multicenter, dose - escalating trial of intravesically administered vb4 - 845 . Eight dose levels were initially evaluated, starting at 0.1 mg once weekly for 6 consecutive weeks and escalating through 0.2, 0.33, 0.66, 1.32, 2.64, 5.28, and 10.56 mg / dose . The maximum tolerated dose (mtd) was not reached; therefore, an additional escalation through 13.73, 17.85, 23.20, and 30.16 mg was undertaken . Each dose was administered to the bladder through a catheter and held for 2 h prior to voiding . Safety data from each dose cohort was evaluated after 3 weeks of treatment before proceeding to the next dose cohort . Dose - limiting toxicity (dlt) was defined as the occurrence of treatment - related aes, including intractable cystitis persisting for more than 1 week associated with severe pain, urgency, and/or frequency not relieved by measures considered to be routine standard of care according to each clinical site; significant hematuria leading to clot obstruction; grade 4 flu - like symptoms; grade 3 or higher hematological toxicity (if it is a two grade increase from baseline); or any other grade 3 nonhematological toxicity (with the exception of alopecia). If two patients experienced a dlt at any dose, that dose would be defined as the mtd specific additional evaluations were performed depending on the timing of the visit, as described below . Pretreatment screening evaluations were conducted within 4 weeks of the baseline visit and included medical history, pregnancy test, 12-lead electrocardiogram, and assessment of karnofsky performance status . Immunohistochemistry on snap - frozen tissue samples was performed at a single facility to determine whether tumors were epcam positive . In addition to the evaluations performed at every study visit, patients were monitored for aes for 3 h following drug administration on days 1, 8, 15, 22, 29, and 36 . The toxicity grade of any ae was classified according to the national cancer institute common toxicity criteria, version 3 . Aes were considered to be treatment related by the investigators if they were possibly, probably, or definitely related to vb4 - 845 administration . On day 2 additional assessments performed included pregnancy test, 12-lead electrocardiogram, and karnofsky performance status . To evaluate tumor response, cystoscopy / photography and cytology were performed . A biopsy was required if the cystoscopy revealed lesions suspicious for malignancy, or cytology results were suspicious or positive for malignancy . Blood samples for pharmacokinetic analysis were taken on day 1 prior to and 1, 2, and 3 h postdosing . Predose samples were also taken on days 8, 15, 22, 29, and 36 . For each sample, 3 ml of venous blood was collected into a tube containing lithium heparinate and placed on ice . Vb4 - 845 plasma levels were measured using a glp - validated, mts - based, potency assay.18 the assay detects intact vb4 - 845 by its ability to kill the epcam - positive cell line cal-27, where the measured ic50 is directly proportional to the concentration of intact drug . The assay was shown to have a lower limit of detection of 14 pg / ml . Blood samples for assessing humoral immune reactivity to vb4 - 845 were taken prior to dosing on days 1, 8, 15, 22, 29, 36, and at the final study visit . Samples were collected as for the pharmacokinetic analysis, and antibody titers to the scfv and eta252608 portions of the fusion protein (human antihuman antibodies (haha) and human anti - pseudomonas antibodies (hapa)) were measured using an enzyme - linked immunosorbent assay.18 responses were expressed as the geometric mean of measurable titers at each time point . Tumor response was assessed on evaluable patients 46 weeks following the last dose of vb4 - 845 . The evaluation was based on cytology, cystoscopy, and, if required, biopsy . Complete response was defined as nonpositive urinary cytology and either normal cystoscopy or abnormal cystoscopy with negative biopsy . All participants were caucasian with a median patient age of 69; 78% were men and 22% were women (table 1). All patients evaluated for efficacy (61/64) had previously received bcg therapy, with 95% of the patients having had two or more bladder cancer recurrences . Based on their previous bcg treatment history, only five patients were considered to have been enrolled as bcg intolerant rather than bcg refractory . All patients had grade 2 or 3, stage ta or t1, tcc, and/or tis, and, except for two cases, all papillary tumors had been previously resected 728 days prior to the first dose of study drug . Of note, as this study was carried out prior to the widespread adoption of re - transurethral resection (re - tur) of tat1, high - grade nmibc in clinical practice, and its inclusion in standard treatment guidelines, re - tur of t1 tumors was not performed . All tumors showed immunohistological evidence of epcam - positive membrane staining (figure 1). Risk factors for progression present in the patient population are summarized in table 2 . At baseline, 98% of patients had at least one factor associated with disease progression, and 64% of these individuals had three or more risk factors . Vb4 - 845 was administered to the bladder via catheter once a week for 6 consecutive weeks, followed by 46 weeks without treatment . An mtd was not determined from the initial dose escalation as no dlt was reported; therefore, additional dose cohorts were added as described above, and, again, all patients received all six scheduled doses without any dlt . Forty - one of the 64 patients (64%) experienced an ae during the course of the study . Twenty - one (33%) patients experienced only aes that were assessed by the investigator as unrelated to study treatment . Classifying the aes as unrelated to the study drug was decided after considering the temporal relationship of the onset of the event to the administration of the study drug, whether the event could be explained by concomitant medications or concurrent disease, the response to withholding the study drug, and the response to rechallenge with the study drug . These included, but were not limited to, urinary tract infection, incontinence, nocturia, pharyngolaryngeal pain, and bladder spasm . The remaining 20 patients (31%) experienced aes judged to be related to vb4 - 845 administration . The most common treatment - related aes were dysuria and hematuria (table 4). Systemic aes included fatigue, fever and chills, loss of appetite, myalgia, dizziness, and nausea . The frequency of treatment - related aes did not increase with dose escalation (figure 2). All treatment - related aes were grade 1 or 2, with the exception of one grade 3 occurrence of hematuria that was reported as possibly related to vb4 - 845 administration . This event was not reported as a dlt as the investigator judged it to be related to aspirin use, and the patient experienced no aes with subsequent vb4 - 845 dosing . There were no cases where the occurrence of aes required discontinuation of treatment, and no patient experienced a serious ae related to the administration of vb4 - 845 . There was one patient death due to cardiac failure, which occurred 3 weeks after the last dose of vb4 - 845 . This death was assessed by the investigator as unrelated to study treatment and attributed to long - standing coronary artery disease, previous myocardial infarction, and hypertension . Postinstillation plasma levels of vb4 - 845 were measured in 63 patients, except at the final visit where samples were provided by 61 patients . In almost all patients, vb4 - 845 plasma levels were below the limit of detection of the assay (14 pg / ml) at all time points examined . Vb4 - 845 was detectable in only two patients: one had levels of 19 and 17 pg / ml at 1 h after vb4 - 845 instillation and on day 8 prior to dosing, respectively, and the other had a level of 18 pg / ml at the 1 h time point . The immunogenicity of vb4 - 845 was examined by analyzing hapa and haha titers in blood samples taken at specified time points during the trial (table 5). Hapa response was more vigorous, as patients developed a measurable titer earlier; the majority of patients exhibited hapa by day 29, with 77% (47/61) having a measurable titer at final visit . In contrast, only 16% (10/61) of patients had haha by the end of the study . Hapa titers were also generally higher, with a mean maximum titer of 20,512 versus 3373 for haha responses . A comparison of mean titers of the hapa and haha responses measured in samples taken on the final visit showed no significant difference between responders and nonresponders (data not shown). The absence of any apparent relationship between antibody titer and response to treatment suggests that the immune response was not detrimental to clinical outcome . Sixty - one patients were considered to be evaluable for efficacy; two patients were excluded from the efficacy analysis due to an absence of bcg treatment prior to the study, and there was one study - unrelated death for whom no final tumor assessment was obtained . Complete response based on tumor classification at baseline and dose group is summarized in table 6 . Overall, a complete response was achieved by 39% (24/61) of the patients . Of the patients with tis, 29% achieved a complete response, while complete responses were observed in 44% and 43% of the patients with t1 and ta, respectively . Of the five patients classified as bcg intolerant, only one (20%) had a complete response . Given the limited number of patients per dose cohort, it was not possible to make definitive individual dose comparisons . In order to examine a potential dose response, patients were classified into three dose groups: (0.1 to <1.0 mg) = lowest dose group; (1.0 to <10.0 mg) = middle dose group; and (10.0 mg) = highest dose group . A comparison of the response rates in the lowest dose group versus the combined middle and highest dose group revealed a statistically significant p - value of 0.0418 . All participants were caucasian with a median patient age of 69; 78% were men and 22% were women (table 1). All patients evaluated for efficacy (61/64) had previously received bcg therapy, with 95% of the patients having had two or more bladder cancer recurrences . Based on their previous bcg treatment history, only five patients were considered to have been enrolled as bcg intolerant rather than bcg refractory . All patients had grade 2 or 3, stage ta or t1, tcc, and/or tis, and, except for two cases, all papillary tumors had been previously resected 728 days prior to the first dose of study drug . Of note, as this study was carried out prior to the widespread adoption of re - transurethral resection (re - tur) of tat1, high - grade nmibc in clinical practice, and its inclusion in standard treatment guidelines, re - tur of t1 tumors was not performed . All tumors showed immunohistological evidence of epcam - positive membrane staining (figure 1). Risk factors for progression present in the patient population are summarized in table 2 . At baseline, 98% of patients had at least one factor associated with disease progression, and 64% of these individuals had three or more risk factors . Vb4 - 845 was administered to the bladder via catheter once a week for 6 consecutive weeks, followed by 46 weeks without treatment . An mtd was not determined from the initial dose escalation as no dlt was reported; therefore, additional dose cohorts were added as described above, and, again, all patients received all six scheduled doses without any dlt . Forty - one of the 64 patients (64%) experienced an ae during the course of the study . Twenty - one (33%) patients experienced only aes that were assessed by the investigator as unrelated to study treatment . Classifying the aes as unrelated to the study drug was decided after considering the temporal relationship of the onset of the event to the administration of the study drug, whether the event could be explained by concomitant medications or concurrent disease, the response to withholding the study drug, and the response to rechallenge with the study drug . These included, but were not limited to, urinary tract infection, incontinence, nocturia, pharyngolaryngeal pain, and bladder spasm . The remaining 20 patients (31%) experienced aes judged to be related to vb4 - 845 administration . The most common treatment - related aes were dysuria and hematuria (table 4). Systemic aes included fatigue, fever and chills, loss of appetite, myalgia, dizziness, and nausea . The frequency of treatment - related aes did not increase with dose escalation (figure 2). All treatment - related aes were grade 1 or 2, with the exception of one grade 3 occurrence of hematuria that was reported as possibly related to vb4 - 845 administration . This event was not reported as a dlt as the investigator judged it to be related to aspirin use, and the patient experienced no aes with subsequent vb4 - 845 dosing . There were no cases where the occurrence of aes required discontinuation of treatment, and no patient experienced a serious ae related to the administration of vb4 - 845 . There was one patient death due to cardiac failure, which occurred 3 weeks after the last dose of vb4 - 845 . This death was assessed by the investigator as unrelated to study treatment and attributed to long - standing coronary artery disease, previous myocardial infarction, and hypertension . Postinstillation plasma levels of vb4 - 845 were measured in 63 patients, except at the final visit where samples were provided by 61 patients . In almost all patients, vb4 - 845 plasma levels were below the limit of detection of the assay (14 pg / ml) at all time points examined . Vb4 - 845 was detectable in only two patients: one had levels of 19 and 17 pg / ml at 1 h after vb4 - 845 instillation and on day 8 prior to dosing, respectively, and the other had a level of 18 pg / ml at the 1 h time point . The immunogenicity of vb4 - 845 was examined by analyzing hapa and haha titers in blood samples taken at specified time points during the trial (table 5). Hapa response was more vigorous, as patients developed a measurable titer earlier; the majority of patients exhibited hapa by day 29, with 77% (47/61) having a measurable titer at final visit . In contrast, only 16% (10/61) of patients had haha by the end of the study . Hapa titers were also generally higher, with a mean maximum titer of 20,512 versus 3373 for haha responses . A comparison of mean titers of the hapa and haha responses measured in samples taken on the final visit showed no significant difference between responders and nonresponders (data not shown). The absence of any apparent relationship between antibody titer and response to treatment suggests that the immune response was not detrimental to clinical outcome . Sixty - one patients were considered to be evaluable for efficacy; two patients were excluded from the efficacy analysis due to an absence of bcg treatment prior to the study, and there was one study - unrelated death for whom no final tumor assessment was obtained . Complete response based on tumor classification at baseline and dose group is summarized in table 6 . Overall, a complete response was achieved by 39% (24/61) of the patients . Of the patients with tis, 29% achieved a complete response, while complete responses were observed in 44% and 43% of the patients with t1 and ta, respectively . Of the five patients classified as bcg intolerant, only one (20%) had a complete response . Given the limited number of patients per dose cohort, it was not possible to make definitive individual dose comparisons . In order to examine a potential dose response, patients were classified into three dose groups: (0.1 to <1.0 mg) = lowest dose group; (1.0 to <10.0 mg) = middle dose group; and (10.0 mg) = highest dose group . A comparison of the response rates in the lowest dose group versus the combined middle and highest dose group revealed a statistically significant p - value of 0.0418 . Tcc is the most common cancer of the bladder, and most patients present with nonmuscle - invasive disease . Currently, bcg is the standard of care for this malignancy . Despite its well - documented therapeutic benefit, bcg therapy is delayed or discontinued in a large proportion of patients due to associated toxicity . In rare instances, patients can develop severe conditions associated with bcg infection, in some cases life - threatening, rendering bcg a nonviable treatment option . In addition, for those patients who fail to respond to bcg therapy or experience early disease recurrence, alternative therapeutic options are limited . Given the potential for intolerance and the high rate of recurrence in patients with nmibc, alternative treatments are under investigation . Targeted therapies limit drug exposure to only diseased cells, thereby minimizing drug - related toxicities . We have described a phase i study of vb4 - 845, an anti - epcam scfv eta, administered intravesically to high - risk patients . Of the 75 patients initially screened for participation, 74 (99%) showed epcam - positive disease . This high rate of epcam expression suggests that epcam represents a significant antigen for vb4 - 845-targeted therapy in patients with advanced nmibc . Although not performed in this study, an examination of the expression levels of epcam in biopsy samples obtained from patients with residual disease following vb4 - 845 treatment might be informative and will be considered for future studies . Aes experienced with vb4 - 845 were mild and easily managed, and no ae required the discontinuation of treatment, even in the highest dose cohort . Although the presence of pre - existing lower urinary tract symptoms may make a determination of the frequency of aes difficult, what is evident is that the relatively mild toxicities experienced with vb4 - 845 are in direct contrast to other intravesical therapies where local and systemic toxicities can be treatment limiting . Vb4 - 845 is a targeted therapy that exerts its effect via specific interaction with the cell surface antigen epcam . Although overexpressed on carcinoma cells, epcam expression is lower on normal tissue and limited to the basal layers rather than luminal surfaces . These features could minimize drug - related toxicities and may explain why related aes were predominantly grade 1 and 2 . It is also interesting to note that failure to reach an mtd for vb4 - 845 over the dose range tested and the apparent absence of any relationship between the appearance of aes and dose are in keeping with its excellent safety profile . Pharmacokinetic analyses indicated that vb4 - 845 was not systemically absorbed and remained within the bladder until elimination by voiding . This is anticipated, given the integrity of the bladder and the molecular size of vb4 - 845 (~69 kda). Vb4 - 845 was shown to be immunogenic with the toxin portion eliciting the more vigorous response . Given the humanization of the scfv antibody fragment and entirely foreign nature of the bacterial toxin, the more intense immune response against the toxin was expected . A similar pattern of immune response was observed with lmb-2, a scfv eta immunotoxin, whereby more patients exhibited a stronger response to the eta moiety than to the scfv portion.20 despite the observed immunogenicity, the development of an immune response was not considered to have any bearing on clinical outcome, as there was no apparent relationship between antibody titer and response to treatment . Rather, the presence of circulating antibodies to vb4 - 845 may, in fact, limit any systemic exposure that may occur by rapidly clearing any drug that may enter circulation . Together, the specificity of vb4 - 845 and its lack of systemic exposure translate to a very acceptable safety profile . Although the phase i trial was primarily designed to determine the mtd, tumor response was assessed as a single efficacy endpoint at 3 months to elucidate any observable trends . Although dosing was not optimized for therapeutic benefit, complete responses were observed across all tumor stages and dose groupings . Although it is not possible to draw definitive conclusions between individual dose and response to treatment, when examined according to dose groups, the data are suggestive of an increased response with higher doses . Although no direct examination of receptor occupancy has been performed, the dose range of vb4 - 845 administered far exceeds the drug concentration required to achieve> 99% cell killing in epcam - positive bladder tumor cell lines . Therefore, it is possible that the tumor response may be saturated at the higher doses . It should be noted that the short duration of follow - up is a significant limitation with respect to assessing efficacy; thus, efficacy results reported in this study are considered preliminary and serve only to support further clinical trials with longer follow - up assessments . Intravesical therapy with the anti - epcam fusion protein vb4 - 845, for the treatment of nmibc was extremely well tolerated . Aes were generally mild and very manageable with no patients experiencing any serious aes related to vb4 - 845 therapy . The lack of detectable postinstillation plasma levels in the vast majority of patients indicates that the drug is effectively held within the bladder and not systemically absorbed . Although most patients developed antibody titers, the data obtained in this study showed no evidence of detrimental effects on clinical outcome associated with the immune response . Preliminary efficacy results from this high - risk, often treatment - refractory, patient population suggest that vb4 - 845 can safely inhibit tumor growth in patients with epcam - positive, high - grade nmibc.
Pneumonia causes almost 1 in 5 under - five deaths worldwide and is responsible for more than 2 million deaths of children each year . Nigeria, with a predicated incidence of 6.1 million pneumonia cases and 0.38 episodes of pneumonia per child - year in 2008, ranks 5th among the 15 countries with highest pneumonia burden in the world . It is a leading cause of childhood morbidity and mortality in developing countries including nigeria . In 2010, pneumonia accounted for 868,000 deaths in under-5 children, which was 14% of all causes of deaths in children . However when detected early, pneumonia can be treated with easily available medication at a low cost . Unfortunately, only one in five caregivers in developing countries can recognize the danger signs of pneumonia like rapid breathing and chest indrawing, and only half of children under 5 with pneumonia are taken to an appropriate healthcare provider . In a population based survey carried out in six countries in sub - saharan africa, only about 18% and 30% of children with suspected pneumonia in nigeria and ethiopia, respectively, were taken to hospital for healthcare . In a systematic review of ninety - one studies that reported recognition and/or care seeking for diarrhoea, pneumonia, and malaria in low and middle income countries (lmics), it was shown that the median sensitivity of recognition of pneumonia among caregivers was low (45.8%) and care seeking from community health worker for pneumonia was 4.2% . The world health organization (who) and unicef have recommended strengthening family's capacity to recognize danger signs and prompt care seeking as one of the interventions for controlling pneumonia in children under five . This study seeks to determine the maternal perception and care seeking behaviour concerning danger signs of childhood pneumonia . It is hoped that the findings of this study will help reduce mortality from pneumonia in children and will contribute to achieving the sustainable development goal on child mortality . This study was conducted in enugu state, the host town of enugu state university of science and technology . It is located in south east nigeria on latitude 6 27n and longitude 7 30e . Enugu state is made up of 17 local government areas with its capital carved from enugu north, enugu south, and enugu east lgas . The majority of the inhabitants are of igbo ethnicity, and christianity is the dominant religion . The minimum monthly income is similar to the national average of 18,000 (90 us dollars; national minimum wage act 2011). Literacy rate is 66%, which is higher than the national literacy rate of 45%, and there are 955 males per 1,000 females . The study was conducted over a 10-month period in 4 of the 17 local government areas (lgas) of enugu state . The lgas were divided into rural and semiurban categories based on level of development and population in the lgas . Simple random sampling using balloting method was used to select 2 lgas from each category . In the second stage, one community was selected from each lga using simple random sampling (i.e., one community from each of the two rural and two semiurban lgas). In each of the communities visited, through adequate mobilization using the community leaders, women who were caring and/or had cared for a child in the past 2 years and consented to participate were consecutively enrolled . Three hundred and nineteen (319) caregivers who met the inclusion criteria and gave consent to participate were interviewed in the rural communities while one hundred and forty - seven (147) were interviewed in the semiurban communities . The following sociodemographic variables of respondents were assessed: (i) age grouped into 2030, 3140, and> 40 years, (ii) highest educational attainment grouped into tertiary, secondary, primary, and no education, (iii) number of living children grouped into none, 14, and> 4 children, and (iv) socioeconomic class: defined as the wealth index of the household derived using maternal and paternal highest educational attainment and occupation based on oyedeji classification . Pneumonia is inflammation of one or both lungs caused by variety of organisms and chemical substances characterized by cough, shortness of breath, fever and difficult breathing, cyanosis, and death in severe cases . Oyi in the local igbo language and this name was used for respondents that do not understand english . The world health organization (who) recognizes four features as danger signs in pneumonia . They include stridor, fast breathing, chest wall indrawing, and difficulty in breathing (labored breathing). Knowledge of danger signs of pneumonia was assessed using open ended questions based on who definition . Based on their responses, the respondents were grouped into no knowledge of danger signs and knowledge of at least one danger sign . Also knowledge and uptake of vaccine against pneumonia the respondents were grouped into yes for those who were aware of the danger signs and had vaccinated their children and no for those who had not heard about pneumonia and had not vaccinated their children . Health seeking behavior of respondents was assessed to determine where care was sought for a child with suspected pneumonia and the treatment given . Quality control check to detect errors in questionnaire administration and data recording was done by researchers on daily basis after enrollment . Where there are errors detected, the interviewers were asked to clarify them accordingly with the interviewed mother or caregiver . Data cleaning to remove grossly incomplete and/or inconsistent data was also done by researcher assistants and the study researchers . Results were presented as percentages and 95% confidence intervals where appropriate . Statistical significance was set at p value <0.05 . Informed consent (written) was obtained from every mother in her own right and on behalf of her child before recruitment . Most of the respondents 166 (37.4%) were 40 years and above while those in the 2030 and 3140 years of age bracket were 158 (35.6%) and 120 (27.0%), respectively . Approximately half of the respondents 278 (49.7%) had primary school or no formal education and 280 (60.1%) in the lower socioeconomic class . Respondents in the middle and upper socioeconomic class made up 103 (22.1%) and 83 (17.8%) of respondents . Almost two - fifth, 166 (39.0%) of the respondents, have more than 4 children and 246 (57.7%) with 14 children . Fourteen (3.3%) of the respondents had no children at all but had cared for one or more children in the past . In all, 211 children 5 years or younger of respondents with a mean age of 13.7 months were involved in the study (table 1). About 95% of the respondents (440/464) had heard of pneumonia and the remaining 24 (5.2%) never heard about it . When asked about the cause, majority of the respondents 394 (88.7%) believe pneumonia is caused by exposure to cold environment and/or ingestion of cold fluid or food while 32 (6.9%) have no idea what the cause of pneumonia is . The correlation (r) between awareness of pneumonia and knowledge of its cause is 0.374 (p = 0.001). For respondents who proffered an answer whether correct or incorrect, source of information was via electronic media (i.e., radio and/or television) in 123 (26.5%), through the internet in 6 (1.3%), and during medical counseling in 226 (48.7%) respondents . Other sources of information include print media in 13 (2.8%), social interactions in 73 (15.7%), schools in 37 (8.0%), churches in 8 (1.7%), and others in 18 (3.9%) respondents . Almost all 426 (97.7%) of the 440 respondents who have heard of pneumonia acknowledged that it is a potentially dangerous illness that could lead to serious complications . Inquiry into preventive measures against pneumonia showed that the vast majority believe that adequate clothing 127 (49.6%) and avoiding cold food and environment 38 (14.8%) were the best strategy to prevent pneumonia . Other preventive measures listed by respondents include cleanliness 24 (9.4%), feeding well 14 (5.5%), drugs 11 (4.3%), vaccination 27 (10.5%), prayer 2 (0.8%), educating caregivers 3 (1.2%), and avoiding crowded areas 3 (1.2%). Knowledge of vaccine against pneumonia was alleged by 218 (46.8%) of respondents out of which 185 (39.7%) claimed they took the pneumococcal vaccine for their index child . Some of the reasons for nonvaccination for the 281 (60.3%) respondents who did not vaccinate their child included lack of knowledge of the availability of pneumococcal vaccine (209/281; 74.3%), nonavailability of the vaccine in health facilities around respondents residence (42/281; 15.0%), religious concerns (8/281; 2.9%), cost of vaccination (12/281; 4.3%), universal concern about ineffectiveness of vaccination (6/281; 2.1%), and side effects (4/281; 1.4%). Further analysis showed that only maternal education (p = 0.00) and place of residence (p = 0.02) were significantly associated with correct knowledge of pneumonia aetiology, knowledge of dangers signs of pneumonia (p = 0.00), and uptake of vaccination (p = 0.01) against pneumonia (table 2). Respondents were asked to list all features that in their opinion signify danger when a child is suspected of having pneumonia . Fever 226 (20.6%), fast breathing 181 (16.5%), continuous cough 180 (16.4%), chest pain 67 (6.1%), and difficulty in breathing 66 (6.0%) were the most listed features . Also listed were cold body (i.e., hypothermia) 63 (5.7%), catarrh / running nose 43 (3.9%), weakness 41 (3.7%), chest wall indrawing 37 (3.4%), convulsion 33 (3.0%), and others 161 (14.7%). The others included but not limited to poor suck 33, excessive cry 28, abdominal distension 27, restlessness 18, stridor, that is, noisy breathing 15, unconsciousness 11, vomiting 10, watery stooling 9, dullness of face and body 6, and pale skin 5 (table 3). Information source on perceived danger signs of respondents followed almost a similar pattern as causes of pneumonia described above except that previous experience of danger signs accounted for close to half, 224 (48.1%) of information source here . Others include electronic media in 99 (21.2%), internet in 11 (2.4%), social interaction in 10 (2.1%), medical counseling in 86 (18.5%), and others in 35 (7.2%) respondents . Two hundred and thirty - two (56.3%) of respondents have experienced their perceived danger sign of pneumonia in one or more of their children . Fever 123 (24.5%), continuous cough 98 (19.5%), fast breathing 75 (14.9%), cold 34 (6.8%), convulsion 22 (4.4%), and difficulty breathing 17 (3.4%) top the list of the most experienced pneumonia danger signs among children of respondents (table 3). The who / unicef recognised danger signs most commonly known by respondents were fast breathing (60.5%) and difficulty in breathing (22.1%) while chest indrawing (12.4%) and stridor, that is, noisy breathing (5.0%), were less known to respondents (table 3). Knowledge of at least one who / unicef recognised danger sign was seen in 304 (65.2%) of respondents while knowledge of 2, 3, and 4 recognised danger signs was seen in 219 (47.0%), 37 (7.9%), and 22 (4.7%) of respondents, respectively . One hundred and sixty - two (34.8%) had knowledge of none of the who / unicef danger signs . Just like cause and vaccine knowledge, maternal level of educational attainment (p = 0.04), and place of residence (p = 0.00) were significantly associated with knowledge of the who / unicef recognised pneumonia danger signs in children among respondents (table 2). Sixty - four percent of the 253 that responded to the question presented to the hospital, 78 (30.8%) either bought drugs over the counter or visited the patent medicine dealer for treatment, 10 (4.0%) consulted the traditionalist, and 3 (1.2%) took the child to the church for prayers and spiritual healing . First - line treatment option in all respondents ranged from antibiotics 52 (20.2%), cough syrups 119 (46.3%), vitamin c 7 (0.03%), drug combinations 46 (17.9%) [i.e., antibiotics + cough syrup, antibiotics + cough syrup + vitamin c, etc . ], herbal concoction 9 (0.04%), and hospital admission 24 (9.4%). Majority of the children 202 (81.1%) recovered fully, 38 (15.3%) did not survive the experience, and 9 (3.6%) recovered but with complications . Caregivers with tertiary education (70.0%) used hospitals more compared to those with secondary (60%), primary (68%), and no education (60%) (p = 0.001). Conversely, caregivers with no formal education (14.0%) and those with primary education (2.0%) used traditional and/or spiritual treatment more compared to those with tertiary (0.0%) and secondary school education (0.0%) (p = 0.001). Finally, caregivers resident in rural area compared to those resident in semiurban areas sought care in a healthcare facility more (65.0% versus 59.0%), self - medicated less (28.0% versus 41.0%), and consulted traditionalist and/or spiritualist more (7.0% versus 0.0%) (p = 0.04) (table 2). This study investigated the knowledge of caregivers and caregivers in enugu about the aetiology and danger signs of pneumonia . It also sought to determine factors that influenced the knowledge and health seeking behaviour of caregivers for their under-5 children with probable pneumonia . The study showed high awareness (95%) and knowledge of potential fatality of pneumonia disease (97.7%) among respondents but poor knowledge of the aetiology (4.1%) and danger signs of probable pneumonia . A similar study in thailand also showed inadequate knowledge of danger signs (7%) and causes (21%) of pneumonia among respondents . The relatively poorer knowledge of causes of pneumonia in this study may be related to the higher number of caregivers without any formal education (29.2%) compared to 4.3% among respondents in the thailand study . It was noted in that study that none of the caregivers surveyed was able to mention the four standard danger signs of childhood pneumonia and only 9.4% of the respondents knew that lower chest wall indrawing was a danger sign in childhood pneumonia . It was also shown in this study that older caregivers, caregivers with higher educational attainment, and those that are resident in semiurban areas had better knowledge of the cause and danger signs of pneumonia . This is hardly surprising as caregivers with these sociodemographic variables are more likely to have more experiences of childhood illnesses and/or are better informed about pneumonia . It showed that caregivers with lower than tertiary education were more likely to have less knowledge on aspects of pneumonia disease . The lagos study in addition showed that caregivers whose information source about pneumonia was from health personnel were more likely to have correct information on the cause of pneumonia compared to those from other sources . This fact was supported by this present study which revealed that respondents who obtained their information from medical personnel were more likely to have correct knowledge of aetiology and prevention of pneumonia compared to respondents that got their information from other sources . Inquiries into the preventive strategies showed that majority of the respondents believed that adequate clothing and avoidance of cold food, drink, or environment prevents pneumonia disease in a child . This belief which is a common misconception among many lay people in nigeria was also corroborated by the study in lagos which found that 75.6% of mothers surveyed believed that cold was the main cause of pneumonia . Furthermore, almost half of respondents in the present survey had knowledge of pneumonia vaccine with only 39.9% of these respondents having vaccinated their child against pneumonia . Similarly, only about a tenth of caregivers in our survey listed vaccination as a preventive strategy for pneumonia . This low uptake rate is expected as pneumococcal vaccines, which are relatively new additions to the national immunization schedules, are not widely available in public health facilities . Furthermore, even when available, they are not free like other routine vaccines in nigeria . Higher maternal educational attainment and residence in semiurban areas were significantly associated with knowledge about pneumococcal vaccine and uptake rate among children of surveyed caregivers . A study conducted in the africa center demographic site in south africa showed that caregivers with secondary and tertiary education were 1.10 and 1.09 times, respectively, more likely to uptake pneumococcal conjugate vaccine (pcv) for their children compared to caregivers with no education . It further showed that those caregivers resident in rural and periurban areas were 0.90 and 0.96 times less likely to uptake pneumococcal vaccine for their child . Though majority of the respondents in our study took their children to hospital on suspicion of pneumonia, nearly 40% either self - medicated and/or consulted traditionalist or spiritualist as the first line of action . The finding that caregivers with lower education were more likely to engage in this behaviour is easily rationalized . Because these caregivers are more likely to be less informed and easily persuaded, they will more likely be influenced by advice from friends and relatives to seek nonmedical solutions for their child's illness (especially in the face of poverty). It was further noted that caregivers in rural area were more likely to visit hospital, less likely to self - medicate, and also more likely to visit traditionalist and/or spiritualist compared to caregivers in urban areas . It could be further rationalized that because of the easy accessibility of various category of drug stores and ease of getting drugs over the counter in urban areas caregivers tended to patronize these drug dealers and self - medicate their children during illnesses . Similarly, because of the traditional belief which is inherent in rural communities and poorer accessibility to healthcare facilities, it is not surprising that caregivers in rural areas may more likely seek traditional and/or spiritualist help for their child's illness . Contrary to this finding, a community based comparative study in dera district of oromia regional state, ethiopia, showed that more caregivers in urban area (75.0%) sought healthcare for acute respiratory tract infection in their children from healthcare institution compared to those in rural areas (34.4%). Although no explanation was proffered by the authors for this finding, one could speculate that the higher number of caregivers with formal education in the urban category (28.7%) compared to caregivers in the rural category (3.6%) may have contributed to the higher healthcare seeking behaviour among caregivers in the urban compared to those in the rural setting unlike our study where there is no significant difference in educational attainment between caregivers' resident in urban and rural area . Finally, we observed in our study that fast breathing and difficulty in breathing (labored breathing) were the most known and experienced who recognized danger signs among respondents . This may be related to the ease of noticing these symptoms in children compared to chest wall indrawing and stridor . The proportion of caregivers who had knowledge of these signs was more than what was observed in a similar study in uganda . On the other hand, fever, fast breathing, and cough were the most perceived and experienced danger sign among respondents in this study . Khas district of pakistan similarly noted that fever and cough (65.2%), followed by fast breathing and chest wall indrawing (59.4%), were the most perceived symptoms of pneumonia by mothers in the district . As noted earlier, this may be related to the ease of picking up these symptoms in children . Selection bias would have resulted due to the point enrollment because prospective study participants were required to gather at a preagreed location for consenting and enrollment . Household sampling and enrollment which could have significantly reduced selection bias could not be done during selection of respondents in the community due to unorganized household locations and difficult terrains in some communities . Also, recall bias could have led to errors in data measurement, classification, and analysis since some caregivers were interviewed on events that took place in the past . This study demonstrated poor knowledge of the aetiology, danger signs, and prevention of childhood pneumonia amongst caregivers . Health education targeted at caregivers will enhance dissemination of information to fill this knowledge deficit and hopefully reduce morbidity and mortality from childhood pneumonia . Therefore the authors intend to embark on workshops to sensitize medical personnel on the need to educate the caregivers on the danger signs and prevention of childhood pneumonia.
In the early 2000s, the grading of recommendations assessment, development and evaluation (grade) working group developed a framework in which the certainty in evidence was based on numerous factors and not solely on study design which challenges the pyramid concept.8 study design alone appears to be insufficient on its own as a surrogate for risk of bias . Certain methodological limitations of a study, imprecision, inconsistency and indirectness, were factors independent from study design and can affect the quality of evidence derived from any study design . For example, a meta - analysis of rcts evaluating intensive glycaemic control in non - critically ill hospitalised patients showed a non - significant reduction in mortality (relative risk of 0.95 (95% ci 0.72 to 1.25)9). The quality of this evidence is rated down due to the methodological imitations of the trials and imprecision (wide ci that includes substantial benefit and harm). Hence, despite the fact of having five rcts, such evidence should not be rated high in any pyramid . The quality of evidence can also be rated up . For example, we are quite certain about the benefits of hip replacement in a patient with disabling hip osteoarthritis . Although not tested in rcts, the quality of this evidence is rated up despite the study design (non - randomised observational studies).10 therefore, the first modification to the pyramid is to change the straight lines separating study designs in the pyramid to wavy lines (going up and down to reflect the grade approach of rating up and down based on the various domains of the quality of evidence). Another challenge to the notion of having systematic reviews on the top of the evidence pyramid relates to the framework presented in the journal of the american medical association user's guide on systematic reviews and meta - analysis . The guide presented a two - step approach in which the credibility of the process of a systematic review is evaluated first (comprehensive literature search, rigorous study selection process, etc). If the systematic review was deemed sufficiently credible, then a second step takes place in which we evaluate the certainty in evidence based on the grade approach.11 in other words, a meta - analysis of well - conducted rcts at low risk of bias cannot be equated with a meta - analysis of observational studies at higher risk of bias . For example, a meta - analysis of 112 surgical case series showed that in patients with thoracic aortic transection, the mortality rate was significantly lower in patients who underwent endovascular repair, followed by open repair and non - operative management (9%, 19% and 46%, respectively, p<0.01). Clearly, this meta - analysis should not be on top of the pyramid similar to a meta - analysis of rcts . After all, the evidence remains consistent of non - randomised studies and likely subject to numerous confounders . Therefore, the second modification to the pyramid is to remove systematic reviews from the top of the pyramid and use them as a lens through which other types of studies should be seen (ie, appraised and applied). The systematic review (the process of selecting the studies) and meta - analysis (the statistical aggregation that produces a single effect size) are tools to consume and apply the evidence by stakeholders . Changing how systematic reviews and meta - analyses are perceived by stakeholders (patients, clinicians and stakeholders) has important implications . For example, the american heart association considers evidence derived from meta - analyses to have a level a (ie, warrants the most confidence). A evidence could have been high, moderate, low or of very low quality.12 the quality of evidence drives the strength of recommendation, which is one of the last translational steps of research, most proximal to patient care . One of the limitations of all pyramids and depictions of evidence hierarchy relates to the underpinning of such schemas . The construct of internal validity may have varying definitions, or be understood differently among evidence consumers . A limitation of considering systematic review and meta - analyses as tools to consume evidence may undermine their role in new discovery (eg, identifying a new side effect that was not demonstrated in individual studies13). Ebm teachers can compare it to the existing pyramids to explain how certainty in the evidence (also called quality of evidence) is evaluated . It can be used to teach how evidence - based practitioners can appraise and apply systematic reviews in practice, and to demonstrate the evolution in ebm thinking and the modern understanding of certainty in evidence.
Osteosarcoma (os) develops most frequently in the extremities, and it is the most common histologic form of the primary bone cancers.1 2 head and neck oss are rare, comprising only 6 to 10% of all oss.3 4 they typically present in the third or fourth decade of life and comprise only 1% of all pediatric head and neck malignancies . The most common craniofacial sites affected by oss are the mandible and maxilla, followed by the calvaria and then the skull base.4 5 6 on cytology, os can be divided into several pathologic types, including the pleomorphic, epithelioid, chondroblastic, small cell, mixed, and osteoclast - like giant cell types.6 in head and neck oss, the chondroblastic type occurs most frequently.7 skull base oss can be challenging to resect and an aggressive surgical approach can result in poor cosmetic outcome.8 imaging plays a crucial role in the diagnosis of each subtype of os and ultimately in patients' survival because the diagnosis is based on a combination of histopathologic and imaging features . The therapeutic options and prognoses for different types of os differ from each other, so correct diagnosis is essential.9 10 magnetic resonance imaging (mri) or computed tomographic (ct) scan should be used to assess the extent of the primary tumor.11 in this case report, we describe a pediatric patient of occipital os of the chondroblastic type . The chondroblastic type of os has an exceedingly poor outcome.12 however, the detailed imaging description of such cases have not been reported in the previous literatures . We present the ct, mri, and enhanced mri features of this case, followed by a brief review of the related cases reported in the previous literatures a 9-year - old boy was admitted to our hospital with a major complaint of a growing mass on his head . Physical examination found a firm and tough mass on the right occipital that showed no tenderness upon palpation . Ct scan showed the right occipital bone to be irregularly thickened with fluffy and cloudy calcification, with a mass deriving from the internal occipital protuberance extending toward the basilar part of the occipital bone, invading the neighboring jugular foramen, the sublingual neural tube, and the mamillary process . On mri, the lesion was 4.5- 5.5- 6.5-cm in size with calcifications areas of hypointensity in t1- and heterogeneous in t2-weighted series . Contrast mri showed peripheral and septal enhancement in the interior side of the tumor (fig . Significant mass effect was present, distorting the cerebellar hemisphere, pons, and the forth ventricle, which led to hydrocephalus, and the oppression of the sigmoid sinus and the transverse sinus . 2), corresponding to the features of chondroblastic os, and occipital bone chondroblastic os was the final definitive diagnosis . (b) t1-weighted image shows a 4- 8- 10-cm mass lesion, isointense to the skull . (c, d) the mass is hypointense in most areas in the t2-weighted series, with focal high signals in the t2-weighted series and reduced signal in flair series. (e) in gd - enhanced mri, most areas show no enhancement or heterogeneous enhancement, with peripheral and atypical septal enhancement on the coronal plane (white arrows). (f) no hyperintensity was observed in both intra- and peritumoral areas in the dwi series . Histopathologic examination (hematoxylin and eosin, 200) shows lace - like osteoid material abutting the neoplastic cells . They typically present in the third or fourth decade of life, account for fewer than 5% of oss in children, and comprise only 1% of all pediatric head and neck malignancies . The most common craniofacial sites are the mandible and maxilla, followed by the calvaria and then the skull base.13 14 15 our case in the right occipital bone of skull base is a very rare location . A search of the english language literature revealed 22 cranial oss previously reported in children (table 1): 12 calvarial tumors and 10 tumors of the skull base . The mean age of the pediatric patients with cranial os was 12.2 years old in this table . Can be divided into pleomorphic, epithelioid, chondroblastic, small cell, mixed, and osteoclast - like giant cell types.6 our case is a chondroblastic subtype, which occurs most frequently in head and neck oss . Abbreviations: gtr, gross total resection; nr, not reported; rt, radiation therapy; str, subtotal resection . The etiology of os is unknown, but the major risk factors for development of os in craniofacial bones may be similar to those of the long skeletal bones, consisting of exposure to radiation, retinoblastoma, li - fraumeni syndrome, and paget's disease . Other bone abnormalities, such as fibrous dysplasia, multiple osteochondromatosis, chronic osteomyelitis, myositis ossificans, and trauma, have also been proposed as risk factors.7 15 16 the presenting symptoms varied with the location of the tumors . The maxillary or cranial lesions usually produced no pain, which was in accordance with our case; however, mandibular tumors frequently presented with focal painful swelling.17 18 other common presenting symptoms include headache, cranial nerve palsies, exophthalmos, and visual impairment due to different location of the tumor.5 13 ct best demonstrates tumor mineralization, especially when minimal, and it is usually able to demonstrate tumor extension into the soft tissues . Hemorrhage, necrosis, and unmineralized, chondroblastic, or fibroblastic components of the tumor will appear as areas of low attenuation on ct if present . Unlike any other conventional oss, we see fluffy calcification in our case, and we believe it is a characteristic of os . The osteoblastic subtype is most common with nearly 90% containing variable amounts of cloudlike opacities.19 bose20 reported an osteoblastic os that appears as a large soft tissue density mass with a few bony densities . Compared with our case, the soft tissue mass is prominent and the calcification is less and diffuse . Mri is the preferred modality for locally staging os, and it should be performed before percutaneous biopsy because it can help identify areas of viable tumor and mineralized matrix . In our case of gadolinium (gd)enhanced mri, we found no enhancement or heterogeneous enhancement in most areas of the tumor, with septonodular and rim enhancement, which is in in accordance with the current literature . Areas that demonstrate either a heterogeneous enhancement pattern or lack enhancement are the preferred sites for biopsy because they are more likely to contain both chondroid and osteoid elements that are necessary for the correct diagnosis.21 22 chondrosarcomas shows similar image characteristic, but they occur in an older age with a mean age of 57 years old . Chondroblastic oss also have significantly higher minimum and maximum apparent diffusion coefficient (adc) values compared with other conventional os subtypes, but they have a lower minimum adc and similar maximum adc value compared with chondrosarcoma.23 skull base oss can be challenging to resect, and an aggressive surgical approach can result in poor cosmetic outcome . Thus, skull base tumors have a poorer prognosis than mandibular or maxillary tumors.3 complete surgical excision is the mainstay of treatment of the primary tumor . Local recurrence is the main reason of treatment failure and mortality in head and neck oss . Positive margins and a high tumor grade correlate with a statistically significant decrease in survival.11 in our case, the tumor could not be completely removed because it invades significant neighboring bone structures, including the jugular foreman and the sublingual neural tube . The patient died after 6 months as a result of local recurrence . In summary, chondroblastic os has been shown to be associated with a poor preoperative chemotherapy response and has a worse prognosis than other variants.24 however, this subtype has some particular image characteristic, which helps surgeons identify before surgery and set early therapeutic regimens.
Usually fungal infections caused by opportunistic and pathogenic fungi were an important cause of morbidity and mortality among in patients having severe basic internal diseases, and also the sufferers did not have any characteristic significant symptoms . And the epidemiology, transport, and pathogenic mechanisms had not been well known . Historically the development of mycosis had been depended primarily on the immune defense competence of the patients . In addition, the clinical pictures of immunocompromised mycotic infections had been different with one another and investigative studies had been diversely accumulated hitherto . However, pathogenic fungal infections in an immunocompetent had been rare and neglected into clinical studies . Especially cryptococcal brainstem abscess mimicking brain tumors had been also much more rare maybe due to blood brain barrier competence and indolent characteristics of such infections in an immunocompetent . This 47-year - old man presented with a history of progressively worsening headache and nausea for 1 month and several days of vomituritions before admission . A computed tomography (ct) scan revealed enhancing mass lesion with central necrosis and peripheral edema in the left pons compressing the fourth ventricle (fig . 1), and subsequent simple, diffusion, and perfusion mri demonstrated a rim - enhancing about 1.81.72.0 cm sized, highly perfusing mass lesion with heterogeneous low or iso - signal intensity (fig . 2), and also positron emission tomogram (pet) scans of the whole body comprising brainstem demonstrated a hot uptake of fluorodeoxy - glucose (fdg) on the brainstem lesion without having any evidences of systemic metastasis (fig . Gross total mass resection was achieved with lateral suboccipital approach with the help of neuronavigation system and somatosensory evoked potential monitoring (fig . This 47-year - old man presented with a history of progressively worsening headache and nausea for 1 month and several days of vomituritions before admission . A computed tomography (ct) scan revealed enhancing mass lesion with central necrosis and peripheral edema in the left pons compressing the fourth ventricle (fig . 1), and subsequent simple, diffusion, and perfusion mri demonstrated a rim - enhancing about 1.81.72.0 cm sized, highly perfusing mass lesion with heterogeneous low or iso - signal intensity (fig . 2), and also positron emission tomogram (pet) scans of the whole body comprising brainstem demonstrated a hot uptake of fluorodeoxy - glucose (fdg) on the brainstem lesion without having any evidences of systemic metastasis (fig . Gross total mass resection was achieved with lateral suboccipital approach with the help of neuronavigation system and somatosensory evoked potential monitoring (fig . Cryptococcus neoformans is a medically important encapsulated pathogenic yeast often responsible for potentially life - threatening meningoencephalitis in humans . Cryptococcosis occurs world wide, and its incidence has increased with the onslaught of the human immunodeficiency virus / acquired immunodeficiency syndrome pandemic . In nature, cryptococcus (c.) neoformans exists in two biochemically distinguishable varieties, c. var . Gatti, and also is subdivided into 5 serotyes: a, d, ad, serotypes corresponding to c. var . Differences among the specific serotypes are based on the antigenic differences resulting from structural variations of the major capsular polysaccharide glucuronoxylomannan . The specific serotypes of clinical cryptococcal isolates can be correlated with differences in epidemiology, pathogenesis, clinical presentations and patient management9). C. neoformans is an opportunistic fungus that typically affects patients who are hiv - positive and other patients with compromised immune systems . Disseminated disease may occur in up to 62% of hiv - seronegative patients with cryptococcosis2). These fungi are present in the air, on medical objects and instrumentations, in the respiratory tract and on the hands of hospital staff, and so transplantations have been the most frequent risk factors7). Cryptococcus neoformans infections usually presents as a meningitis with t - lymphocyte defects but not as a cerebral mass lesion3). However, cryptococcal mass lesion (cryptococcoma) rarely occurred even in apparently immunocompetent child in one series4). In a diagnostic study we performed perfusion - weighted mr imaging, hakyemez et al.7) previously reported 105 patients with lesion [high - grade glioma (n=23), low - grade glioma (n=11), meningioma (n=23), metastasis (n=25), hemangioblastoma (n=6), pyogenic abscess (n= 4), schwannoma (n=5), and lymphoma (n=5)]. The regional cerebral blood volume (rcbv) ratio of the lesions were obtained by dividing the values obtained from the normal white matter . The rcbv ratio was 5.76 in high - grade glioma, 8.02 in meningioma, 5.27 in metastasis, 11.36 in hemangioblastoma, 0.76 in abscesses, 1.10 in lymphoma, and 3.23 in schwannomas . In conclusion, rcbv ratios on perfusion mri scan can be used to discriminate various intracranial space occupying lesions by comparing vascularities . With this rcbv tool we can discriminate between ring enhancing abscess and high - grade glioma with hemorrhagic necrosis and single metastasis1) historically lesions of the pons and brachium pontis have long been considered as a difficult diagnostic and surgical challenge . A presumptive diagnosis can often be made based upon characteristic ct and mr imaging appearances therefore, patients have been treated purely on the basis of radiologic findings in the absence of a diagnostic tissue specimen . Patients are frequently administered inappropriate empirical therapy because their diagnoses were based upon clinical and radiographic impressions not on histological grounds . And surgical approaches to the pons have generally been one of the following: 1) the suboccipital approach via the fourth ventricle; 2) an approach through a retromastoid suboccipital craniectomy via the cerebellar pontine angle; 3) the subtemporal transtentorial technique; or 4) a ct - guided stereotactic suboccipital transcerebellar biopsy . The open conventional surgical methods carry inherent risks in compromised patients and are associated with a low diagnostic yield . Stereotaxic biopsy has been shown effective and consistent in obtaining diagnostic tissue samples through a transcortical frontal trajectory or by a suboccipital transcerebellar approach6). There have been only three previously reported cases of pontine biopsies obtained by the suboccipital transcerebellar method5,6). Abernathey et al.1) present 26 cases of pontine mass lesions biopsied through a suboccipital transcerebellar approach traversing the middle cerebellar peduncle . Interestingly in this series we safely performed direct gross total mass resection simply with lateral suboccipital approach with the help of neuronavigation and ssep monitoring system . Generally immunocompromised brain abscess medication plan is amphotericin b 0.7 mg / kg intravenous+flucytosine 100 mg / kg per os (po) daily for 2 weeks, then fluconazole 400 mg po daily for 8 weeks, then fluconazole 200 mg po daily till stable, however, we prescribed amphotericin or fluconazole 5 mg / kg po for 2 - 3 mo8). This is a previously unreported rare case of brainstem cryptococcal abscess mimicking brain tumors in an immunocompetent host without having any apparent typical cryptococcal meningeal symptoms and signs with resultant good neurosurgical recovery.
Thromboembolism in patients with nephrotic syndrome is a well - known complication because of hypercoagulable state and many other factors like hemoconcentration, increased blood viscosity, hypoalbuminemia, alteration in platelet function, and possibly administration of steroids and/or diuretics . The thromboembolic complication of peripheral and deep veins is more common in adults as compared to children though lower incidence in children, it tended to be more severe in children . Cerebral venous sinus thrombosis (cvst) in children is a rare complication and only few cases have been reported. [37] following two cases demonstrate this rare complication . Sinovenous thrombosis is probably less recognized and the symptoms and clinical course are highly variable . As in the present reported cases, symptoms may be very non - specific like headache, vomiting, drowsiness and altered behavior . The cvst should be suspected in any patient with nephrotic syndrome who develops any neurologic symptoms like headache, vomiting and cranial nerve palsy, altered sensorium or seizures . Easy availability of imaging techniques like magnetic resonance imaging (mri) and magnetic resonance venography (mrv) along with increasing awareness of condition should make it more frequently diagnosed, with scope of its reversal with timely treatment . A 4-year - old boy, weighing 14 kg with steroid - responsive nephrotic syndrome of 1-year duration developed vomiting and drowsiness for 2 days, with fresh re - appearance of edema (relapse of nephrotic state). He had vomiting, 5 - 6 times a day, after each feeding attempt . He was on tapered treatment with prednisolone 5 mg per day, but developed fresh relapse . On admission, past medical history was negative for migraine headaches, head trauma, febrile illness, substance abuse, vitamin intake or thrombophlebitis . Family history was unremarkable . On admission, his vital signs were normal with pulse rate of 80/min, regular, and blood pressure of 110/80 mmhg . Neurologic examination revealed uncooperative child, conscious but resenting interference with normal cranial nerve examination and good tone and power in all four limbs with normal sensation and normal deep tendon reflexes . There was no neck rigidity, fundus examination revealed blurring of disc margin with early papilledema . Investigations showed hemoglobin 11.1 gm / dl with packed cell volume of 36%, total leucocytes count of 12,600/mm, platelet count 2.8 lac / mm, serum creatinine of 0.42 mg / dl, blood urea of 18 mg / dl and serum albumin of 2.1 . Coagulation profile showed prothrombin time of 11 s with international normalized ratio (inr) of 1.12 and activated partial thromboplastin time of 28 s. protein c and protein s levels were normal . Urine analysis showed 2 + proteinuria, 24 h urine protein excretion was 2.0 gm / day . Computer tomography scan brain without contrast was normal, so mri brain and mr venography was done which demonstrated thrombosis of superior sagittal, left transverse and left sigmoid sinuses . Magnetic resonance imaging brain (left) and magnetic resonance venography (right) showing thrombosis of superior sagittal sinus, left transverse and left sigmoid sinuses he was treated with daltaparin, a low molecular weight heparin 1000 u (75 - 100 u / kg each dose 12 h) subcutaneous twice daily for 5 days, and oral anticoagulant warfarin was added on fourth day in dose of 0.75 mg twice a day . Dose of warfarin was adjusted to keep inr between 2.0 to 2.5 (in dose range of 0.05 - 0.1 mg / kg). The nephrotic state was treated with 50 ml of 20% human albumin i / v daily for four days with prednisolone 20 mg / day and patient had increased dieresis on 4 day of admission and child had nil proteinuria after 10 days . The child responded well to treatment, his drowsiness and vomiting improved in 2 days, and he recovered completely in 2 weeks . Repeat mri brain with mrv performed after 2 months showed complete resolution of thrombus from all the cerebral venous sinuses . His warfarin dose was reduced after resolution, and was continued in lower dose 0.5 mg twice a day for further 6 months, though inr was difficult to keep at two . At present, patient is off anticoagulant treatment without any neurological complaints [figure 2]. Repeat magnetic resonance imaging brain with magnetic resonance venography performed after 2 months showed complete resolution of thrombus from all the cerebral venous sinuses a 3 and a 1/2-year - old boy weighing 12.5 kg with new onset nephrotic syndrome of 15 days duration presented with continuous frontal headache for 3 days at the time of admission . Past medical history and family history was unremarkable . On admission, patient was conscious, cooperative, and well oriented . His pulse rate was 86/min and blood pressure was 100/60 mmhg . On general examination, investigations showed hemoglobin 11.6 gm / dl, total leucocyte count of 11800/mm, platelet 2.3 lac / mm, serum creatinine of 0.50 mg / dl, blood urea of 22 mg / dl, and serum albumin of 1.7 gm / dl, s. serum cholesterol was 537 mg / dl . Coagulation profile showed prothrombin time of 10 s with inr of 1.1, and activated partial thromboplastin time of 29 s cranial mri and mrv showed thrombosis of superior sagittal, bilateral transverse and bilateral sigmoid sinuses [figure 3]. Magnetic resonance imaging and magnetic resonance venography showed thrombosis of superior sagittal, bilateral transverse, and bilateral sigmoid sinuses his steroid dose was continued at 15 mg per day and daily intravenous human albumin (20%) 50 ml was given for 5 days till his serum albumin rose to 3 gm / dl . He was started on deltaparin (lmwh) 1000 u (75 - 100 u / kg each dose 12 h) subcutaneous twice daily for 5 days and warfarin was added on the fourth day with a dose of 0.75 mg (in dose range of 0.05 - 0.1 mg / kg) twice a day . Sixth day onwards, patient was maintained on warfarin only to keep inr between 2.0 and 2.5 . His mri with mrv done 3 weeks later showed significant dissolution of thrombosis in all affected sinuses . Presently, the boy is on warfarin 1.5 mg for venous thrombosis, prednisolone 15 mg / day, and mycophenolate mofetyl 125 mg twice a day with his nephrotic state in remission . A 4-year - old boy, weighing 14 kg with steroid - responsive nephrotic syndrome of 1-year duration developed vomiting and drowsiness for 2 days, with fresh re - appearance of edema (relapse of nephrotic state). He had vomiting, 5 - 6 times a day, after each feeding attempt . He was on tapered treatment with prednisolone 5 mg per day, but developed fresh relapse . On admission, past medical history was negative for migraine headaches, head trauma, febrile illness, substance abuse, vitamin intake or thrombophlebitis . Family history was unremarkable . On admission, his vital signs were normal with pulse rate of 80/min, regular, and blood pressure of 110/80 mmhg . Neurologic examination revealed uncooperative child, conscious but resenting interference with normal cranial nerve examination and good tone and power in all four limbs with normal sensation and normal deep tendon reflexes . There was no neck rigidity, fundus examination revealed blurring of disc margin with early papilledema . Investigations showed hemoglobin 11.1 gm / dl with packed cell volume of 36%, total leucocytes count of 12,600/mm, platelet count 2.8 lac / mm, serum creatinine of 0.42 mg / dl, blood urea of 18 mg / dl and serum albumin of 2.1 . Coagulation profile showed prothrombin time of 11 s with international normalized ratio (inr) of 1.12 and activated partial thromboplastin time of 28 s. protein c and protein s levels were normal . Urine analysis showed 2 + proteinuria, 24 h urine protein excretion was 2.0 gm / day . Computer tomography scan brain without contrast was normal, so mri brain and mr venography was done which demonstrated thrombosis of superior sagittal, left transverse and left sigmoid sinuses . Magnetic resonance imaging brain (left) and magnetic resonance venography (right) showing thrombosis of superior sagittal sinus, left transverse and left sigmoid sinuses he was treated with daltaparin, a low molecular weight heparin 1000 u (75 - 100 u / kg each dose 12 h) subcutaneous twice daily for 5 days, and oral anticoagulant warfarin was added on fourth day in dose of 0.75 mg twice a day . Dose of warfarin was adjusted to keep inr between 2.0 to 2.5 (in dose range of 0.05 - 0.1 mg / kg). The nephrotic state was treated with 50 ml of 20% human albumin i / v daily for four days with prednisolone 20 mg / day and patient had increased dieresis on 4 day of admission and child had nil proteinuria after 10 days . The child responded well to treatment, his drowsiness and vomiting improved in 2 days, and he recovered completely in 2 weeks . Repeat mri brain with mrv performed after 2 months showed complete resolution of thrombus from all the cerebral venous sinuses . His warfarin dose was reduced after resolution, and was continued in lower dose 0.5 mg twice a day for further 6 months, though inr was difficult to keep at two . At present, patient is off anticoagulant treatment without any neurological complaints [figure 2]. Repeat magnetic resonance imaging brain with magnetic resonance venography performed after 2 months showed complete resolution of thrombus from all the cerebral venous sinuses a 3 and a 1/2-year - old boy weighing 12.5 kg with new onset nephrotic syndrome of 15 days duration presented with continuous frontal headache for 3 days at the time of admission . His pulse rate was 86/min and blood pressure was 100/60 mmhg . On general examination, investigations showed hemoglobin 11.6 gm / dl, total leucocyte count of 11800/mm, platelet 2.3 lac / mm, serum creatinine of 0.50 mg / dl, blood urea of 22 mg / dl, and serum albumin of 1.7 gm / dl, s. serum cholesterol was 537 mg / dl . Coagulation profile showed prothrombin time of 10 s with inr of 1.1, and activated partial thromboplastin time of 29 s cranial mri and mrv showed thrombosis of superior sagittal, bilateral transverse and bilateral sigmoid sinuses [figure 3]. Magnetic resonance imaging and magnetic resonance venography showed thrombosis of superior sagittal, bilateral transverse, and bilateral sigmoid sinuses his steroid dose was continued at 15 mg per day and daily intravenous human albumin (20%) 50 ml was given for 5 days till his serum albumin rose to 3 gm / dl . He was started on deltaparin (lmwh) 1000 u (75 - 100 u / kg each dose 12 h) subcutaneous twice daily for 5 days and warfarin was added on the fourth day with a dose of 0.75 mg (in dose range of 0.05 - 0.1 mg / kg) twice a day . Sixth day onwards, patient was maintained on warfarin only to keep inr between 2.0 and 2.5 . His mri with mrv done 3 weeks later showed significant dissolution of thrombosis in all affected sinuses . Presently, the boy is on warfarin 1.5 mg for venous thrombosis, prednisolone 15 mg / day, and mycophenolate mofetyl 125 mg twice a day with his nephrotic state in remission . Cerebral sinovenous thrombosis is a rare disorder in children, but increasingly diagnosed now because of greater clinical awareness, sensitive neuroimaging techniques . In follow - up of 700 children with nephrotic syndrome over a period of 17 years, divekar et al . It is surprising that case 2 in the present study had persistant headache at the time of initial presentation of nephrotic syndrome of 15 days duration . Cvst in nephrotic syndrome is associated with a hypercoagulable state arising due to alteration in blood levels of various factors involved in coagulation, fibrinolytic system, alteration in platelet function, hemoconcentration, increased blood viscosity, and possibly administration of steroids and diuretics . Loss of antithrombin iii (physiological anticoagulants) and plasminogen in urine and their low level in blood has been correlated with low serum albumin level . Mri with magnetic resonance venography is the preferred modality over ct scan for diagnosis and follow - up of cerebral venous thrombosis because of its high sensitivity . In addition to treating relapse of nephrotic state, correction of hypoalbuminemia by intravenous fresh frozen plasma to replace deficient physiologic anticoagulant factors and or i / v human albumin in small aliquots daily till serum albumin is reasonably normalized . The most commonly used treatment is unfractionated heparin or low - molecular - weight heparin to arrest the thrombotic process for initial few days to a week with overlap of oral anticoagulants in the last 2 days of parentral heparin. [37] the optimal duration of oral anticoagulant (warfarin) treatment after the acute phase is unknown in this particular situation, but in the absence of recurrent thrombosis, according to the guidelines of seventh accp conference on antithrombotic therapy in children, anticoagulant therapy is to be continued at least for 6 months, using vitamin k antagonists (warfarin) with targeted inr range 2.0 - 3.0 fibrinolytic agents have not been fully investigated in nephrotic children . The signs and symptoms of cerebral venous thrombosis are occasionally non - specific making clinical diagnosis difficult . Hence, a high degree of suspicion is required, and a diagnosis of cvst should be considered in any child of nephrotic syndrome presenting with features of central nervous system involvement . Mri and mr venography is a better way of imaging as compared to ct scan . Prompt treatment with anticoagulation is very useful and should be instituted as early as possible to improve the outcome . Not allowing serum albumin to fall below 2 gm / dl could be a preventive measure to such complications.
. Some of these valves can be reconstructed; however this is not always feasible due to tissue degeneration and therefore replacement is needed . Several suitable prostheses are available to replace the aortic valve, with specific advantages and disadvantages . Biological heart valves, of either allogenic or xenogenic origin, show excellent hemodynamic behavior and low risk of thromboembolic complications, but their use is limited due to tissue deterioration . Mechanical heart valves have extended durability, but permanent anticoagulation is mandatory . Therefore a new generation heart valve is needed to overcome these limitations, showing the advantages of a healthy viable tissue valve with remodelling, regeneration and growth potential . A concept to create a patient specific viable aortic heart valve can be performed with tissue engineering (te) techniques, in which three essential components are included . First of all a sufficient extracellular matrix, the so - called scaffold, is needed to utilize a three - dimensional structure . These scaffolds are generally made out of polymers or decellularized allo- or xenogenic materials . A sufficient scaffold should fulfil mechanical and biological integrity, provide dynamic and biochemical signals, showing cell attachment and migration, secure diffusion of vital cell nutrients and expressed factors and allow dynamic changes of the scaffold s architecture . The generally used synthetic scaffolds are simple tubes created by polyglycolic acid (pga) or pga in combination with poly-4-hydroxybutyrate (p4hb). The recently developed scaffolds were created from p4hb, including sinuses to support the leaflet closure . Nevertheless none of these te aortic heart valves have yet been clinically implanted and evaluated . The important difference between aortic valves and pulmonary valves is the discrepancy between the thickness of the aortic wall and the pulmonary valve in relation with the leaflet of the valves . Complete decellularization of the aortic wall is therefore more demanding since the leaflets will be more easily decellularized but should not show structural deterioration and need to withstand the systemic blood pressure . Additionally, validated sterilization should be performed on the decellularized scaffolds . In the past allograft irradiation unfortunately, cohen et al . Showed that irradiation completely destroyed the extracellular matrix, with a minimal valve survival and early deterioration of these treated allografts . This study compared the implantation of irradiated allografts (n=41) versus -propiolactone (n=39) treated allografts . At a 15 year follow - up 84% of the irradiated and -propiolactone irradiated heart valves needed to be replaced and therefore only 22% and 12.5% respectively remained implanted . In these patients the dosage of irradiation was 22.5 kgy; however the generally excepted dose to irradiate to sterilize tissue is 25 kgy . . Showed in a study the changes of decellularized tissue matrices by low dosage of irradiation: increase of tensile strength and a decrease of elasticity . The reason for this is the cross - linking of 1 and 2 subunits of the collagen . . Studied the dosage influence of irradiation on tissue, with significant stiffness increase at 1 gy . Leaflet deterioration showed a statistically significant increase in calcium content after 2 weeks of implantation in the subcutaneous rat model, showing 100 gy irradiation compared with 10 gy irradiation, respectively 65157 g / mg vs. 11854 g / mg; p=0.005 . Additionally the optical density values of igg antibodies were also in linear relation with the radiation dose . Important to notice in this study are the 100 times lower doses compared with doses used to sterilize tissue . When an appropriate scaffold is available, valvular cells are needed to allow the heart valve to remodel, regenerate and eventually grow . Compared in vitro seeded and non - seeded heart valves in the sheep model . In this study no evidence was shown to seed te heart valves in the laboratory; however, if decellularized scaffolds were seeded preoperatively the repopulation of the tissue was accelerated . In some studies only vascular endothelial cell were used for seeding; in other studies also interstitial valvular cells were used . Endothelial cells are demanding to multiply in vitro and therefore alternative cell sources are being investigated, such as bone marrow derived cells or umbilical cord cells, to improve the growth rate without compromising cell function . Studies are still going on to identify the optimal cell source if in vitro cell seeding is needed, mostly in synthetic scaffolds . Meanwhile there are some experimental studies performed on te heart valves implanted in the aortic position . Since this is a demanding surgical procedure, with a high mortality rate, there were several implantation techniques performed . . Started implanting decellularized equine pericardium into the descending aorta to investigate the stability of this material under systemic circulation conditions . There was no operative mortality and the authors could prove that there was not only no tissue deterioration but also complete ingrowth of interstitial cells and an overgrowth of endothelial cells . These initial studies were performed to develop a te heart based on decellularized pericardium . Implanted decellularized ovine aortic heart valves in a juvenile sheep model as a root replacement with a maximum follow - up of 9 months . Significant differences between the two groups were seen, since control valves showed massive degeneration and thrombotic formation as early as 3 months after implantation, compared with the decellularized with only minimal calcification at the anastomosis side and micro - thrombotic formation in only one leaflet surface . Functional analyses showed significant differences in aortic valve regurgitation between the decellularized and control group (0.50.5 versus 2.50.0 respectively; p=0.002). Implanted decellularized porcine heart valves in the aortic position, using a subcoronary technique . At explantation, gross examination showed smooth and pliable leaflets with complete recellularization as early as 4 months after implantation . During this study was also able to show that decellularized valve leaflets are able to withstand the systemic circulation . As a control valve a commercially available gluteraldehyde treated carpentier - edwards (edwards lifesciences, irvine, ca) valve was used and compared after 6 months of implantation . The porcine model was used since the anticoagulation system is more similar to the human compared with the juvenile sheep model . The reason for using a stented heart valve is that today 90% of all bioprosthetic valves implanted are stented and in adult patients no growth potential is needed . The study showed no stenosis and no calcification or regurgitation in the decellularized stented aortic heart valves; however the carpentier - edwards was completely destroyed at 6 months of implantation . This study confirmed superiority of the decellularized stented aortic heart valve compared with the commercially available carpentier - edwards valve . Emmert et al . Demonstrated, in an initial report, a transapical approach of implanting a te heart valve transcatheter in the descending aortic position . In the next years meanwhile a limited number of experiences have been published of clinically implanted te aortic valves . These results show not only the resistance of the systemic pressure but additionally regeneration and remodelling potential of these valves . No reoperations due to valve dysfunction were needed at a mean follow - up of 30.35.2 months . Echocardiographic examination showed a mean gradient of 8.86.3 mmhg and maximum aortic valve regurgitation was trivial in all patients . Furthermore panel reactive antibody testing was negative in 19 out of 20 at 1 year follow - up . This group reports no valve related reoperation up to 53 months of follow - up; however, one patient was reoperated on due to severe mitral valve regurgitation . At this time histological examination showed a well preserved extracellular matrix at 18 months of follow - up but also some degree of intimal hyperplasia was seen . Hemodynamic evaluation showed no or trivial regurgitation in all except one patient who showed mild to moderate regurgitation . The mean pressure gradient at discharge (n=34) was 5 mmhg, (range 1 - 17 mmhg) and at the latest follow - up (n=31) 2 mmhg (range 1 - 11 mmhg). Experimental data support the feasibility to implant a te heart valve, based on a decellularized scaffold, into the systemic circulation.
Y - tzp (yttria stabilized tetragonal zirconia polycrystals) are commonly used core materials that are manufactured from fine zirconia (zro2) particles and 1.75 - 3.5 mol.% yttrium oxide (y2o3).1,2,3 y2o3 is commonly used for stabilizing zirconia - based ceramics at room temperature.3,4 however, cerium oxide can also be used to stabilize the tetragonal phase of zirconia.3,5 adding cerium oxide to the zirconia stabilizes its tetragonal phase under chemical and thermal conditions.6 because of the low strength of ce - tzp (ceria stabilized tetragonal zirconia polycrystals), a combination of alumina - zirconia is decided to improve the strength of the material.6,7 recently, a new type of zirconia ceramic, which is called ceria - stabilized tetragonal zirconia / alumina (ce - tzp / al2o3) nanocomposite, has been developed.7,8,9 ce - tzp / al2o3 nanocomposite has an interpenetrated nanostructure and consists of 10 mol% ce - tzp and 30 vol.% al2o3.7,10,11 the homogeneously dispersed al2o3 phase increases the hardness, elasticity modulus, and hydrothermal stability.12,13 evaluating the characteristics under thermocycling and mechanical loading is crucial for the success of zirconia - based restorations because they are exposed to both thermal and mechanical fatigue in the oral environment.14 thermal and mechanical stresses may affect the materials' strength and clinical performance . On the other hand, phase transformations may be triggered by mechanical2 and thermal fatigue;15 and fatigue causes progression of subcritical cracks.2,16 the effect of different aging procedures on ce - tzp / al2o3 has been studied in several investigations.11,15,17,18,19,20 after storing in water and artificial saliva,15 physiological saline solution,11,19 acetic acid11,19 and autoclave,11,15,17,19 ce - tzp / al2o3 showed satisfactory phase and mechanical durability versus to aging.11,19 these experimental conditions do not fully reflect the oral environment . Restorative materials are exposed to saliva, acidification, thermal and mechanical cyclic stresses in the oral cavity . The reported comfortable temperature ranges between 15 and 55.21 fontijn - tekamp et al.22 reported that physiological forces range from 60 to 75 n in the anterior dentition and from 110 to 125 n in the posterior dentition . However, maximum forces ranged from 140 to 170 n, and 250 to 400 n in anterior and posterior regions, recpectively.22 investigating the mechanical properties of the newly developed materials under simulated oral cavity conditions and comparing the results with commonly used core materials are needed for defining their clinical acceptance . The purpose of this study was to evaluate the effect of thermocycling and mechanical loading on the biaxial flexural strength and the phase transformation of ce - tzp / al2o3 and compare the results with two y - tzp core materials . Three groups were designed to investigate the effect of thermocycling and mechanical loading on zirconia - based core materials . Two yttria - stabilized zirconias; cercon base (cercon, degudent, hanau, germany) and lava plus (lava, 3 m espe, seefeld, germany) and one ceria - stabilized zirconia; nanozr (panasonic electric works, osaka, japan) were used in this study (table 1). The dimensions were selected according to iso 687223 and were verified using an electronic digital micrometer (powertechtools, zhejiang, china). Three experimental groups (n = 10) were created from each kind of material . Control specimens were in group 1 and were abbreviated as cc for cercon base, lc for lava plus, and nc for nanozr . Thermocycled specimens were in group 2 and were abbreviated as ct, lt, and nt, for cercon base, lava plus, and nanozr, respectively . Thermocycling was subjected in distilled water at 5 and 55 for 10000 cycles in a thermocycling machine (nve, ankara, turkey). During a cycle, specimens were stored for 30 seconds in each bath.14,15,21 the mechanical loading group was group 3 . For mechanical loading, the specimens were positioned on the supporting balls which were described in iso 6872.23 specimens in group 3 were abbreviated as cm, lm, and nm for cercon base, lava plus, and nanozr, respectively . The mechanical loading was subjected with 200 n loads and a frequency of 2 hz for 100000 times with a mechanical cycler (instron 8801, instron, canton, ma) at the room conditions (22 1, and 60 5% relative humidity).2 the test was carried out after thermocycling and mechanical loading with a tension - compression test machine (middle east technical university, department of metallurgy, ankara, turkey). The specimens were tested with a technique of piston on three balls, which was identified in the standard of iso 6872.23 three hardened steel balls (turkish aerospace industries inc ., ankara, turkey), 3.2 mm in diameter, were placed at an angle of 120 degrees relative to each other to support the specimens . Each specimen was located on these supports of the testing machine and the centers of the specimens were loaded (fig . The load was applied with a flat punch (1.4 mm in diameter) until a fracture occurred . The biaxial flexural strengths of the specimens were calculated with the following formula:23 s = -0.2387 p (x - y) / d, where s is the flexural strength at fracture (mpa); p is the total load causing fracture (n), x = (1 +)ln(r2 / r3) + [(1 -) / 2](r2 / r3), y = (1 +)[1 + ln(r1 / r3)] + (1 -)(r1 / r3), and is poisson's ratio (if the value for the ceramic being studied is not known, poisson's ratio of 0.25 is used); r1 is the radius of the support circle, r2 is the radius of the loaded area (mm), r3 is the radius of the specimen (mm), and d is the specimen thickness at the origin of fracture (mm). For this study, = 0.25, r1 = 5 mm, r2 = 0.7 mm, and r3 = 7.5 mm were used . The weibull modulus, characteristic strength, 10%, 5% and 1% probabilities of failure were calculated using the biaxial flexural strength data . Phase transformation was determined by xrd patterns of the control, thermocycling and mechanical loading specimens before the biaxial flexural strength test . The xrd patterns were recorded with an x - ray diffractometer (d / max 2200pc, rigaku - geirflex x - ray difraktometer, tokyo, japan) by using cu - k -radiation . Specimens were scanned at 40 kv, 40 ma, 0.018/step interval from 20 - 40, and 2 degrees . The relative amount of the monoclinic phase (xm) was calculated with the following formula described by garvie and nicholoson24 for detecting the phase composition of zirconia: xm = (im1 + im2) / (im1 + im2 + it), where i is the intensity detected by the detector, t is the tetragonal peak, and m1 and m2 are the two major monoclinic peaks . The monoclinic phase content was determined by calculating the areas under the t, m1, and m2 peaks with matlab (matlab 2010 a, mattworks, natick, ma, usa). Control, thermocycled, and mechanically loaded specimens were examined by a raman spectrometer (senterra; bruker optics gmbh, ettlingen, germany) before the strength test . The raman laser was focused on center of the specimen (p1), the center of the radius of the specimen (p2), and the edge of the radius of the specimen (p3)(fig . 2) at a wavelength of 520 nm and 20 mw power, 3 cm resolution, and 16 spectral integration times . The raman intensity monoclinic ratio (xm) was calculated with the following formula: xm= i m (180 cm) + i m (190 cm) / i m (180 cm) + i m (190 cm) + it (147 cm) where i corresponds the net peak intensities at the raman shifts . By using the formula of kim et al.,25 the monoclinic fraction (m) was calculated: m = (0.19 - 0.13 / (xm - 1.01)) - 0.56 . The flexural strength data were analyzed by using the two - parameter cumulative weibull distribution, which is often used for ceramic materials because of their asymmetrical distribution . The weibull moduli were calculated with the formula:23,26 p() = 1 - exp [-(/ 0)], where p is the fracture probability, is the fracture strength, 0 is the characteristic strength at the fracture probability of 63.2%, and m is the weibull modulus . In addition, the 10%, 5%, and 1% probabilities of failure were calculated . The relative amount of monoclinic phase was analyzed by two - factor factorial anova (analysis of variance). Significant differences with a significance level of =0.01 (spss 18, spss inc ., the results of the monoclinic fraction were analyzed by three - factor anova with repeated measures on each factor . Significant differences with a significance level of =0.05 (spss 18, spss inc ., the weibull statistical analyses, the characteristic strength, and the 10%, 5%, and 1% probabilities of failure are summarized in table 2 . The characteristic strengths of nanozr were significantly higher in all materials (p<.001), and the differences among the groups in nc, nt, and nm groups were not statistically significant (p>.001). The characteristic strength values of lava plus were higher than cercon base and lower than nanozr . The characteristic strength values increased in the lm group, and it showed significant difference when compared with the lc and lt groups (p<.001). However, the values were not statistically different within the cercon base groups (p>.001).the weibull moduli of nanozr were higher when compared with the other materials . The lava plus values were higher than the cercon base values and lower than the nanozr values . The values increased in the lm group in comparison with the lc and lm groups . The monoclinic phase contents of the materials are shown in table 3 and table 4 . The xrd patterns of one of the specimens in experimental groups are shown in fig . Statistical results of xrd showed that thermocycling and mechanical loading did not affect the monoclinic phase content . The monoclinic phase contents were not statistically different between the control, thermocycled, and mechanically loaded specimens . However the monoclinic phase content of the cercon base, lava plus and nanozr specimens were statistically different (p<.01). 37 - 1484) were detected in all groups except the control and thermal - aged specimens of lava plus . The highest monoclinic phase content was observed in nanozr and it was significantly different from other materials (p<.01). The raman spectroscopy results showed that material type (c, l, and n), the point raman spectra taken from (p1, p2, and p3), and the thermocycling and mechanical loading methods (c, t, and m) affected the monoclinic phase fraction . When comparing the phase fraction of materials at the same point and the same method, except nm at point p1 and all groups in nanozr at points p2 and p3, the differences were not significant in all materials (p>.05). When comparing the points raman spectra taken from the same material and the same method, except for nc and nt groups at point p2, the nm group at point p3, and lm group at points p1 and p2, the differences were not significant in all materials (p>.05). When comparing the experimental groups at the same point and the same material, the mechanical loading groups showed significant differences in all materials (p<.05). Zirconia - based restorations are exposed to temperature changes and cyclic stresses in the oral cavity.14 fatigue of these materials can cause detrimental effects on the mechanical properties of the materials.27 therefore, fatigue tests are essential to define mechanical properties and ensure the clinical success of these materials.27 some studies have stated that fatigue affects the mechanical properties of the zirconia - based core materials.2,11,14 within the limitation of the present study, there were no significant differences in the strength of the core materials after thermocycling and mechanical loading . Nanozr had significantly higher characteristic strength values than the two other y - tzp ceramics and there were no significant differences in the characteristic strength of nc, nt, and nm groups . Nanozr showed a high weibull modulus (m) and demonstrated a low variability ranging from 20.67 to 21.82 . A high weibull modulus defines better clinical reliability of the materials.26,28 the characteristic strength of lm group was significantly higher than that of the lc and lt groups (p<.001). The authors observed a similar increase in the strength of bilayered lava specimens when subjected to 20000 mechanical loadings in a previous study.2 the observed m values for lava plus were 12.36 for the control group and 14.99 for the mechanical loading group . The characteristic strengths of cercon base were not significantly different in all groups (p>.001); the m values of the cercon base were between 5.6 and 7.24 . Nano - meter - sized ce - tzp / al2o3, which has strong mechanical properties and stability against aging, has been developed by nawa et al.10 and its properties have been demonstrated in some studies.9,11,15 ban et al.11 found that nanostructured ce - tzp / al2o3 had durability against aging and there were no significant phase transformation under different storage conditions . Stress - induced phase transformation is an important factor for strengthening tzp.29 in the present study, the biaxial flexural strengths of nanozr groups were not statistically significant from each other and the values in the lm group were significantly higher than in the lt and lc groups (p<.001). Borchers et al.14 researched the influence of different environmental and loading conditions on the biaxial strength of two different 3y - tzp . It was concluded that y - tzp ceramics showed phase transformations after different hydrothermal treatments . However, their strengths were not significantly influenced because the transformation depths did not progress enough from the surfaces into the materials.14 cattani - lorente et al.1 demonstrated similar results and stated that the transformation occurred in a 6 m thick subsurface layer . In the present study, the specimens were subjected to 10000 thermocycles or 100000 mechanical cycles . The specimens were not affected negatively by the cycling conditions; this result was consistent with the fact that the transformation was not deep enough to extend into the material under these thermal and mechanical cycles . In the present study, results of both thermocycling and mechanical loading methods did not significantly affect the phase transformation of the materials . Ylmaz et al.2 reported that the monoclinic phase content of the lava and cercon specimens significantly increased after 20000 mechanical cycles . The cc and lc groups consisted of tetragonal zirconia but the monoclinic contents of the specimens increased after mechanical loading . In the present study, the difference in the monoclinic phase contents of the materials was not statistically significant after thermocycling and mechanical loading . However, the monoclinic phase content of the lm group was significantly higher than that of the lc and lt groups . The difference in the amount of monoclinic phase between the two y - tzp core ceramics may be a result of the difference in the intrinsic structure or sintering schedule.15 the amount of the monoclinic phase of nanozr ranged from 5.917 0.34% to 5.969 0.39% . A similar monoclinic content, ranging from 4.8 to 5.5%, was reported by ban et al.11 perdigo et al.15 evaluated the effect of hydrothermal fatigue on zro2-based (lava, ips and nanozr) materials . The specimens were stored in water, autoclaved or thermocycled in artificial saliva for 30000 thermal cycles . The observed values in the present study were lower than the values presented by perdigo et al.15 although both studies used the same thermocycling conditions, the difference may originate from the higher number of cycles used in the study of perdigo et al.15 the monoclinic phase contents of the zirconia - based materials can be detected with raman spectroscopy . Raman spectroscopy is able to detect very small regions on a specimen surface without preparation30 and can be useful for defining the amount of monoclinic phase.25 sato et al.18 evaluated the mechanical properties of ce - tzp / al2o3 and y - tzp after sandblasting and heat treatment and detected the phase changes with raman spectroscopy . Raman analyses showed that the transformation zone depth was approximately less than 10 m and that the biaxial flexural strength increased after sandblasting but decreased after heat treatment.18 in the present study, the transformation zone depths were not detected by raman spectroscopy but the raman spectra were taken from three different points on the surface of the specimens . In the present study, according to xrd results, the monoclinic phase contents of the materials were not affected by the experiments . However, it was detected that the phase fraction of the materials was significantly affected by thermocycling and mechanical loading when the raman spectra were obtained from different points on the surface of the specimens (p<.05). Furthermore, it was observed that the results were significantly lower in mechanical loading groups at the same point and the same material (p<.05). During mechanical loading, behrens et al.31 stated that applied loads triggered phase transformation and that compressive stresses were higher in the indentation center . The monoclinic phase fractions were found lower at the center of the indent when compared to edge of the indent.31,32 in the present study, specimens were subjected to 100000 mechanical cycles under 200 n and the lower monoclinic phase fraction may be a result of the concentrated compressive stresses on the specimens during mechanical loading . Ce - tzp / al2o3 is a novel material with a high mechanical strength and resistance to fatigue . The results of the study showed that both materials have reliability and can be used clinically . However, further experiments related to the effect of the firing processes, surface treatments on the porcelain connection values of bilayered specimens, and clinical applications are needed to evaluate the long - term behavior of this new material . Within the limitations of this study, the x - ray diffraction results showed that thermocycling and mechanical loading did not have a significant effect on the phase transformation of the tested materials . However, raman spectroscopy results showed that monoclinic phase fraction of the mechanically loaded specimens were lower at the point p1 where the load was applied . Furthermore, it was concluded that the characteristic strengths of all the tested materials were not affected negatively by thermocycling and mechanical loading and all the tested materials have reliability in clinical use.
This plant grows in the first days of summer and used for various attempt (1, 2). Nitrogenic materials, glucose, protein, ferolic acid, quercetin, daucosterol, cirsiliol, steroids and glucomannan are some materials found in salep (3, 4). This plant is used in preparation of drinks, confections and ice cream . On the other hand, it is used as a traditional medicine in various countries including iran . In traditional medicine, it is used for the treatment of diarrhea, cough and impotency (5, 6). Nevertheless, we know that some herbal drugs may cause some adverse effects in some organs, especially liver and kidneys . A systematic review in korea presented the possibility of increased risk of hepatotoxicity by administration of some herbal drugs (7). Despite the fact, some studies showed that some plants may have protective effect for liver such as garlic in nonalchoholic steatohepatitis (nash) (8), or aubutilon indicum in paracetamol induced hepatotoxicity (9). Considering that salep is widely used in some communities, evaluation of the effects of this plant on liver seems to be necessary . The aim of this study was to assess the effect of salep on liver for the first time . Samples of the plant harvested from farmlands around yasouj (a city in the southwest of iran) were obtained . The roots of the plant washed and dried in the laboratory (away from direct light of sun). The powder mixed by ethylic alcohol in 1 to 5 proportions and mixed for 24 hours to yield a uniform solution . In the next stage, the solution filtered and dried for 48 hours to yield the solid extract without alcohol . The final extract mixed with distilled water in 20, 40 and 80 mg / ml and maintained in refrigerator (10). In this study, all the ethic points in working by animals were considered in all steps . The proposal of this study was approved in the research ethic committee of jahrom university of medical sciences . In this study, 50 wistar rats weighting 180 to 200 grams were used . Before starting the study, rats cared in animal house in jahrom university of medical sciences for adaptation to environment . During the study, these animals were in 12 hours light and 12 hours darkness with temperature of 20 - 25c . The first group underwent no intervention; the second group received 1 ml distilled water intraperitoneally daily, each rat in 3rd, 4th and 5th groups received 20, 40 and 80 mg / kg prepared hydroextract, respectively daily ., blood tests for liver function were performed . For this purpose after anesthesia, 5 ml blood received from each rat and after centrifugation in 3000 r / m for 15 minutes serum was separated and used for tests (11). The levels of alt (alanine aminotransferase) and ast (aspartate aminotransferase) were measured by dgkc buffer phosphate method and the level of alp (alkaline phosphatase) measured by -nitrophenyl phosphate amp . Serum albumin and total bilirubin and total protein were measured by the methods of bromocresol green, diazo with sulphanilic acid, and biuret reaction end point, respectively . The level of mad was evaluated by elisa method (biospes italy) and the levels of tac and toc were also measured by elisa (ldn italy) (12). After drawing the blood, liver was separated, weighted and fixed by formalin solution . Statistical analysis was performed using spss software (ibm) and p value below 0.05 considered to be significant . Samples of the plant harvested from farmlands around yasouj (a city in the southwest of iran) were obtained . The roots of the plant washed and dried in the laboratory (away from direct light of sun). The powder mixed by ethylic alcohol in 1 to 5 proportions and mixed for 24 hours to yield a uniform solution . In the next stage, the solution filtered and dried for 48 hours to yield the solid extract without alcohol . The final extract mixed with distilled water in 20, 40 and 80 mg / ml and maintained in refrigerator (10). This study was an experimental study on wistar rats . In this study, all the ethic points in working by animals were considered in all steps . The proposal of this study was approved in the research ethic committee of jahrom university of medical sciences . In this study, 50 wistar rats weighting 180 to 200 grams were used . Before starting the study, rats cared in animal house in jahrom university of medical sciences for adaptation to environment . During the study, these animals were in 12 hours light and 12 hours darkness with temperature of 20 - 25c . The first group underwent no intervention; the second group received 1 ml distilled water intraperitoneally daily, each rat in 3rd, 4th and 5th groups received 20, 40 and 80 mg / kg prepared hydroextract, respectively daily . In 29th day of study, blood tests for liver function were performed . For this purpose after anesthesia, 5 ml blood received from each rat and after centrifugation in 3000 r / m for 15 minutes serum was separated and used for tests (11). The levels of alt (alanine aminotransferase) and ast (aspartate aminotransferase) were measured by dgkc buffer phosphate method and the level of alp (alkaline phosphatase) measured by -nitrophenyl phosphate amp . Serum albumin and total bilirubin and total protein were measured by the methods of bromocresol green, diazo with sulphanilic acid, and biuret reaction end point, respectively . The level of mad was evaluated by elisa method (biospes italy) and the levels of tac and toc were also measured by elisa (ldn italy) (12). After drawing the blood, liver was separated, weighted and fixed by formalin solution . Statistical analysis was performed using spss software (ibm) and p value below 0.05 considered to be significant . Liver enzymes evaluation; the levels of ast, alt and alp significantly decreased with administration of higher doses of salep (80 mg / kg). This effect was found in bilirubin mda and toc levels . On the other hand, it seems that with administration of salep the level of total serum protein and albumin elevated, especially with the highest dose . Abbreviations: alt, alanine transferase; alp, alkaline phosphatase; ast, aspartate transferase; g / dl, gram per deciliter; iu / l, internation unit per liter; mda, malondialdehyde; tac, total antioxidation capacity; and toc, total oxidation capacity . Pathology of liver tissue evaluated and there was no sign of edema, irregularity of the hepatocells, vein engorgement, necrosis, accumulation of kupfer cells, change in portal area and infiltration of inflammatory cells . In short, no significant effect on liver tissue was revealed by various doses (20, 40, or 80 mg / kg) of salep (figure 1). Pathology of liver tissue evaluated and there was no sign of edema, irregularity of the hepatocells, vein engorgement, necrosis, accumulation of kupfer cells, change in portal area and infiltration of inflammatory cells . In short, no significant effect on liver tissue was revealed by various doses (20, 40, or 80 mg / kg) of salep (figure 1). Injection of salep extract could decrease the level of liver function enzymes including ast, alt and alp . In liver damage, the level of these enzymes would be increased (13). This effect may be due to the presence of anti - oxidant material in this plant . Polyphenols and flavonoid components such as quercetin are important anti - oxidants in this plant (14). These components have protective effect on the liver against toxins and free radicals (15, 16). It is shown that ferolic acid has protective effect for liver against toxic materials such as alcohol and high fat foods . In these studies it was shown that ferolic acid can effectively reduce the level of alt, ast and alp in rats, which received alcohol and high fat foods (17). Glucomannan is a fiber, which is water soluble and can inhibit stress oxidation and reduce ast and alt levels (18, 19). This fiber is also effective on reducing the blood sugar, cholesterol and body weight (20, 21). One of the signs of progression of chronic liver disease is decreased level of total protein and albumin . The level of this deduction is proportionate with the severity of liver damage (22, 23). In this study, we showed that the level of total protein and albumin could be elevated by salep . Mad is one of the important criteria for lipid peroxidation and is an indicator for liver damage (24). Tac (total antioxidant capacity) is a better criterion than gpx, cat and sod for evaluating the anti - oxidation condition in the body (25), this capacity has a reverse correlation with toc (26). Augmentation of tac and reduction of toc and mad by salep may be due to antioxidants in this plant . This action is performed by elevation of antioxidative enzymes such as glutathione, glutathione reductase, glutathione peroxidase and catalase (27). Reduced glutathione can diminish the oxidized form of glutathione peroxidase, which in turn can diminish hydrogen peroxide (h2o2) as a dangerous reactive component within the cell . This component can elevate the level of sod (supper oxide dismutase) and catalase enzymes ., salep may have a protective effect on liver in animal models and more studies are needed to evaluate its effect in human.
When evaluating patients for clinical trial, intrauterine pregnancy is commonly considered an exclusion criteria, however guidelines do not exist to determine what to do should a pregnancy test result as positive . The patient underwent standard - of - care therapy upfront with progression of disease and was referred to investigational therapeutics for phase i treatment . Pelvic ultrasound and -hcg heterophilic antibody testing were negative ruling out intrauterine and ectopic pregnancy or phantom hcg . Her elevated -hcg appeared consistent with paraneoplastic syndrome reflected in a decline in -hcg that correlated with a response to treatment . A 34-year - old african american female with metastatic mucinous ovarian carcinoma presented with a pelvic mass discovered during her annual gynecological examination . An ultrasound showed a 7.7 8.7 cm left ovarian mass with complex appearance . Laparoscopic left salpingo - oophorectomy showed a moderately differentiated mucinous ovarian carcinoma, stage ic1, due to intraoperative rupture of the ovarian mass . Tumor cells were diffusely positive for keratin 7 and very focally positive for keratin 20 . The patient was taken for diagnostic laparoscopy with appendectomy, omentectomy, and multiple peritoneal biopsies by a gynecologic oncologist; all of which were negative for malignancy . Eight weeks later the patient started adjuvant chemotherapy with one cycle of capecitabine and oxaliplatin (xelox), transitioned to four months of fluorouracil and oxaliplatin (folfox) due to insurance issues . The ct abdomen and pelvis post folfox chemotherapy showed an enhancing 2.6 cm nodule in rectus abdominis muscle . The patient underwent an exploratory laparotomy with excision of a 5 6 cm abdominal mass involving fascia and muscle, consistent with metastatic adenocarcinoma . After recovery, the patient received chemotherapy with carboplatin and paclitaxel for 6 months . The ct abdomen and pelvis performed following 6 months of carboplatin and paclitaxel showed progression of disease with new nodal and perihepatic implants . The patient subsequently consulted with the md anderson cancer center department of investigational cancer therapeutics for phase i clinical trial options . The patient was treated with a phosphatidylinositol-4,5-bisphosphate 3-kinase (pi3k) inhibitor plus a mitogen / extracellular signal - regulated kinase (mek) inhibitor with progression of disease after 5 months . She then started on a second phase i trial with a polymeric micellar nanoparticle of the substance dach - pt (1,2-diaminocyclohexane platinum), an in vivo metabolite of oxaliplatin, with progression following 1 cycle of therapy . The patient was then enrolled in a trial with paclitaxel, bevacizumab and temsirolimus (pat). On the day of clearance to start treatment, she was found to have a positive urine and serum pregnancy test . The left ovary was surgically absent however the right ovary was enlarged with complex cystic mass with solid components measuring 9.5 6.6 cm . The work - up for false positive elevation of -hcg including serial dilutions, incorporation of animal serum and outside institutional validation testing for -hcg heterophile antibody, was negative . Therefore, the -hcg elevation was not thought to be related to a phantom hcg . Dilation and curettage to rule out trophoblastic disease was planned, but not completed due to discovery of a deep vein thrombosis and initiation of therapeutic anticoagulation . As no intrauterine or extrauterine pregnancy could be identified, after several weeks, the patient returned to clinic and was cleared to start treatment . At that time, her -hcg was 210.3 . Three weeks post treatment with pat, the -hcg decreased to 27.5 and ct scans showed partial response (fig ., the patient was admitted to the intensive care unit for weakness due to guillain barre syndrome . Nearly all female patients undergoing enrollment on clinical trial are evaluated for pregnancy as part of standard exclusion criteria . An elevated -hcg in the absence of viable pregnancy can occur for multiple reasons and has a broad differential diagnosis including miscarriage, ectopic pregnancy, pituitary hcg production, trophoblastic disease and phantom hcg . Ectopic pregnancy is frequently suspected when -hcg levels plateau or fail to double within 48 h without evidence of intrauterine pregnancy with ultrasound . When intrauterine and extrauterine pregnancy are ruled out, pituitary hcg may be produced in perimenopausal or postmenopausal women . As estrogen and progesterone production decreases, releasing gonadotropin releasing hormone (gnrh) from negative feedback, luteinizing hormone (lh) and follicular stimulating hormone (fsh) rise . The subunit gene of lh is found in a sequence of 7 hcg subunit genes and therefore uncontrolled gnrh stimulation may lead to hcg production by pituitary gonadotrope cells (cole, 2005). Using an fsh level of 45.0 miu / ml identified hcg of perimenopausal women, ages 4155 years, with mildly increased serum hcg concentrations (5.014.0 miu / ml), an fsh cutoff of 45.0 miu / ml identified hcg of placental origin with 100% sensitivity and 75% specificity . Fsh levels greater than 45 miu / ml were never observed when hcg was of placental origin . When hcg is less than 5.0 miu / ml, fsh testing is not performed as pregnancy is unlikely . When hcg is greater than 14.0 miu / ml, fsh testing is not performed as this is consistent with pregnancy (gronowski et al ., 2008). In one study, two weeks of estrogen - progesterone therapy suppressed pituitary hcg and may help confirm diagnosis (cole et al ., 2007). Gestational trophoblastic disease is a group of conditions that may be classified as either premalignant, in the case of a partial or complete hydatidiform mole, or malignant, including invasive mole, choriocarcinoma, placental - site trophoblastic tumor and epithelioid trophoblastic tumor . Although not always the case, -hcg is commonly found to be elevated in these conditions and these diseases are considered high on the differential diagnosis list when found . Phantom hcg is a phenomenon first described in 1998 by laurence cole, ph.d ., where patients have persistent mild elevations of -hcg, occasionally following miscarriage . The elevated -hcg often falsely interpreted and misleads physicians to evaluate or incorrectly treat patients with cytotoxic agents for trophoblast disease (cole, 1998). Heterophilic antibodies against mouse, goat, rabbit, cow, horse, or sheep antigens can interfere with two - site immunoassays by creating complexes between the two anti--hcg antibodies without -hcg being present . This leads to a false - positive result (vladutiu et al ., 1982). People can develop heterophilic antibodies by exposure to the serum, tissue, or other antigens of nonhuman species . Heterophilic antibodies do not appear to be excreted in the urine and therefore urine assays can be used to identify false - positive serum results . Serial dilutions may also be performed to assess for interference . To avoid false positives, the excess of nonspecific antibodies saturates heterophilic antibodies in human serum and usually eliminates their interference with the assay (johnson et al ., 2000). Additionally, serum samples can be sent to outside laboratories with different assay systems to validate results . In our patient's case, the patient continued to have normal menstrual cycles and was using two forms of contraception with her husband who had history of vasectomy . The work up, including pelvic ultrasound and ct scans, was negative for pregnancy and trophoblastic disease . It was therefore believed that the patient's -hcg production was due to the patient's tumor, consistent with a paraneoplastic syndrome . Paraneoplastic syndromes occur in about 8% of cancer patients and frequently develop with advanced disease . They may appear earlier than symptoms of the primary tumor itself, leading to new diagnosis of cancer (pelosof and gerber, 2010). It can also be found in the testis, liver, lung, colon, and stomach in small amounts . Malignancies in these organs may lead to an increased serum levels (demirtas et al . A gastric origin is the most frequent, ranging from 11% to 17% of this rare subset (germann et al ., 2002). In our case, the patient had a primary ovarian neoplasm with metastases within the abdomen . -hcg levels rose while the patient was on a treatment break, nearly impeding her from further treatment . With re - initiation of therapy, the -hcg declined, corresponding to a partial response per response evaluation criteria in solid tumors (recist) on radiographic imaging . Urine hcg remained positive despite decline in serum hcg as the level was still over the detection threshold of 25 interesting enough, other tumor markers (ca 125, ca 15 - 3, ca 19.9 and cea) all increased . The decreased size of tumor within the pelvis corresponded with decreasing -hcg levels while increased hepatic metastasis corresponded to increases in other markers . This lends evidence to the idea that the patient had tumoral heterogeneity with different markers produced by tumors in different areas of the body . This is the first case of paraneoplastic -hcg production from an ovarian adenocarcinoma reported in the literature . We provide a treatment algorithm to evaluate for paraneoplastic hcg production when assessing for clinical trial eligibility (fig . Paraneoplastic syndrome should remain in the differential diagnosis in patients found to have elevated levels of -hcg without evidence of intra- or extra - uterine pregnancy . Informed consent to publish the information was granted from the patient . None of the authors have conflict of interest or competing interest . Jg participated in the design of the manuscript, analyzed the biomarker data and drafted the manuscript . Pp jg participated in the design of the manuscript, analyzed the biomarker data and drafted the manuscript . Sp conceived of the study, and participated in its design and coordination and helped to draft the manuscript.
Epidemiological studies during the past decade have shown a positive correlation between the general consumption of cruciferous vegetables and decreased incidence of some cancers, including lung, stomach, colon, breast,[23] bladder,[45] prostate,[67] and non - hodgkin lymphoma . Lung cancer risk is reduced especially by dietary intake of isothiocyanates or cruciferous vegetables in persons with genotypes of glutathione s - transferase mu 1-null and glutathione s - transferase theta 1-null, highlighting an important interplay between genetic susceptibility factors and chemopreventive agents. [1910] the cancer chemopreventive effect of cruciferous vegetables is due to glucosinolates . Sulforaphane (sfn) is formed by enzymatic transformation of a glucosinolate, glucoraphanin, present in cruciferous vegetables, during chewing or through conversion through the gut microflora . Sfn acts as a chemopreventive agent by inducing phase - ii enzymes by activating the nuclear factor erythroid-2 related factor 2 (nrf2) pathway. [1213] evidence is mounting that sfn acts through other chemopreventive mechanisms such as blocking cancer initiation via inhibition of cytochrome p-450-dependent monoxygenases that convert procarcinogens into active carcinogens and modulating signaling pathways that regulate cell growth and apoptosis to suppress cancer progression. [1415] several groups have reported that sfn is effective in preventing chemical carcinogen - induced breast, stomach, pancreas, and colon cancer, as well as decreased polyp formation in apc mice . In the lungs, sfn and its n - acetylcysteine conjugate inhibit malignant progression of adenomas induced by tobacco carcinogens . In addition to its anticarcinogenic effect, sfn treatment inhibits the growth of established prostate,[2123] breast, pancreas,[2527] and lung cancer xenografts in mice . Preclinical study results have also shown that sfn - mediated activation of nrf2 improves bacterial clearance and corticosteroid sensitivity in mice with chronic cigarette smoke exposure. [2930] several clinical trials are underway for evaluating sfn for treatment of various diseases (www.clinicaltrials.gov). Somatic loss of function mutations in kelch - like ech - associated protein 1 (keap1) and oncogenic gain of function mutations in nrf2 have been reported in lung, prostate, skin, esophagus, gallbladder, and ovarian cancer . More recently, oncogenic activation of k - ras, myc, and b - raf have been shown to activate nrf2 signaling, and this cross talk has been shown to be important for oncogene - induced senescence evasion and tumorigenesis. [3637] for future development of sfn as a chemopreventive or therapeutic agent, we have evaluated the risk of long - term sfn administration on lung tumorigenesis by using oncogenic k - ras mouse model and human lung cancer xenograft mouse models . D, l - sulforaphane was purchased from toronto research chemicals, ontario (cat#s699115) and stored at -20c . The sfn was freshly diluted in phosphate - buffered saline (pbs) before treatment . A549 and h1975 cells were purchased from american type culture collection (manassas, virginia, united states) and cultured under recommended conditions . Cells were cultured in dulbecco modified eagle medium (invitrogen, carlsbad, california, united states) containing 10% fetal bovine serum and 1% penicillin streptomycin . Ad - cmv - cre (1 10 pfu / ml) was obtained from vector biolabs . Kras (strain number 01xj6) mice were obtained from a mouse repository (nci, frederick) and were bred in our facility . Athymic ncr - nu / nu (strain number 01b74) nude mice (6 - 8 weeks) were obtained from nci . Mice were fed the 2018sx diet (harlan teklad) and had access to water ad libitum; they were housed under controlled conditions (232c; 14-h light/ 10-h dark cycles). All experimental protocols conducted on the mice were performed in accordance with national institutes of health guidelines and were approved by the johns hopkins university animal care and use committee . Eight - week - old kras mice were anesthetized with ketamine (100 mg / kg of body weight) and xylazine (10 mg / kg of body weight). Five million adenovirus cre - recombinase particles diluted in 100 l of minimal essential medium were administered by oropharyngeal aspiration (50 l, twice at an interval of 5 min) according to the standard protocol . Five days after adenovirus cre treatment, mice were randomly placed into one of the two groups . Sfn (0.5 mg) suspended in 50 l of pbs was administered 5 d / wk for 14 weeks by using a nebulizer (aeroneb lab) in the sfn group (n = 9). The lesions were assessed subjectively for the percentage of lesion area to total lung section area . The lesions were categorized as mild hyperplasia, severe hyperplasia, adenoma, or adenocarcinoma . Either a549 cells (5 10) or h1975 cells (1 10) were injected subcutaneously into the right flank of the anesthetized athymic nude mice . When the volume of the tumor reached 50 20 mm, the mice were separated randomly into different treatment groups, and sfn treatment was started . The mice were weighed and the tumor dimensions were measured with calipers once a week until 1 week before harvesting of the tumor . The tumor volumes were calculated by using the following formula: length (mm) width (mm) width (mm) 0.52 . The subcutaneous sfn injections were administered 0.51 cm adjacent to the left side of the tumor . The control mice in all the experiments received the same volume of pbs as did their respective cohorts . The mice were euthanized by means of cervical dislocation, and the tumors were removed without skin and weighed . Design for xenograft experiments total rna was extracted from tissues and cells by using the rneasy kit (qiagen) and was quantified by means of ultraviolet absorption spectrophotometry . The reverse transcription reaction was performed by using a high - capacity cdna synthesis kit (applied biosystems, foster city, california, united states) and the multiscribe first strand synthesis system (applied biosystems, foster city, california, united states) in a final volume of 20 l containing 1 g of total rna, 100 ng of random hexamers, 1 reverse transcription buffer, 1 mm deoxyribonucleotide triphosphate, multiscribe reverse transcriptase, and nuclease - free water . A quantitative real - time polymerase chain reaction analysis of nad(p)h dehydrogenase, quinone 1 (nqo1), heme oxygenase 1(ho-1), and glutamate - cysteine ligase, catalytic subunit (gclc) was performed by using assay - on - demand primers and probe sets from applied biosystems . Tumor volume, tumor weight, and body weight were measured and presented as mean plus or minus standard error and compared by means of a t - test or anova . D, l - sulforaphane was purchased from toronto research chemicals, ontario (cat#s699115) and stored at -20c . The sfn was freshly diluted in phosphate - buffered saline (pbs) before treatment . A549 and h1975 cells were purchased from american type culture collection (manassas, virginia, united states) and cultured under recommended conditions . Cells were cultured in dulbecco modified eagle medium (invitrogen, carlsbad, california, united states) containing 10% fetal bovine serum and 1% penicillin streptomycin . Ad - cmv - cre (1 10 pfu / ml) was obtained from vector biolabs . Kras (strain number 01xj6) mice were obtained from a mouse repository (nci, frederick) and were bred in our facility . Athymic ncr - nu / nu (strain number 01b74) nude mice (6 - 8 weeks) were obtained from nci . Mice were fed the 2018sx diet (harlan teklad) and had access to water ad libitum; they were housed under controlled conditions (232c; 14-h light/ 10-h dark cycles). All experimental protocols conducted on the mice were performed in accordance with national institutes of health guidelines and were approved by the johns hopkins university animal care and use committee . Eight - week - old kras mice were anesthetized with ketamine (100 mg / kg of body weight) and xylazine (10 mg / kg of body weight). Five million adenovirus cre - recombinase particles diluted in 100 l of minimal essential medium were administered by oropharyngeal aspiration (50 l, twice at an interval of 5 min) according to the standard protocol . Five days after adenovirus cre treatment, mice were randomly placed into one of the two groups . Sfn (0.5 mg) suspended in 50 l of pbs was administered 5 d / wk for 14 weeks by using a nebulizer (aeroneb lab) in the sfn group (n = 9). The lesions were assessed subjectively for the percentage of lesion area to total lung section area . The lesions were categorized as mild hyperplasia, severe hyperplasia, adenoma, or adenocarcinoma . Either a549 cells (5 10) or h1975 cells (1 10) were injected subcutaneously into the right flank of the anesthetized athymic nude mice . When the volume of the tumor reached 50 20 mm, the mice were separated randomly into different treatment groups, and sfn treatment was started . The mice were weighed and the tumor dimensions were measured with calipers once a week until 1 week before harvesting of the tumor . The tumor volumes were calculated by using the following formula: length (mm) width (mm) width (mm) 0.52 . The subcutaneous sfn injections were administered 0.51 cm adjacent to the left side of the tumor . The control mice in all the experiments received the same volume of pbs as did their respective cohorts . The mice were euthanized by means of cervical dislocation, and the tumors were removed without skin and weighed . Total rna was extracted from tissues and cells by using the rneasy kit (qiagen) and was quantified by means of ultraviolet absorption spectrophotometry . The reverse transcription reaction was performed by using a high - capacity cdna synthesis kit (applied biosystems, foster city, california, united states) and the multiscribe first strand synthesis system (applied biosystems, foster city, california, united states) in a final volume of 20 l containing 1 g of total rna, 100 ng of random hexamers, 1 reverse transcription buffer, 1 mm deoxyribonucleotide triphosphate, multiscribe reverse transcriptase, and nuclease - free water . A quantitative real - time polymerase chain reaction analysis of nad(p)h dehydrogenase, quinone 1 (nqo1), heme oxygenase 1(ho-1), and glutamate - cysteine ligase, catalytic subunit (gclc) was performed by using assay - on - demand primers and probe sets from applied biosystems . Tumor volume, tumor weight, and body weight were measured and presented as mean plus or minus standard error and compared by means of a t - test or anova . We determined whether chronic sfn administration affects oncogenic k - ras (k - ras)-driven lung tumorigenesis . The k - ras mutant mouse develops progressively less differentiated lung tumors after the administration of adenoviral cre - recombinase . In this study, we evaluated the effect of sfn on k - ras mediated lung tumorigenesis post oncogenic k - ras activation . One week after administration of a high titer adenoviral cre to induce mutant k - ras expression, 1 cohort of mice was treated with sfn (0.5 mg, 5 d / wk) for 3 months by means of a nebulizer [figure 1a]. We have reported that sfn (0.5 mg / mice) administered using nebulizer significantly induces nrf2-dependent gene expression in lungs . Fourteen weeks after adenovirus - cre - recombinase administration and mutant k - ras activation, mice were sacrificed, and lung sections were screened for neoplastic foci . Histological analyses of the lung sections from the k - ras mutant mice showed the presence of atypical adenomatous hyperplasia and adenomas with infiltration of inflammatory cells . Although tumor number, histological subtypes, and grades were not significantly different between the two groups, we noticed a trend toward reduced tumor burden in the sfn - treated cohort of mice [figure 1b]. Extended exposure to sfn transcriptionally induced the expression of the nrf2 target genes nqo1, gclc, and ho-1 in the lungs [figure 1c]. Thus, our data demonstrate that prolonged exposure to sfn via aerosol inhalation does not promote or increase tumorigenesis in the mutant k- ras - driven mouse model of lung tumorigenesis . (a) schematic of the experimental design . (b) bar graph showing relative number of neoplastic lesions (hyperplasia, adenoma, adenocarcinoma) and percentage of the area of the lung covered by the tumor cells . (c) real - time rt - pcr data showing induction of nrf2-dependent gene expression in the lungs of the sfn - treated cohort of mice . P - values are calculated using t test to evaluate the effect of prolonged sfn treatment on the growth of established lung cancer xenografts, we selected two cell lines, a549 and h1975 . A549 cells with a mutant k - ras and keap1 allele, and h1975 with a mutant epidermal growth factor receptor (egfr) allele, are the two commonly used lung cancer xenograft models . A549 and h1975 cells were injected into the flanks of nude mice, and change in tumor volume was recorded . Once the tumor volume reached 50 20 mm, mice were randomly allocated into two groups, and sfn treatment was initiated [figures 2a and 3a]. Mice were treated with vehicle (pbs) or sfn by means of intraperitoneal injection (5 d / wk), and tumor volume was measured weekly . Sfn dose was selected based on previously published results where sfn administration at a dose 250400 mol / kg body weight, has been reported to be well tolerated . We did not notice a significant reduction in the tumor growth rate / weight of a549 xenografts treated with 3 mol dose of sfn, and thus, we increased the sfn dose and repeated the experiment with 3 and 6 mol . Again, sfn treatment at a dose of 3 or 6 mol did not inhibit the growth of a549 tumors significantly, although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [figures 2b and c and 3b and c). For h1975 cells expressing mutant egrf allele, we increased the sfn dose further (9 mol) since these tumors proliferate rapidly in vivo . Similar to our results with a549 tumors, sfn at a dose 9 mol did not attenuate the growth of h1975 xenografts significantly, although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [figures 3b and 3c]. To ascertain whether systemic sfn administration causes weight loss, we recorded body weights of the control and treated mice . Average body weights of the control and sfn - treated mice did not differ significantly throughout the treatment protocol [figures 2d and 3d]. Systemic delivery of sfn does not promote growth of keap1 and k - ras mutant, a549, lung cancer xenografts . Tumor volume is not significantly different between the three cohorts of mice (anova). (e) real - time rt - pcr showing expression of the nrf2-dependent gene, nqo1, in the liver and tumor tissues. p <0.05 (t test). Systemic delivery of sfn does not promote growth of egfr mutant (h1975) lung cancer xenografts . Tumor volume is not significantly different between the two cohorts of mice (t test). (c) tumor weights (day 30) are not significantly different between the two groups (t test). (e) real - time pcr showing expression of the nrf2-dependent gene, nqo1, in the liver and tumor tissues. p <0.05 (t test) to demonstrate that systemic delivery of sfn induced nrf2-dependent target gene expression in mice, we measured nqo1 expression in the liver [figures 2e and 3e]. In addition to the liver, we also measured the induction of nrf2 signaling in a549 and h1975 tumors . However, sfn treatment did not upregulate nrf2 signaling in tumors [figures 2e and 3e]. These data indicate that systemic sfn treatment induces nrf2 signaling but does not promote growth of lung cancer xenografts . To determine whether localized delivery of sfn that results in higher accumulation of sfn in the tumor and neighboring areas can retard the growth of subcutaneous tumors, we selected a549 cells as the model system . A549 cells were injected into the flanks of nude mice, and change in tumor volume was recorded . Once the tumor volumes reached 50 20 mm, mice were randomly allocated into 2 groups, and treatment groups were administered sfn (1 mol, 5d / wk) by means of subcutaneous injection adjacent to the tumor [figure 4a]. The average change in tumor volume was significantly different between the vehicle- and sfn - treated tumors (p <0.05) [figure 4b]. Tumor weights were significantly higher in the vehicle - treated tumors than in the sfn - treated tumors (p = 0.05) [figure 4c], with no apparent difference in total body weight [figure 4d] between the two groups . Graph showing relative growth of a549 tumors treated with 1000 nmol of sfn . * p <0.05 (t test). (c) tumor weights were significantly lower in the sfn - treated group . * p <0.05 (t test). (e g) relative tumor growth, average tumor weight, and body weight in the cohort of mice treated with 500 nmol of sfn . (h j) relative expression of nrf2-dependent genes in the liver and tumor tissues . * significant inhibition of tumor growth by sfn at a dose of 1 mol prompted us to determine whether sfn exerts a similar effect at a lower dose (0.5 mol) as well . Treatment of a549 xenograft tumors with a lower dose of sfn significantly retarded the growth of a549 tumors in nude mice . Average tumor volume and weight were significantly lower in the sfn - treated cohort of mice [figures 4e and 4f]. There was no adverse effect of localized sfn treatment on animal health or body weight, although we noticed slight thickening of skin in the area surrounding the injection spot [figure 4 g]. To demonstrate that localized delivery of sfn induced nrf2-dependent target gene expression, we measured nqo1, ho-1, and gclc expression in the skin and liver . In addition to these two tissues, we measured the induction of nrf2 signaling in a549 tumors . Sfn treatment significantly induced the expression of ho-1 in the skin with no apparent effect on nrf2-dependent gene expression in the tumors [figures 4h j]. Since sfn was administered locally, we did not notice significant change in nrf2 dependent gene expression in the liver . These data indicate that localized sfn treatment probably results in greater accumulation of sfn in subcutaneous tumors leading to significant growth inhibition . We determined whether chronic sfn administration affects oncogenic k - ras (k - ras)-driven lung tumorigenesis . The k - ras mutant mouse develops progressively less differentiated lung tumors after the administration of adenoviral cre - recombinase . In this study, we evaluated the effect of sfn on k - ras mediated lung tumorigenesis post oncogenic k - ras activation . One week after administration of a high titer adenoviral cre to induce mutant k - ras expression, 1 cohort of mice was treated with sfn (0.5 mg, 5 d / wk) for 3 months by means of a nebulizer [figure 1a]. We have reported that sfn (0.5 mg / mice) administered using nebulizer significantly induces nrf2-dependent gene expression in lungs . Fourteen weeks after adenovirus - cre - recombinase administration and mutant k - ras activation, mice were sacrificed, and lung sections were screened for neoplastic foci . Histological analyses of the lung sections from the k - ras mutant mice showed the presence of atypical adenomatous hyperplasia and adenomas with infiltration of inflammatory cells . Although tumor number, histological subtypes, and grades were not significantly different between the two groups, we noticed a trend toward reduced tumor burden in the sfn - treated cohort of mice [figure 1b]. Extended exposure to sfn transcriptionally induced the expression of the nrf2 target genes nqo1, gclc, and ho-1 in the lungs [figure 1c]. Thus, our data demonstrate that prolonged exposure to sfn via aerosol inhalation does not promote or increase tumorigenesis in the mutant k- ras - driven mouse model of lung tumorigenesis . (a) schematic of the experimental design . (b) bar graph showing relative number of neoplastic lesions (hyperplasia, adenoma, adenocarcinoma) and percentage of the area of the lung covered by the tumor cells . (c) real - time rt - pcr data showing induction of nrf2-dependent gene expression in the lungs of the sfn - treated cohort of mice . To evaluate the effect of prolonged sfn treatment on the growth of established lung cancer xenografts, we selected two cell lines, a549 and h1975 . A549 cells with a mutant k - ras and keap1 allele, and h1975 with a mutant epidermal growth factor receptor (egfr) allele, are the two commonly used lung cancer xenograft models . A549 and h1975 cells were injected into the flanks of nude mice, and change in tumor volume was recorded . Once the tumor volume reached 50 20 mm, mice were randomly allocated into two groups, and sfn treatment was initiated [figures 2a and 3a]. Mice were treated with vehicle (pbs) or sfn by means of intraperitoneal injection (5 d / wk), and tumor volume was measured weekly . Sfn dose was selected based on previously published results where sfn administration at a dose 250400 mol / kg body weight, has been reported to be well tolerated . We did not notice a significant reduction in the tumor growth rate / weight of a549 xenografts treated with 3 mol dose of sfn, and thus, we increased the sfn dose and repeated the experiment with 3 and 6 mol . Again, sfn treatment at a dose of 3 or 6 mol did not inhibit the growth of a549 tumors significantly, although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [figures 2b and c and 3b and c). For h1975 cells expressing mutant egrf allele, we increased the sfn dose further (9 mol) since these tumors proliferate rapidly in vivo . Similar to our results with a549 tumors, sfn at a dose 9 mol did not attenuate the growth of h1975 xenografts significantly, although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [figures 3b and 3c]. To ascertain whether systemic sfn administration causes weight loss, we recorded body weights of the control and treated mice . Average body weights of the control and sfn - treated mice did not differ significantly throughout the treatment protocol [figures 2d and 3d]. Systemic delivery of sfn does not promote growth of keap1 and k - ras mutant, a549, lung cancer xenografts . Tumor volume is not significantly different between the three cohorts of mice (anova). (e) real - time rt - pcr showing expression of the nrf2-dependent gene, nqo1, in the liver and tumor tissues. p <0.05 (t test). Systemic delivery of sfn does not promote growth of egfr mutant (h1975) lung cancer xenografts . Tumor volume is not significantly different between the two cohorts of mice (t test). (c) tumor weights (day 30) are not significantly different between the two groups (t test). (e) real - time pcr showing expression of the nrf2-dependent gene, nqo1, in the liver and tumor tissues. p <0.05 (t test) to demonstrate that systemic delivery of sfn induced nrf2-dependent target gene expression in mice, we measured nqo1 expression in the liver [figures 2e and 3e]. In addition to the liver, we also measured the induction of nrf2 signaling in a549 and h1975 tumors . However, sfn treatment did not upregulate nrf2 signaling in tumors [figures 2e and 3e]. These data indicate that systemic sfn treatment induces nrf2 signaling but does not promote growth of lung cancer xenografts . To determine whether localized delivery of sfn that results in higher accumulation of sfn in the tumor and neighboring areas can retard the growth of subcutaneous tumors, we selected a549 cells as the model system . A549 cells were injected into the flanks of nude mice, and change in tumor volume was recorded . Once the tumor volumes reached 50 20 mm, mice were randomly allocated into 2 groups, and treatment groups were administered sfn (1 mol, 5d / wk) by means of subcutaneous injection adjacent to the tumor [figure 4a]. The average change in tumor volume was significantly different between the vehicle- and sfn - treated tumors (p <0.05) [figure 4b]. Tumor weights were significantly higher in the vehicle - treated tumors than in the sfn - treated tumors (p = 0.05) [figure 4c], with no apparent difference in total body weight [figure 4d] between the two groups . (b) graph showing relative growth of a549 tumors treated with 1000 nmol of sfn . * p <0.05 (t test). (c) tumor weights were significantly lower in the sfn - treated group . * p <0.05 (t test). (e g) relative tumor growth, average tumor weight, and body weight in the cohort of mice treated with 500 nmol of sfn . (h j) relative expression of nrf2-dependent genes in the liver and tumor tissues . * p <0.05 . Significant inhibition of tumor growth by sfn at a dose of 1 mol prompted us to determine whether sfn exerts a similar effect at a lower dose (0.5 mol) as well . Treatment of a549 xenograft tumors with a lower dose of sfn significantly retarded the growth of a549 tumors in nude mice . Average tumor volume and weight were significantly lower in the sfn - treated cohort of mice [figures 4e and 4f]. There was no adverse effect of localized sfn treatment on animal health or body weight, although we noticed slight thickening of skin in the area surrounding the injection spot [figure 4 g]. To demonstrate that localized delivery of sfn induced nrf2-dependent target gene expression, we measured nqo1, ho-1, and gclc expression in the skin and liver . In addition to these two tissues, we measured the induction of nrf2 signaling in a549 tumors . Sfn treatment significantly induced the expression of ho-1 in the skin with no apparent effect on nrf2-dependent gene expression in the tumors [figures 4h j]. Since sfn was administered locally, we did not notice significant change in nrf2 dependent gene expression in the liver . These data indicate that localized sfn treatment probably results in greater accumulation of sfn in subcutaneous tumors leading to significant growth inhibition . Since the early 1980s, naturally occurring antioxidants and vitamins have been investigated and promoted as chemopreventive agents . Antioxidants have been popular as chemopreventive agents because there is strong scientific evidence supporting reduced incidence of epithelial cancers associated with high dietary intake of fruits and vegetables . Because of this evidence, major clinical trials were launched to test -carotene, alone or in combination with vitamin e or vitamin a. the largest trials for chemoprevention of lung cancers in high - risk smoker populations were atbc, the alpha tocopherol (vitamin e), beta - carotene trial in finland, and caret, the beta - carotene and retinol efficacy trial smokers in the united states . The results were devastatingly similar, with excess lung cancer incidence and mortality rates in the active treatment arm, compared with those in the placebo arm of each trial . The unexpected and unwanted outcomes from the caret and atbc trials prompted us to reconsider issues related to both the effectiveness and the safety of micronutrient supplementation and careful evaluation of chemopreventive agents in preclinical cancer models . Sfn derived from broccoli sprouts is currently under clinical evaluation in diseases in which oxidative stress and inflammation play important roles in disease pathogenesis, namely, pulmonary diseases, such as chronic obstructive pulmonary disease, asthma, and cystic fibrosis, and cardiovascular disease, and protection against radiation dermatitis . In addition to these, sfn is being evaluated as a potential chemopreventive agent in melanoma and breast cancer and as an anticancer agent in patients with recurrent prostate cancer . At present, there are 17 clinical trials listed at www.clinicaltrials.gov that are using sfn or an enriched broccoli sprout preparation for treatment of a variety of diseases although sfn is known to modulate several signaling pathways, it is a potent inducer of nrf2-keap1 signaling, and the anticarcinogenic and anti - inflammatory activities of sfn are mediated primarily by nrf2-dependent induction of phase - ii detoxification and antioxidant enzymes. [13142930] results from recent studies suggest that sfn offers protection against tumor development and progression during the post - initiation phase by modulating diverse cellular processes that inhibit the growth of transformed cells . The ability of sfn to induce reactive oxygen species production, apoptosis, and cell cycle arrest is associated with regulation of many molecules, including the bcl-2 family of proteins, caspases, p21, cyclins, cyclin - dependent kinases, and histone deacetylases and nuclear factor - kappa - b pathways . Sfn also suppresses angiogenesis and metastasis by downregulating hypoxia - inducible factor 1 alpha, matrix metallopeptidase 2, matrix metallopeptidase 9, and vascular endothelial growth factor in various preclinical models of cancer however, in the past few years, loss of function mutations in keap1 and activating mutations in nrf2 leading to gain of nrf2 have been reported in lung cancer . In addition to lung cancer, gain of nrf2 function has been reported in other cancers such as prostate, gallbladder, esophagus, breast, skin, and ovarian cancer . In addition to mutations, results from recent studies have demonstrated that the oncogenes k - ras, b - raf, and myc upregulate nrf2 signaling to evade senescence and apoptosis and promote tumorigenesis . A comprehensive profiling of sfn bioavailability and tissue distribution kinetics revealed a dose - dependent increase in tissue concentrations after oral administration of sfn in mice . Sfn accumulation peaked at 2 h in lung, liver, kidney, brain, and plasma, but in small intestine, colon, and prostate, the highest concentrations were recorded at 6 h. maximum sfn accumulation was detected in small intestine . Furthermore, tissue concentrations of sfn metabolites varied as much as 100-fold between different tissues suggesting that peak plasma concentrations do not always align precisely with target tissues . Thus, route of administration may have a critical impact on sfn tissue distribution and accumulation . In this study, we compared three routes of sfn administration; (a) direct delivery to the target organ (lung) using nebulizer, (b) localized delivery to tumor bearing area (subcutaneous injection), and (c) systemic delivery with minimal risk of potential side effects due to systemic distribution (intraperitoneal injection). To evaluate the effect of prolonged sfn treatment on growth of lung cancer xenografts, we selected two models: (1) a549 cells harboring mutant k - ras oncogene and mutant keap1 gene and (2) h1975 cells harboring mutant egfr oncogene but wild - type keap1 and nrf2 . Prolonged systemic sfn treatment did not promote the growth of these tumors in vivo . On the contrary, localized delivery of sfn significantly abrogated the growth of keap1 mutant a549 tumors in vivo suggesting that the tumor suppression effects of sfn are mediated by non - nrf2-dependent signaling mechanisms . Importantly, significant tumor growth inhibition and a trend toward reduction with localized and systemic route of sfn administration, respectively, suggests that localized delivery of sfn probably results in better target tissue distribution and accumulation as compared to the systemic route . Next, we investigated whether prolonged sfn administration affects oncogenic k - ras (k - ras)-driven lung tumorigenesis . A study by denicola et al . Showed that oncogenic k - ras signaling upregulates nrf2 expression and its transcriptional network . Interestingly, although sfn administration via aerosol inhalation only modestly affected the nrf2 signaling, we still noticed a trend toward reduced tumor burden in sfn treated cohort of mice . This raises a possibility that alternative route of administration resulting in localized accumulation and robust induction of nrf2 signaling may significantly inhibit k - ras mediated lung tumor growth . In summary, sfn treatment however, one limitation of our study is that we only evaluated the effect of sfn on k - ras - mediated lung tumorigenesis post oncogenic k - ras activation and tumor foci formation . It remains to be investigated if sfn administration prior to cre - recombinase mediated oncogenic k - ras activation may impact the overall tumor burden . Pharmacological activators of nrf2 (dithiolethiones, isothiocyanates, and triterpenoids) including sfn have been evaluated as modifiers of multistage carcinogenesis in animal models and no protumorigenic effects have been reported to date . Our data from three preclinical mouse models of lung cancer suggest that prolonged sfn treatment does not promote tumor growth in mice . These findings, along with those from previously published studies on the chemopreventive properties of sfn against chemical carcinogens, support its future clinical development as a drug for chronic obstructive pulmonary disease and other pulmonary diseases . Dr . Anju singh, department of environmental health sciences, bloomberg school of public health, johns hopkins university, baltimore, maryland dr . Shyam biswal, department of environmental health sciences, bloomberg school of public health, johns hopkins university, baltimore, maryland department of oncology, sidney kimmel comprehensive cancer center, school of medicine, johns hopkins university, baltimore, maryland mr . Shengbin yang, department of environmental health sciences, bloomberg school of public health, johns hopkins university, baltimore, maryland dr . Edward gabrielson, department of oncology, sidney kimmel comprehensive cancer center, school of medicine, johns hopkins university, baltimore, maryland dr . Jinfang ma, department of environmental health sciences, bloomberg school of public health, johns hopkins university, baltimore, maryland dr . Rajesh k thimmulappa, department of environmental health sciences, bloomberg school of public health, johns hopkins university, baltimore, maryland dr . Ponvijay kombairaju, department of environmental health sciences, bloomberg school of public health, johns hopkins university, baltimore, maryland
Surgical excision of abnormal parathyroid tissue whether adenoma, carcinoma, or hyperplasia is considered as the definitive treatment of hyperparathyroidism . Surgery is the standard of care due to potential long - term skeletal, cardiovascular, and neuropsychiatric effects of even mild hyperparathyroidism . Nuclear medicine imaging techniques have played an important role in the detection and localization of parathyroid adenomas, with 99mtc - methoxy - isobutyl - isonitrile (mibi) scan currently being the standard test used . The addition of single photon emission computed tomography / computed tomography (spect / ct) has had an incremental benefit in exact localization of the adenomas . Although f - choline has been used as a photon emission tomography (pet) tracer for some years now, its utility in the detection of parathyroid adenomas has only recently been explored . We are currently studying the role of this modality vis - a - vis 99mtc - mibi scan with early spect / ct in patients with primary hyperparathyroidism . A 45-year - old female patient presented with complaints of bone pain and difficulty in walking for last 3 months . A plain radiograph of bilateral hip joints ordered by a local practitioner showed multiple lytic areas in the pelvis and femora . She was referred to a tertiary care institute for a fluorodeoxyglucose (fdg) pet / ct, in which multiple poorly fdg avid skeletal lesions were reported with no evidence of primary malignant pathology . The patient was referred to our institution for further management . A review of the fdg pet / ct revealed a rounded lesion measuring 2.2 cm with mild fdg uptake superior to the upper pole of the left lobe of thyroid just inferior to the left submandibular gland lateral to and abutting the left lamina of thyroid cartilage . Her renal function was deranged for at least last 1 year, and last serum creatinine was 1.9 mg% ., we ordered a parathyroid hormone (pth) assay which showed a value of more than 3000 pg / ml from two different laboratories . Her serum calcium was 10.4 mg / dl (laboratory normal 8.8010.60) and serum phosphorus was 3.60 mg / dl (2.404.40), and vitamin d levels were consistently in deficient range . We proceeded with a 99mtc - mibi scan and early, delayed and early spect / ct images were taken which revealed high uptake in the above lesion . This was interpreted as an ectopic left superior parathyroid adenoma [figure 1a and b]. As part of our ongoing study, she underwent a f - choline pet / ct study post 3 mci intravenous injection of the tracer, which showed intense uptake in the above lesion [figure 2a and b]. We found the lesion to thyroid ratio much higher than that seen in the mibi scan, making the images easier to interpret . A diagnosis of ectopic left superior parathyroid adenoma arising in the probable setting of tertiary hyperparathyroidism was made . One month after surgery, the patient reported a significant improvement in her symptoms and pth levels have returned to near normal . (a) planar early and delayed images of 99mtc - methoxy - isobutyl - isonitrile scan showing a focus of increased tracer uptake superior to the left lobe of thyroid (black arrow). (b) single photon emission computed tomography / computed tomography done after the early image reveals the same lesion (white arrow) measuring 2.2 cm 1.5 cm 2.3 cm inferior to the left submandibular gland, closely abutting the left lamina of thyroid cartilage (a and b) maximum intensity projection (posterior view) and transaxial images of f - choline photon emission tomography / computed tomography image showing the ectopic left superior parathyroid adenoma with intense uptake (white arrow) in the era of pet / ct, various tracers have been tried for accurate localization of parathyroid adenomas . However, the efficacy of fdg, fluorodopa, and c - methionine has not been established so far . A search for a pet - based tracer is ongoing that can be as good as mibi scan or better . F - choline has recently been reported as a possible novel pet agent for parathyroid localization preoperatively . The possible mechanism for choline uptake is its conversion into phosphatidylcholine by the enzyme choline kinase and subsequent incorporation into the cell membrane . A recent study by lezaic et al . In 24 patients revealed that the sensitivity and specificity of f - choline pet / ct were 92% and 100%, and sensitivity was much higher than any of the conventional scintigraphic techniques . Cazaentre et al . Reported incidental uptake of f - choline in parathyroid hyperplasia in a case of prostate cancer . In a study of 12 patients by michaud et al ., the authors found a detection rate of 92% for f - choline pet / ct and it also solved discrepant results between mibi scan and ultrasound . Reported a case where f - choline pet / ct enabled a minimally invasive surgery for parathyroid adenoma in a patient with negative ultrasound and mibi scan . No larger studies have been performed in this regard yet . At our institution, we do not have c - choline, and synthesis of f - choline has only recently started . In this particular case, the diagnosis was reliably made on mibi scan, but f - choline pet / ct proved to be as good as mibi scan . Furthermore, unlike mibi scan, the choline pet / ct in our case showed a very high lesion to thyroid ratio . Thus, it may be easier to report smaller lesions very close to the thyroid with this modality . It appears to provide additional hope specially in cases of low gland mass and multi - gland involvement due to better resolution of pet / ct as compared to spect / ct coupled with good image contrast . We feel that f - choline is, therefore, a promising tracer requiring further investigation.
While the senate version of the bill reversed certain elements of the employee retirement income security act (erisa), by allowing patients to sue in state and federal courts for denials of care by managed care organizations, the house version of the bill did not provide such a right and president bush was reported to have threatened to veto the bill if it included such a provision.1 the bill was moved to a house senate conference to work out differences between house - passed and senate - passed bills, but these negotiations failed . Despite this, many states enacted patients bill of rights laws.2,3 the concept of patients rights represents a cultural shift that began to emerge 40 years ago when notions of informed consent and autonomy were first endorsed by court opinion and institutional policy.4,5 in 1973, the american hospital association (aha) presented the first patients bill of rights.6 the 12 themes addressed in this initial document (e.g., right to respectful care, right to refuse treatment, right to confidentiality, right to refuse participation in research) have remained in subsequent versions (table 1), and in the 1990s the joint commission phased in a requirement to inform every patient about their rights as a national standard for hospital accreditation (ri.2.20). Table 1frequency of american hospital association patients bill of rights themes in state statutes and hospital documentsthemestate statutes (= 23),% hospital documents (= 240),% the patient has the right to:1 . Be informed of charges as well as policies for patient responsibilities and resolution of conflicts7457 frequency of american hospital association patients bill of rights themes in state statutes and hospital documents unfortunately, efforts to advance patients rights can be thwarted by inadequate attention to the complexity and language of the materials presented to patients . Adult reads at an 8th grade reading level, informed consent documents and notices of privacy practices typically require the reading capacity of a high school graduate.7,8 we hypothesized that pbor texts are also written at a level of complexity that far exceeds patients average capacity . We therefore undertook a survey to determine the readability of pbor texts in the united states . Hospitals and all pbor texts designated by state law to be given to all patients . We performed the following three additional analyses of state pbor statutes: 1) comparison of the rights delineated in state law to the themes advanced in the 2002 version of the american hospital association pbor; 2) abstraction of any enforcement powers that are delineated within the statute; and 3) evaluation of the presence of pbor texts in languages other than english for those states with mandatory language defined within the statute . We obtained state pbor statutes by searching all 50 state government web sites and legal codes in the lexis - nexus data base . If this information was unclear, we contacted the legal counsel for the state department of public health and welfare and/or the legal counsel for the state legislature . As such, pbor legislation intended for specific patient populations (e.g., psychiatric patients) or special circumstances (e.g., long - term care) were not included . To obtain a sample of hospital pbor documents, we used the u.s . News and world report 2006 alphabetical state listing of the nation s best hospitals; in each state we searched the publicly available web sites for every fourth general hospital on the list with the goal of obtaining 5 different pbor documents from each state . We designated a document as different from other documents in the state sample if the language, excluding institutional names, was not exactly the same . In addition, documents had to be at least 300 words long to be included . This served to exclude documents that are merely advertisements or outlines of actual pbor texts and ensured an adequate word count for readability analysis . In circumstances where multiple hospitals on the list had identical pbor documents, we retained one copy of the pbor and continued to search for additional documents . We continued to search the list until we found five unique documents of sufficient length per state or the list was exhausted by cycling through the list four times . Readability analyses were conducted on each hospital pbor using three software programs; prose: the readability analyst, grammatik 6.0, and wstyle: writing style analyzer (1992).9 for any state that designated the specific pbor text to be presented to patients, the readability of such text was evaluated in the same fashion . In addition, for each state that designated the specific pbor text to be presented to patients, we searched relevant web sites for approved text in other languages . The upper limit for most readability formulas is grade 17, which represents a 1st year graduate school reading level . Wstyle categorizes writing style as very poor, poor, weak, satisfactory, good, very good, and excellent . The specific rights that are protected in each state statute were abstracted and compared with the 12 themes in the 2002 version of the american hospital association pbor . This process was conducted independently by two coders (mpo and dj), who designated each aha theme as present, present but altered, or not present . In addition, state pbor themes not included in the aha pbor document were documented . Each instance of disagreement among reviewers was reevaluated in a joint conference for final classification until agreement was reached . We also noted instances where the statute specifically limits a person s options to pursue legal remedies for breach of the rights delineated in the statue . We used the wilcoxon signed - rank test to compare the average reading grade level of documents required by state statutes to the average reading grade level of the hospital sample in those states . The reading grade levels of pbor documents of hospitals in states with a pbor text defined by statute were compared to the reading grade levels for pbor documents of hospitals in other states with use of the wilcoxon rank sum test . We obtained state pbor statutes by searching all 50 state government web sites and legal codes in the lexis - nexus data base . If this information was unclear, we contacted the legal counsel for the state department of public health and welfare and/or the legal counsel for the state legislature . As such, pbor legislation intended for specific patient populations (e.g., psychiatric patients) or special circumstances (e.g., long - term care) were not included . To obtain a sample of hospital pbor documents, we used the u.s . News and world report 2006 alphabetical state listing of the nation s best hospitals; in each state we searched the publicly available web sites for every fourth general hospital on the list with the goal of obtaining 5 different pbor documents from each state . We designated a document as different from other documents in the state sample if the language, excluding institutional names, was not exactly the same . In addition, documents had to be at least 300 words long to be included . This served to exclude documents that are merely advertisements or outlines of actual pbor texts and ensured an adequate word count for readability analysis . In circumstances where multiple hospitals on the list had identical pbor documents, we retained one copy of the pbor and continued to search for additional documents . We continued to search the list until we found five unique documents of sufficient length per state or the list was exhausted by cycling through the list four times . Readability analyses were conducted on each hospital pbor using three software programs; prose: the readability analyst, grammatik 6.0, and wstyle: writing style analyzer (1992).9 for any state that designated the specific pbor text to be presented to patients, the readability of such text was evaluated in the same fashion . In addition, for each state that designated the specific pbor text to be presented to patients, we searched relevant web sites for approved text in other languages . The upper limit for most readability formulas is grade 17, which represents a 1st year graduate school reading level . Wstyle categorizes writing style as very poor, poor, weak, satisfactory, good, very good, and excellent . The specific rights that are protected in each state statute were abstracted and compared with the 12 themes in the 2002 version of the american hospital association pbor . This process was conducted independently by two coders (mpo and dj), who designated each aha theme as present, present but altered, or not present . In addition, state pbor themes not included in the aha pbor document were documented . Each instance of disagreement among reviewers was reevaluated in a joint conference for final classification until agreement was reached . We also noted instances where the statute specifically limits a person s options to pursue legal remedies for breach of the rights delineated in the statue . We used the wilcoxon signed - rank test to compare the average reading grade level of documents required by state statutes to the average reading grade level of the hospital sample in those states . The reading grade levels of pbor documents of hospitals in states with a pbor text defined by statute were compared to the reading grade levels for pbor documents of hospitals in other states with use of the wilcoxon rank sum test . Pbor laws existed exclusively for the protection of specific patient populations . Of the 23 states with pbor legislation for general patient populations, nine states laws presented a specific pbor document for distribution to patients . We analyzed a total of 240 hospital pbor documents from all 50 states; we did not find five unique hospital pbor documents in delaware (4), hawaii (3), north dakota (2), south dakota (2) and utah (4). The average reading grade level for the 240 hospital pbor texts was 14.1 (95% confidence interval 13.9 to 14.3, range 8.2 to 17.0). The average reading grade level for each state s hospital sample of pbor texts was 14.1 (95% confidence interval 13.8 to 14.4; range, 12.0, maine, to 16.6, minnesota). Nine states stipulated within their statute the actual pbor text to be distributed to patients . The average reading grade level for these nine documents was 15.2 (95% confidence interval 13.8 to 16.7; range 11.6, new york, to 17, minnesota) as seen in table 3 . Hospitals in these nine states rarely presented the text exactly as prescribed by state law (1 of 45). The reading grade level of hospital pbor texts in these nine states was lower than the language specified by state law (14.7 vs. 15.2, = 0.14) and higher than the average reading grade level of hospital pbor documents in other states (14.7 versus 14.0, = 0.05). Table 4 presents examples of excerpts from hospital pbor texts for four common themes . In six of the nine states that present statutory pbor texts, the state presented the mandatory text exclusively in english; three of these states presented a pbor document in spanish and two of these states also presented documents in additional languages (new york: italian, russian, greek, chinese, yiddish, and creole; minnesota: hmong, somali, russian, and laotian). Of the 12 aha themes, state statutes included an average of 7.4 themes and hospital documents included an average of 9.8 themes . As seen in table 1, the aha theme that is least commonly presented is the right to be informed of business relationships that influence care . In the 23 state statutes and the 240 hospital documents there were 95 themes not addressed in the aha document (e.g., pain management including opiates, receiving an itemized bill, and freedom from restraints). Table 2most common non - american hospital association patients bill of rights themes in state statutes and hospital documentsnon - aha themesstate statutes (= 23),% hospital documents (= 240),% the patient has the right to:1 . File a grievance100712 . Examine and receive an explanation of the itemized bill regardless of source of payment57753 . Respect for dignity and worth despite diagnosis50464 . Exercise their rights without regard to sex, race, economic status, educational background, color, religion, ancestry, nation origin, sexual orientation or marital status, or the source of payment for care29678 . Freedom from seclusion and restraint, unless clinically required or necessary to protect hospital staff29429 . Receive care in a safe setting and help accessing protective services215810 . Consideration of the ethical, cultural, spiritual, or psychosocial issues that arise in provision of care1433table 3readability statistics for patients bill of rights as codified in state lawstatereading grade levelflesch reading easesentence complexityvocabulary complexitywriting stylenew york11.652: fairly difficult2555satisfactorypennsylvania12.948: difficult4356weakcalifornia15.035: difficult4567weakflorida15.236: difficult7550poortexas16.127: very difficult5066poornew jersey16.329: very difficult5566very poormassachusetts16.518: very difficult7055poornew hampshire16.623: very difficult7858poorminnesota17.015: very difficult8466pooraverage15.331: difficult5860poorreading grade level is the average of eight readability formulas as calculated by prose: the readability analyst software (1988 - 1991)flesch reading ease as calculated by prose: the readability analyst software (1988 - 1991).sentence complexity (100 = most complex) as calculated by grammatik 6.0 software (1994). Score is based on the number of words and clauses in a document.vocabulary complexity (100 = most complex) as calculated by grammatik 6.0 software (1994). Score is based on the number of syllables in a document and a comparison to a word list of unusual or difficult words.writing style as calculated by wstyle . Portion of sentences using only active verbs; 2) word economy ratio of words that convey meaning (verbs, nouns, adjectives, and adverbs) to supporting words (propositions, articles, etc . ); 3) readability difference between the document s readability grade and the target - reader s grade; 4) word choice ratio of direct, active verbs and concrete nouns to abstract nouns and verbs transformed to nouns . Most common non - american hospital association patients bill of rights themes in state statutes and hospital documents readability statistics for patients bill of rights as codified in state law reading grade level is the average of eight readability formulas as calculated by prose: the readability analyst software (1988 - 1991) flesch reading ease as calculated by prose: the readability analyst software (1988 - 1991). Sentence complexity (100 = most complex) as calculated by grammatik 6.0 software (1994). Score is based on the number of words and clauses in a document . Vocabulary complexity (100 = most complex) as calculated by grammatik 6.0 software (1994). Score is based on the number of syllables in a document and a comparison to a word list of unusual or difficult words . Portion of sentences using only active verbs; 2) word economy ratio of words that convey meaning (verbs, nouns, adjectives, and adverbs) to supporting words (propositions, articles, etc . ); 3) readability difference between the document s readability grade and the target - reader s grade; 4) word choice ratio of direct, active verbs and concrete nouns to abstract nouns and verbs transformed to nouns . Each state s statute established an internal and external grievance policy . In most of these states, complaints may be directed to the state department of health and in several states complaints are directed to the board of registration . For example, in vermont complaints are directed to the board of medicine and failure to comply with any provision of the patients bill of rights law may constitute a basis for disciplinary action against a physician . In one state, illinois, the law stipulated fines for violations and in four states (arizona, massachusetts, maine, and texas), the statute protects a private civil right of action . A plaintiff who prevails in a suit under this section may recover actual damages, including damages for mental anguish even if an injury other than mental anguish is not shown.10 in contrast, the florida statute included language to explicitly restrict patients legal options: this section shall not be used for any purpose in any civil or administrative action and neither expands nor limits any rights or remedies provided under any other law.11 the average reading grade level for the 240 hospital pbor texts was 14.1 (95% confidence interval 13.9 to 14.3, range 8.2 to 17.0). The average reading grade level for each state s hospital sample of pbor texts was 14.1 (95% confidence interval 13.8 to 14.4; range, 12.0, maine, to 16.6, minnesota). Nine states stipulated within their statute the actual pbor text to be distributed to patients . The average reading grade level for these nine documents was 15.2 (95% confidence interval 13.8 to 16.7; range 11.6, new york, to 17, minnesota) as seen in table 3 . Hospitals in these nine states rarely presented the text exactly as prescribed by state law (1 of 45). The reading grade level of hospital pbor texts in these nine states was lower than the language specified by state law (14.7 vs. 15.2, = 0.14) and higher than the average reading grade level of hospital pbor documents in other states (14.7 versus 14.0, = 0.05). Table 4 presents examples of excerpts from hospital pbor texts for four common themes . In six of the nine states that present statutory pbor texts, the state presented the mandatory text exclusively in english; three of these states presented a pbor document in spanish and two of these states also presented documents in additional languages (new york: italian, russian, greek, chinese, yiddish, and creole; minnesota: hmong, somali, russian, and laotian). Of the 12 aha themes, state statutes included an average of 7.4 themes and hospital documents included an average of 9.8 themes . As seen in table 1, the aha theme that is least commonly presented is the right to be informed of business relationships that influence care . In the 23 state statutes and the 240 hospital documents there were 95 themes not addressed in the aha document (e.g., pain management including opiates, receiving an itemized bill, and freedom from restraints). Table 2most common non - american hospital association patients bill of rights themes in state statutes and hospital documentsnon - aha themesstate statutes (= 23),% hospital documents (= 240),% the patient has the right to:1 . Examine and receive an explanation of the itemized bill regardless of source of payment57753 . Respect for dignity and worth despite diagnosis50464 . Exercise their rights without regard to sex, race, economic status, educational background, color, religion, ancestry, nation origin, sexual orientation or marital status, or the source of payment for care29678 . Freedom from seclusion and restraint, unless clinically required or necessary to protect hospital staff29429 . Consideration of the ethical, cultural, spiritual, or psychosocial issues that arise in provision of care1433table 3readability statistics for patients bill of rights as codified in state lawstatereading grade levelflesch reading easesentence complexityvocabulary complexitywriting stylenew york11.652: fairly difficult2555satisfactorypennsylvania12.948: difficult4356weakcalifornia15.035: difficult4567weakflorida15.236: difficult7550poortexas16.127: very difficult5066poornew jersey16.329: very difficult5566very poormassachusetts16.518: very difficult7055poornew hampshire16.623: very difficult7858poorminnesota17.015: very difficult8466pooraverage15.331: difficult5860poorreading grade level is the average of eight readability formulas as calculated by prose: the readability analyst software (1988 - 1991)flesch reading ease as calculated by prose: the readability analyst software (1988 - 1991).sentence complexity (100 = most complex) as calculated by grammatik 6.0 software (1994). Score is based on the number of words and clauses in a document.vocabulary complexity (100 = most complex) as calculated by grammatik 6.0 software (1994). Score is based on the number of syllables in a document and a comparison to a word list of unusual or difficult words.writing style as calculated by wstyle . Portion of sentences using only active verbs; 2) word economy ratio of words that convey meaning (verbs, nouns, adjectives, and adverbs) to supporting words (propositions, articles, etc . ); 3) readability difference between the document s readability grade and the target - reader s grade; 4) word choice ratio of direct, active verbs and concrete nouns to abstract nouns and verbs transformed to nouns . Most common non - american hospital association patients bill of rights themes in state statutes and hospital documents readability statistics for patients bill of rights as codified in state law reading grade level is the average of eight readability formulas as calculated by prose: the readability analyst software (1988 - 1991) flesch reading ease as calculated by prose: the readability analyst software (1988 - 1991). Sentence complexity (100 = most complex) as calculated by grammatik 6.0 software (1994). Vocabulary complexity (100 = most complex) as calculated by grammatik 6.0 software (1994). Score is based on the number of syllables in a document and a comparison to a word list of unusual or difficult words . Portion of sentences using only active verbs; 2) word economy ratio of words that convey meaning (verbs, nouns, adjectives, and adverbs) to supporting words (propositions, articles, etc . ); 3) readability difference between the document s readability grade and the target - reader s grade; 4) word choice ratio of direct, active verbs and concrete nouns to abstract nouns and verbs transformed to nouns . Each state s statute established an internal and external grievance policy . In most of these states, complaints may be directed to the state department of health and in several states complaints are directed to the board of registration . For example, in vermont complaints are directed to the board of medicine and failure to comply with any provision of the patients bill of rights law may constitute a basis for disciplinary action against a physician . In one state, illinois, the law stipulated fines for violations and in four states (arizona, massachusetts, maine, and texas), the statute protects a private civil right of action . A plaintiff who prevails in a suit under this section may recover actual damages, including damages for mental anguish even if an injury other than mental anguish is not shown.10 in contrast, the florida statute included language to explicitly restrict patients legal options: this section shall not be used for any purpose in any civil or administrative action and neither expands nor limits any rights or remedies provided under any other law.11 our findings suggest that pbor documents presented in u.s . Hospitals far exceed the reading capacity of the majority of adults . In addition, these documents commonly fail to include themes designated by state law and by the american hospital association . While close to half of the states in the u.s . Have patients bill of rights legislation for the general public, the specific rights named in these laws vary and few of these laws incorporate remedies other than a mechanism to file complaints . Furthermore, in nine states statutory language to be presented to patients is very complex and is usually exclusively presented in english . These observations may not be surprising for people who know that other documents such as informed consent forms and notices of privacy protection have also been shown to be overly complex . Similarly, efforts to cultivate communication skills and inculcate the importance of patient education in trainees are hampered by the mixed message presented by patients rights documents that patients cannot read . Students may be taught that they should care about health literacy and low english proficiency while simultaneously observing what may appear as institutional indifference in the domain of patients rights documents . There are several reasons why clinicians and other patient advocates should particularly care about the readability and language accessibility of pbor documents . Complex public documents may serve to train patients to be more passive in their care and may instill fear in patients with limited literacy or english proficiency . Many clinicians probably view the pbor as a health system issue that does not directly impact clinical practice or their relationships with patients . However, a well - presented pbor document has the capacity to encourage patient activation and trust in those providing services . The current research, which demonstrates that pbor documents are frequently not understandable to patients, reveals a missed opportunity to present the patient care mission in a clear manner . In the 1970s, the patients rights movement was advanced because physicians were perceived as too powerful.12 at that time, patients had to advocate for the right to be given information about their diagnosis and prognosis.13 by the 1990s, when the concept of a patients bill of rights was introduced in congress, the topic was advanced by a consumer rights movement due to a sense that managed care companies and insurers were too powerful.14 instead of protecting a right to refuse treatment from paternalist physicians, consumers wanted to secure a right to choose their providers and have access to treatments being denied by payors . The american hospital association, which has long been an advocate for a patients bill of rights, changed their format in 2006 to a brochure called the patient care partnership, which contains the same themes and informs patients about what they should expect during their hospital stay with regard to their rights.15 while the brochure is a clear departure from the legal jargon of prior pbor documents advanced by the american hospital association (and is presented on their web site in arabic, chinese, english, russian, spanish, tagalog, and vietnamese), the english text is still written at an 11th grade reading level . As seen in table 4, where we present examples written at a 5th grade level, the themes of the pbor can be written in plain english . In most states, hospitals are free to revise their pbor documents; however, in nine states (ca, fl, ma, mn, nh, nj, ny, pa and tx) statutes should be amended either to allow hospitals to write their own language or to present the official state pbor in plain english . A note of caution is warranted . According to robert gunning, developer of the fog readability formula: " like all good inventions, readability yardsticks can cause harm in misuse . They are handy statistical tools to measure complexity in prose but they are not formulas for writing. "16 authors who replace long words with short words that are similarly arcane have not improved the actual readability, even if they do reduce their readability score.17,18 different formulas report grade levels that vary by two to four grades, partly because they are based on different levels of reader comprehension . Because the smog formula is based on 100% reader comprehension, it tends to score higher than other formulas which are based on 35%70% reader comprehension . Rather than using a single formula that might bias the results by scoring we used prose software because it provides the average grade level estimates of eight readability formulas . In addition, we provide further analyses to exhibit the level of complexity of the pbor documents . There are limitations to readability software programs . First, the same formula in different programs may give different grade levels due to variations in algorithms used to count sentences and syllables.17 second, formulas do not take into account a pbor s organization, font size, font family, etc . Third, these formulas cannot account for the background knowledge of the readers, their motivation, cultural experiences, etc . Despite these limitations, the formulas do provide a reasonable and cost - effective way of assessing how clearly pbors are written . Interested hospitals and legislatures may benefit from consulting specialists in adult basic education, readability, and improving patient care systems in this process . Similarly, hospitals can improve patients comprehension of their rights by supplementing their print material with other educational methods such as video or interactive multimedia that can be developed . A promising proposal for a national health literacy act, to establish a national center for health literacy at the agency for healthcare research and quality as well as provide funding for state health literacy offices, is currently being vetted.19 resources of this kind could help avoid future instances of legislatures compelling hospitals to present unreadable legal jargon to patients . The strengths of this study that lend weight to our conclusions are the amount of text analyzed, the blind sampling within every state, and the complete evaluation of state statutes . First, we surveyed only hospital pbor texts that were available through institutional web sites . Although it is likely that the materials presented on institutional web sites accurately reflect local practices, additional materials were not examined . Third, we evaluated readability using the average of eight readability formulas and three measures of syntax and semantics: sentence complexity, vocabulary complexity, and writing style . While this represents a significant advance over the vast majority of published analyses which are based simply on the flesch kincaid scale, or other single metrics of readability, additional factors that affect legibility and understandability, such as the type font, layout, and length, were not evaluated in this project . Similarly, we were not able to evaluate the readability of pbor documents in languages other than english to determine, for example, if the minnesota state pbor, which is at a graduate school level in english, is also at a 17th grade level in hmong, somali, russian and laotian . Fourth, we report the remedies offered within statutes; however, this does not reflect the volume or types of complaints that these statutes have actually generated . We made multiple attempts to determine details of these programs, but were not able to obtain records on complaints or otherwise assess the consequences of pbor statutes . It would be valuable to know how patients and states use these programs.20 when a hospital pbor document is missing a theme that is recommended by the aha or required by state statute, it is unclear if this represents an accidental lapse or a purposeful departure . The absence of themes from pbor documents, however, does not change clinical standards . For example, the least common aha pbor theme presented in hospital documents and state statutes relates to the disclosure of business relationships that may influence care . Nonetheless, professional standards dictate disclosure of such relationships.21 promoting patients rights has had many years of regulatory support from the aha and the joint commission . Similarly, almost half the states in our country have shown legislative support for a bill of rights to protect all patients ., patients rights statutes are designed to promote the ethical and humane treatment of patients . These goals will not be realized by presenting patients with documents they are not able to read and understand . Table 4examples of patients bill of rights text in four common domainsreadability leveldomainright to refuse careright to privacy of records5th gradetell us what medical care you want and what medical care you do not want.we do not share you records unless you give us permission.8th gradelet you choose whether to accept or refuse treatments.keep your hospital and medical records private.12th gradeyou have the right to consent to or refuse treatment, as permitted by law, throughout your hospital stay . If you refuse a recommended treatment, you will receive other needed and available care.you have the right to expect that treatment records are confidential unless you have given permission to release information or reporting is required or permitted by law . When the hospital releases records to others, such as insurers, it emphasizes that the records are confidential.16th gradethe patient has the right to make decisions about the plan of care prior to and during the course of treatment and refuse a recommended treatment or plan of care to the extent permitted by law and hospital policy and to be informed of the medical consequences of this action . In case of such refusal, the patient is entitled to other appropriate care and services that the hospital provides or be transferred to another hospital . The hospital should notify patients of any policy that might affect patient choice within the institution.the patient has the right to expect that all communications and records pertaining to his / her care will be treated as confidential by the hospital, excepting cases such as suspected abuse and public health hazards when reporting is permitted or required by law . The patient has the right to expect that the hospital will emphasize the confidentiality of this information when it releases it to any other parties entitled to review information in these records.right to know names of providersright to see bill5th gradethe doctors and nurses must tell you their names.you have the right to see your bill.8 gradetell you the names and roles of the people caring for you.show you your bill and explain it to you, no matter how it is paid.12th gradebe informed of the name and position of the doctor who will be in charge of your care in the hospital.you have the right to an examination and explanation of your bill, regardless of how it is paid.16th gradeupon request, to obtain from the facility in charge of his care the name and specialty, if any, of the physician or other person responsible for his care or the coordination of his care.every such patient or resident of said facility in which billing for service is applicable to such patient or resident, upon reasonable request, shall receive from a person designated by the facility an itemized bill reflecting laboratory charges, pharmaceutical charges, and third party credits and shall be allowed to examine an explanation of said bill regardless of the source of payment . This information shall also be made available to the patient s attending physician . quotations denote verbatim excerpts from hospital documents . The readability level represents the overall reading level of the document from which the excerpt was taken . Text that is not in quotations and presented in grey scale was written by the authors examples of patients bill of rights text in four common domains * * quotations denote verbatim excerpts from hospital documents . The readability level represents the overall reading level of the document from which the excerpt was taken . Text that is not in quotations and presented in grey scale was written by the authors
There are 209,000 cases of and 102,000 deaths due to renal cell carcinoma (rcc) per year worldwide . Rcc had been treated with cytokines with a modest response rate and some survival benefit . Since 2005, the u.s . Food and drug administration and european medicines agency have approved novel agents targeting the vascular endothelial growth factor pathways for patients with metastatic rcc (mrcc) on the basis of the results of large randomized clinical trials . Single - agent interferon (ifn) is no longer regarded as a standard option for first - line systemic treatment of mrcc in western countries . In a large cohort in a retrospective japanese study, the median survival time was approximately twice as long as that in previous studies from north america and europe in the cytokine era . One of the reasons for the difference was considered to be related to varying individual sensitivities to cytokine treatments . Racial differences might also affect biological characteristics of the tumors, leading to differences in frequencies of metastatic lesions and pathological features . Previous reports demonstrated positive response rates of 10% to 20% in response to cytokine treatments . However, some patients with favorable - risk disease achieved a complete and long - lasting remission . Recent studies suggest that stat3 polymorphism predicts a favorable response and survival benefit of ifn - alpha in japanese patients with mrcc . Thus, cytokine treatments may be useful for some japanese patients with mrcc, even in the era of targeted therapy . The present study investigated outcomes in japanese patients with favorable - risk mrcc according to the memorial sloan kettering cancer center (mskcc) criteria who had been treated with ifn or tyrosine kinase inhibitor (tki) therapy as a first - line systemic therapy . A total of 48 japanese mrcc patients with favorable - risk disease as defined by the mskcc criteria who had been treated with immunotherapy or tki therapy at chiba university graduate school of medicine hospital (cu) or chiba cancer center (ccc), japan, from 1995 to 2014 were retrospectively enrolled in this study . Ten patients were treated with tki therapy as a first - line therapy at ccc; the others were treated at cu . The mskcc criteria included karnofsky performance status <80%, elevated lactate dehydrogenase, low hemoglobin, elevated serum corrected calcium, and time from diagnosis to starting systemic therapy <1 year . Data regarding clinical characteristics, including age, gender, clinical stage, histology of the primary tumor, metastasectomy, radiation, and radiofrequency ablation (rfa), were collected from 48 patients . If necessary, we performed metastasectomy, rfa, and radiation before and during systemic treatment . In principle, we performed metastasectomy when the patient would be a surgical complete response (cr). Because systemic treatment response in liver metastasis was low in many cases, we tried to perform rfa for liver metastasis if possible . First - line systemic ifn therapy included ifn - alpha and ifn - gamma in 29 and 2 cases, respectively . First - line systemic tki therapy included sorafenib, sunitinib, and axitinib in five, eight, and four cases, respectively . First - line progression - free survival (pfs), overall survival (os), and first - line response rate were evaluated in all 48 patients . After sorafenib was approved for clinical use in 2008, we began to examine its clinical application for other potential molecular targets . We assessed the tumor response according to the recist (response evaluation criteria in solid tumors). Statistical analysis was performed by using the student t - test, chi - square test, or mann - whitney u test, and survival curves (pfs and os) were created by using the kaplan - meier method with the log - rank test . Comparisons of the clinical and pathological features of all 48 patients according to first - line therapy are summarized in table 1 . The mean age at diagnosis was 60 years in the ifn group and 58 years in the tki group (no significant difference). There was no significant difference between the two groups in distribution of gender . Before 2008, all patients were treated by immunotherapy . Since 2008, 10 patients were treated with ifn and 17 patients were treated with tki as a first - line therapy . The initial clinical stage was 1 in 13 cases (42%), 2 in 7 cases (23%), 3 in 10 cases (32%), and 4 in 1 case (3%) in the ifn group . The initial clinical stage was 1 in six cases (35%), 2 in three cases (18%), 3 in four cases (24%), and 4 in four cases (24%) in the tki group . Stage 4 was much more frequent in the tki group than in the ifn group (p=0.0276). Histology was the clear - cell type in 26 cases (84%), sarcomatoid in 2 cases (6%), collecting duct in 2 cases (6%), and chromophobe in 1 case (3%) in the ifn group . Metastasectomy from any organ was performed in 12 patients (39%) in the ifn group and in 8 patients (48%) in the tki group . Radiation of any site was performed in three (10%) and one (6%) patient, respectively, and rfa to any organ was performed in two (6%) and two (12%) cases, respectively . Duration from nephrectomy to systemic therapy was 34.1 months in the ifn group and 47.3 months in the tki group . There was no significant difference in the duration from nephrectomy to systemic therapy when comparing the two groups . In the ifn group, responses included cr in 3 cases (10%), partial response (pr) in 6 cases (19%), stable disease (sd) in 18 cases (58%), and progressive disease (pd) in 4 cases (13%). In the tki group, responses included cr in one case (6%), pr in seven cases (41%), sd in nine cases (53%), and pd in 0 cases (fig . The cr rate was higher in the ifn group than in the tki group, and the objective response rate (orr) and clinical benefit were higher in the tki group than in the ifn group, but these differences did not reach the level of statistical significance (p=0.649, p=0.212 and p=0.122, respectively). 2a shows os using a kaplan - meier curve, in which os was superior in the ifn group than in the tki group . Median os in the ifn and tki groups was 71 and 47 months, respectively (p=0.014). Median pfs was 20 and 16 months in the ifn and tki groups, respectively . In the first year after initial systemic therapy, pfs was superior in the tki group when compared with the ifn group (fig . The superior pfs of tki in the first year might influence the response rate, as 13% of the ifn group had pd in response to initial systemic therapy . Median pfs in the first - line ifn group (n=14) and tki group (n=10) was 7 months and 7 months, respectively (p=0.380). Four patients from the first - line ifn group had been treated using interleukin-2 as a second - line therapy . One patient from the first - line tki group had been treated with ifn as a second - line therapy . There was no significant difference in the number of favorable - risk mrcc patients receiving a second - line therapy on the basis of the selection of ifn or tki as a first - line therapy in this study . 4a presents os on the basis of metastatic sites . In patients with lung or lymph node metastasis median os in the first - line ifn group (n=23) and tki group (n=4) was 70 months and 16 months, respectively (p=0.230). 4b presents os in patients with metastatic disease at sites other than the lung or lymph nodes . A significant difference was found in os when comparing ifn or tki as a first - line therapy . Median os in the first - line ifn group (n=8) and tki group (n=13) was 57 months and 47 months, respectively (p=0.032). These data suggest that first - line ifn therapy was not inferior to tki therapy when evaluated according to metastatic sites . This retrospective study showed that ifn was effective for mskcc - defined favorable - risk mrcc patients . Median os in the ifn group (71 months) was longer than that in the tki group (47 months). Median pfs in the ifn group (20 months) was not significantly different from that in the tki group (16 months). There was no significant difference in pfs after second - line therapy on the basis of the selection of ifn or tki as the first - line therapy . Three patients (10%) in the ifn group and two patients (12%) in the tki group suffered from toxicities and could not continue ifn . Because they could not continue their primary systemic treatment, this study demonstrated a higher response rate in the ifn group (29%) than in the tki group (47%). Response rates in the range of 30% to 40% have been observed in response to recent tki treatment . Furthermore, the response to ifn treatment seen in the present study is also superior to that seen in a previous report (10%-20%). It is possible that patients in previous reports included those with mskcc - defined intermediate- and poor - risk disease as well as patients with a different distribution of ethnicity than in the present study . A previous study of mskcc risk classification in japanese patients with mrcc showed that median os was not reached and that 3-year os was 80% among mskcc - defined favorable - risk patients . Studies on japanese mrcc patients have showed varying survival times compared with studies conducted on north american or european patients . However, there were some differences in the distribution of patients among the different risk groups and in the survival time according to risk group when comparing japanese studies with other studies . Another japanese retrospective study of 1,467 patients with mrcc showed that os was about 2 times longer than that seen in previous studies in north america and europe in the cytokine era . Therefore, japanese mrcc patients might have better outcomes than do north american and european mrcc patients . Ifn - alpha monotherapy is associated with an improvement in survival among patients with advanced rcc . However, previous trials have not shown the superiority of cytokine treatment monotherapy over other therapies . Ifn - alpha was chosen as the comparator in several trials on the basis of data from previous studies and the widespread use of this agent . Because ifn therapy is associated with a low response rate and substantial adverse effects, identification of reliable predictive markers for a favorable response to ifn is needed to establish optimal treatment strategies for patients with mrcc . A japanese genetic study was the first prospective study to demonstrate that a stat3 polymorphism can predict favorable response to treatment with ifn - alpha in patients with mrcc . Another japanese study demonstrated that the sensitivity to ifn - alpha is increased by yb-1 suppression and that this suppression does not down - regulate ifn - alpha activation of t lymphocytes . Further study should be performed to clarify the difference between japanese mrcc patients and those from other geographic regions and ethnicities . This study demonstrated that ifn was not inferior to tki therapy according to metastatic sites . A previous japanese study showed that it is possible to improve the success rate in treating advanced rcc patients, especially those with lung metastases, if combination therapy with interleukin-2 and ifn - alpha is chosen as the firstline treatment . That study showed an orr of 35.5% and a clinical benefit rate of 80.6% in patients with lung metastasis alone; those values were 60.0% and 80.0%, respectively, in patients with lung plus lymph node metastasis . On the other hand, in patients with lung plus bone metastatic sites, endpoint results from specific surveillance of sunitinib treatment of japanese patients showed that the orr for all mskcc - defined risk groups was 22.8% for patients with lung metastasis, 18.9% for patients with liver metastasis, and 14.9% for patients with bone metastasis . Endpoint results from specific surveillance of sorafenib treatment of japanese patients showed that the orr of all mskcc - defined risk groups was 33.5% for patients with lung metastasis, 16.8% for patients with liver metastasis, 11.7% for patients with bone metastasis, and 12.5% for patients with brain metastasis . These data demonstrated that patients with lung metastasis experience a better response than do other patients when treated with immunotherapy or tki therapy . In the present study, the number of patients with metastatic disease was low, because the patient population consisted of those with mskcc - defined favorable - risk disease, and this phenomenon might have affected the results . The present study and previous reports showed that tki produces insufficient benefit in patients with metastatic disease at sites other than the lung or lymph nodes . Because this was a retrospective study with a small number of patients treated with tki for first - line systemic treatment, our understanding of the use of a single tki agent for first - line systemic treatment is limited . In this study, recently, sunitinib and pazopanib have been recommended for use as single tki agents for first - line therapy of good- to intermediate - risk clear cell carcinoma on the basis of the results of large randomized clinical trials . Further study is needed to determine appropriate first - line tki agents in favorable - risk patients . This retrospective study included patients with non - clear - cell histology in the ifn group . Patients with sarcomatoid variants and collecting duct rcc have poor survival . In this group of enrolled patients, one patient with sarcomatoid carcinoma was alive and being treating with ifn 10 months after systemic treatment, whereas the other patient died 17 months after ifn treatment and pfs was 2.8 months . One patient with collecting duct carcinoma was dead 13 months after ifn treatment and pfs was 1.5 months, the other patient was alive at 68 months after ifn treatment and pfs was 50 months . In this study, some of the patients with a poor prognosis histology received a survival benefit . This seemed to be affected by good general condition and performance status . In the target therapy era, we might be able to achieve effectiveness in japanese patients with non - clear - cell, favorable - risk disease treated with ifn . Further clinical study with a large number of patients is needed to clarify the treatment effects of ifn in non - clear - cell, favorable - risk patients . There is the possibility of bias in the initial stage and in the era of starting systemic therapy . Our understanding of this study in mskcc favorable - risk mrcc is limited and needs to be developed further . Further research about the molecular differences between japanese patients and those of other ethnicities may improve our understanding of why some mrcc patients in this study had markedly better responses to immunotherapy . Further clinical study is needed to evaluate favorable - risk patients with regard to os and pfs in the selection of a first - line systemic therapy . Collaborative group studies might help to boost the numbers of patients with this rare favorable - risk profile who are available for study . Ifn is associated with a survival benefit in japanese patients with favorable - risk metastatic rcc in the era of targeted therapy . However, because the present study was a retrospective analysis, there is the possibility of outside factors influencing the results at the initial stage and in the era of starting systemic therapy.
Non communicable diseases are taking an epidemic form and will be a major cause of death in developing countries by the year 2020 . Studies conducted in india show that every second person above 35yrs of age has periodontal pockets and 30% of total teeth extracted after 35yrs of age are due to periodontal disease . As a result of high prevalence of both coronary artery disease (cad) and periodontal disease framingham heart study has identified a list of classical risk factors (hypertension, male gender, age, smoking, diabetes mellitus, and obesity). However, they were not sufficient to account for the etiology of this multifactorial pathological process . Although major improvements have been made in primary and secondary prevention of major risk factors of cad, it continues to be highly prevalent . Around 40% cases of atherosclerosis cannot be attributed to the classical risk factors . Due to these facts, there is an increased interest for considering chronic infections as a risk factor for atherosclerosis . As inflammatory mechanisms play a central role in mediating all phases of atherosclerosis and destructive periodontal disease, when left untreated they become a chronic inflammatory condition leading to an increase in local and systemic inflammatory mediators periodontal associated inflammatory process contributes to an increase in inflammatory mediators including tumor necrosis factor- (tnf-), c reactive proteins (crp) and interleukin- 6 (il- 6). Epidemiological studies have associated plasma levels of il-6 and tnf - with cardio vascular risk factors and have associated il- 6levels with a risk of cad . Thus, the purpose of this interventional survey was to see the effect of periodontal treatment on levels of serum inflammatory markers crp, tnf- and white blood cell (wbc) counts . Twenty subjects were recruited from the department of cardiology, csm medical university, lucknow, india, for the survey . Subjects with a chronic generalized periodontitis having coronary artery disease confirmed by clinical findings and ecg as interpreted by a cardiologist were included in the study . Exclusion criteria included current smoking, smoking within last 6 months, diabetes and acute / chronic systemic diseases (e.g. Influenza, rheumatoid arthritis, copd, or kidney disease), antibiotics or anti inflammatory drug administration within the last 2 months, and pregnancy / lactation . Probing depth was recorded at six sites per tooth (mesio buccal, mid buccal, disto buccal, mesio lingual, mid lingual, and disto lingual). The presence or absence of bleeding was recorded using sulcular bleeding index (sbi, muhlemann and son 1971). Oral hygiene instructions were given and the subjects were recalled 1 month after the last treatment visit . Serum high - sensitivity c reactive protein (hscrp) was measured by using particle- enhanced turbidimetric assay (hscrp, latex, cobas, integra 400 plus, roche, basel, switzerland). Tnf- serum levels were determined by sensitive enzyme- linked immunosorbent assay (elisa) using commercially available kit (r and d systems inc, minneapolis, mn, usa). The plates were read using an automated microplate reader (bio rad, hercules, california, usa). Wbc count (/10) was obtained . All the parameters (pd, bop, hscrp, tnf-, wbc counts) were obtained at the baseline and 1 month post treatment . Probing depth was recorded at six sites per tooth (mesio buccal, mid buccal, disto buccal, mesio lingual, mid lingual, and disto lingual). Probing depth was recorded to the nearest millimeter using unc 15 probe . The presence or absence of bleeding oral hygiene instructions were given and the subjects were recalled 1 month after the last treatment visit . Serum high - sensitivity c reactive protein (hscrp) was measured by using particle- enhanced turbidimetric assay (hscrp, latex, cobas, integra 400 plus, roche, basel, switzerland). Tnf- serum levels were determined by sensitive enzyme- linked immunosorbent assay (elisa) using commercially available kit (r and d systems inc, minneapolis, mn, usa). The plates were read using an automated microplate reader (bio rad, hercules, california, usa). All the parameters (pd, bop, hscrp, tnf-, wbc counts) were obtained at the baseline and 1 month post treatment . Serum high - sensitivity c reactive protein (hscrp) was measured by using particle- enhanced turbidimetric assay (hscrp, latex, cobas, integra 400 plus, roche, basel, switzerland). Tnf- serum levels were determined by sensitive enzyme- linked immunosorbent assay (elisa) using commercially available kit (r and d systems inc, minneapolis, mn, usa). The plates were read using an automated microplate reader (bio rad, hercules, california, usa). All the parameters (pd, bop, hscrp, tnf-, wbc counts) were obtained at the baseline and 1 month post treatment . Bop was reduced by 28% and pd was reduced by 41% at the end of 1 month [table 1]. Data on periodontal disease markers before and after treatment hscrp and wbc levels were significantly reduced post treatment . The hscrp, tnf- levels and wbc counts reduced by 18, 2, and 14%, respectively [table 2]. The present study was conducted with the purpose of detecting the role of non surgical periodontal therapy in subjects with coronary artery disease . The study clearly showed that there was a significant reduction in the levels of serum inflammatory markers (hscrp and wbc counts) after periodontal therapy . The study is in agreement with the results of previous studies[610] that stated a reduction in systemic inflammatory markers of inflammation associated with periodontitis post treatment . In this study, bop is an informative parameter to estimate the severity of gingival inflammation as well as the response to treatment, and it is a clinical indicator of disease progression and stability . Bop 20% of sites is associated with a lower risk for periodontal attachment loss . In the present study, bop was significantly reduced 1 month after treatment, and the number of subjects with bop> 20% of sites at baseline was reduced by almost half after therapy . Similarly, all subjects experienced significant reductions in pd after the treatment . In this study, 1month after mechanical therapy, the circulating levels of hscrp and wbc counts significantly reduced in all subjects . Crp is a prognostic marker for future cardiovascular events . The release of bacteria and proinflammatory mediators such as bacterial endotoxins and cytokines in the bloodstream that causes the release of acute phase reactants (such as c reactive protein) leading to increased inflammatory activity in atherosclerotic lesions may represent the link between periodontal infection and cad . Wbc count, a crude marker of systemic inflammation associated with the prediction of future cardiovascular events also significantly decreased for subjects with cad . Tnf- has a role in apoptosis, bone resorption, matrix metalloproteinase (mmp) and il- 6 production . However, the role of tnf - remains disputed as several studies have not been able to recognize its association to cardiovascular events . Also, in our study there was no significant reduction in the levels of tnf- . Large sample size is required to justify the association of tnf-. The observation that cardiovascular risk factors might be influenced by periodontitis may have important clinical consequences . First, as inflammation plays an important role in the pathophysiology of various conditions (metabolic syndrome, bp, vascular health). The association of mild chronic inflammation with future serious events in observational studies may be influenced by an underlying severe periodontal infection . Second, periodontitis may increase the risk of future cardiovascular events because of the pro atherogenic changes (increased cholesterol) and increased systolic blood pressure induced in affected individuals . Cigarette smoking represents the major influential factor with regard to the association between periodontal infections and systemic inflammation, and this preliminary investigation raises the hypothesis of a possible interaction of smoking, periodontal infection and systolic blood pressure on systemic health.third, if periodontitis were the major inflammatory stimulus in at least some patients with periodontitis, severe periodontal infections may represent a major etiologic factor for atherosclerosis, metabolic syndrome, and their sequelae . Still, the causal relationship between periodontitis and cad cannot be established as the sample size was small and the role of other risk factors was not explored . On the basis of the data obtained, a large scale interventional study could be conducted in future . Poor oral health is detrimental to systemic health.inflammatory markers are significantly associated with periodontitis.periodontitis is a modifiable risk factor, which can be prevented and treated.
Gestational age (ga) was calculated according to the first date of the last menstrual period, and confirmed by the first - trimester sonographic measurements . The study population consisted of pregnancies which fulfilled the following criteria: 1) history of regular menses with a known date of the beginning of the last menstrual period, 2) a discrepancy of less than seven days between the ga predicted by the last menstrual period and that estimated by first - trimester sonography, 3) singleton pregnancy, 4) absence of maternal disease known to affect normal fetal growth, 5) a clinically - normal fetus at birth . To maintain a properly designed cross - sectional study, our institutional review board approved the study, and verbal informed consent was obtained from each woman evaluated . Ultrasounds were conducted using a voluson 730,logiq 400, 500, or 5 (ge medical systems, milwaukee, wi), or an ultramark 9 system (philips medical systems, bothell, wa). The ultrasound evaluations were performed by seven experienced sonographers certified by the american registry of diagnostic medical sonographers, two of which had four years experience and one each with 5, 6, 7, 9 and 11 years experience . Evaluation of the fetal gall bladder was conducted as a standard part of prenatal sonographic examinations . Identification of the fetal gall bladder was done in the axial plane just below the level of the stomach . Color doppler imaging was employed as necessary to confirm that the gall bladder had not been confused with the intraabdominal umbilical vein . If the fetal gall bladder was not found during the initial search, the fetal abdomen was intermittently rescanned in an attempt to observe the fetal gall bladder . If the fetal gall bladder was identified, the transducer was tilted to visualize the gall bladder in its maximal length, and length and width of the fetal gall bladder were measured using electronic calipers at an increment of 0.1 mm . The height of the fetal gall bladder was measured along the transverse section of the gall bladder, which was acquired after the transducer was rotated 90 degrees perpendicular to the long axis of the fetal gall bladder (fig . 1). To evaluate the reliability of us, the three dimensional diameters, width and volume of the gall bladder was measured in the fetuses of 20 randomly - sampled pregnant women between 20 and 36 weeks of gestation by two observers with 11 years and four years of experience, respectively . Each observer performed each measurement twice and the mean value was taken as the representative values of each observer . Area and volume were calculated using the following formulas; area = 3.14 * ([width + length]/2) 2, volume = 0.523 * width * height * length . When three measurements of the same fetal gall bladder were available, the obtained data were used to establish reference values based on the gestational age . Statistical analyses were performed using commercially available software packages (spss version 10.0, spss, chicago, il). Scatter plots of the analyzed parameters (length, width, height, area, and volume) as a function of gestational age were generated and their relationships were assessed using the pearson correlation analysis . Pearson correlation coefficients were calculated to evaluate the strength of the relationship between measured parameters and gestational age . Reference values were acquired by stratifying the fetuses into eight subgroups on the basis of their gestational age, and then calculating reference values (median, 5th percentile, 95th percentile) for each subgroup . The gall bladder lengths in our study were compared with those of caucasian and african - american populations using the wilcoxon signed rank sum test . In the latter, the median values were 1.0 cm at 15 - 19 weeks, 1.5 cm at 20 - 22 weeks, 1.9 cm at 23 - 24 weeks, 2.1 cm at 25 - 26 weeks, 2.1 cm at 27 - 30 weeks, 2.6 cm at 31 - 34 weeks, and 2.7 cm at 35 - 40 weeks (2). The reliability of our measurements was evaluated using the bland and altman plot, with the repeatability coefficient being used to demonstrate interobserver agreement (4). The coefficient of variation was calculated to express the ratio of the standard deviation (sd) to the mean value . We prospectively evaluated the fetal gall bladder in low - risk pregnant women who visited our unit for routine antenatal sonography between february and april 2003 . Gestational age (ga) was calculated according to the first date of the last menstrual period, and confirmed by the first - trimester sonographic measurements . The study population consisted of pregnancies which fulfilled the following criteria: 1) history of regular menses with a known date of the beginning of the last menstrual period, 2) a discrepancy of less than seven days between the ga predicted by the last menstrual period and that estimated by first - trimester sonography, 3) singleton pregnancy, 4) absence of maternal disease known to affect normal fetal growth, 5) a clinically - normal fetus at birth . To maintain a properly designed cross - sectional study, our institutional review board approved the study, and verbal informed consent was obtained from each woman evaluated . Ultrasounds were conducted using a voluson 730,logiq 400, 500, or 5 (ge medical systems, milwaukee, wi), or an ultramark 9 system (philips medical systems, bothell, wa). The ultrasound evaluations were performed by seven experienced sonographers certified by the american registry of diagnostic medical sonographers, two of which had four years experience and one each with 5, 6, 7, 9 and 11 years experience . Evaluation of the fetal gall bladder was conducted as a standard part of prenatal sonographic examinations . Identification of the fetal gall bladder was done in the axial plane just below the level of the stomach . Color doppler imaging was employed as necessary to confirm that the gall bladder had not been confused with the intraabdominal umbilical vein . If the fetal gall bladder was not found during the initial search, the fetal abdomen was intermittently rescanned in an attempt to observe the fetal gall bladder . If the fetal gall bladder was identified, the transducer was tilted to visualize the gall bladder in its maximal length, and length and width of the fetal gall bladder were measured using electronic calipers at an increment of 0.1 mm . The height of the fetal gall bladder was measured along the transverse section of the gall bladder, which was acquired after the transducer was rotated 90 degrees perpendicular to the long axis of the fetal gall bladder (fig . 1). To evaluate the reliability of us, the three dimensional diameters, width and volume of the gall bladder was measured in the fetuses of 20 randomly - sampled pregnant women between 20 and 36 weeks of gestation by two observers with 11 years and four years of experience, respectively . Each observer performed each measurement twice and the mean value was taken as the representative values of each observer . Area and volume were calculated using the following formulas; area = 3.14 * ([width + length]/2) 2, volume = 0.523 * width * height * length . When three measurements of the same fetal gall bladder were available, the obtained data were used to establish reference values based on the gestational age . Statistical analyses were performed using commercially available software packages (spss version 10.0, spss, chicago, il). Scatter plots of the analyzed parameters (length, width, height, area, and volume) as a function of gestational age were generated and their relationships were assessed using the pearson correlation analysis . Pearson correlation coefficients were calculated to evaluate the strength of the relationship between measured parameters and gestational age . Reference values were acquired by stratifying the fetuses into eight subgroups on the basis of their gestational age, and then calculating reference values (median, 5th percentile, 95th percentile) for each subgroup . The gall bladder lengths in our study were compared with those of caucasian and african - american populations using the wilcoxon signed rank sum test . In the latter, the median values were 1.0 cm at 15 - 19 weeks, 1.5 cm at 20 - 22 weeks, 1.9 cm at 23 - 24 weeks, 2.1 cm at 25 - 26 weeks, 2.1 cm at 27 - 30 weeks, 2.6 cm at 31 - 34 weeks, and 2.7 cm at 35 - 40 weeks (2). The reliability of our measurements was evaluated using the bland and altman plot, with the repeatability coefficient being used to demonstrate interobserver agreement (4). The coefficient of variation was calculated to express the ratio of the standard deviation (sd) to the mean value . Sonographic visualization and measurement of the fetal gall bladder was possible at as early as 12 weeks gestation . Our study population consisted of 1,911 of the 2,170 fetuses enrolled in this study (88.1%) that produced results meeting the acceptable criteria . Of these, the gall bladders of 1,417 fetuses (74.1%) were visualized using antenatal sonography . Visualization of the gall bladder was achieved in greater than 90% of women at gestation periods between 16 and 34 weeks, however the gall bladder was identified in less than 90% of study population after 35 weeks (fig . Measurements of the length, width, and height of the fetal gall bladder were obtained from 1,292 of 1,417 fetuses (91.2%), however the scanning plane could not be adjusted to depict the maximal length, width, or height of the gall bladder in the remaining 125 fetuses . Table 1 shows fetal gall bladder length, height, width, area and volume according to gestational age . The measured parameters of the fetal gall bladder had a significant positive relationship with gestational age (table 2). The correlation of length and area to gestational age was better than that of width, height, and volume, therefore, reference values based on the gestational age were only calculated for the length and area (table 3). The median values of the gall bladder lengths in the korean population were not significantly different from those in the caucasian and african - american populations (p = 0.915). The mean differences, repeatability coefficients, and coefficients of variation between the two operators were 1.85 2.84 mm (mean sd), 5.56 mm, and 12.9% for the length, 0.56 1.14 mm, 2.23 mm, and 21.0% for the width, 0.25 0.83 mm, 1.63 mm, and 16.6% for the height, 125.66 175.56 mm, 344.11 mm, and 33.52% for the area, and 110.07 217.54 mm, 426.37 mm, 97.36% for the volume . It has been demonstrated that growth of fetal organs varies between ethnicities, even when environmental and socio - economic factors are considered (5 - 8). The fetal gall bladder can commonly be seen as a fluid filled structure during antenatal sonography and the normative data of the fetal gall bladder throughout gestation has been established in caucasian and african - american populations (1 - 2). The application of those normative data may be inappropriate for evaluation of the fetal gall bladder in other populations, however, if ethnic differences between populations have not been compared . In our larger study of 1,911 fetuses, we provided reference values for the growth of the fetal gall bladder throughout gestation in the korean population . The lengths of the fetal gall bladder were not statistically different from those of the caucasian and african - american populations, which suggests that there is no need to consider ethnicity when interpreting the size of fetal gall bladder in asian, african - american and caucasian populations . Some investigators have asserted that the fetal gall bladder can always be observed after the early part of the second trimester, therefore non - visualization of the fetal gall bladder after this point may indicate cystic fibrosis, gall bladder atresia, and biliary atresia (9 - 10). Hertzberg et al . Have refuted this assertion, however, in a study that assessed the prognostic importance of non - visualization of the gall bladder during gestation (11). In their study, the fetal gall bladder was not seen in approximately 5% of fetuses at 24 - 32 weeks gestation, however a normal outcome occurred in most of the cases in which the gall bladder of the fetus could not be visualized . In our study, the gall bladder was not seen in 84 (6.8%) fetuses at 16 - 34 weeks gestation, which is similar to the findings of hetzberg et al . Moreover, the visualization rate of the fetal gall bladder decreased in fetuses scanned after 35 weeks of gestation, which might be attributable to either technical difficulty or increased gall bladder contractility with advanced gestation (11, 12). Therefore, our findings support hertzberg's opinion (11) that the rate of non - visualization of the fetal gall bladder is sufficiently high, indicating that non - visualization of the gall bladder may not be a useful screen for fetal anomalies such as biliary atresia . It should be noted that our study was limited because it lacked a true reference standard for validating normal neonatal outcomes in the study population . While we excluded all fetuses with abnormalities that were believed to have a potential influence on the normality of the fetal gall bladder, we cannot be completely certain that this exclusion was effective . However, we believe that such a limitation is unavoidable when conducting prenatal imaging research to provide reference values for the general population . Another limitation of this study is that the analysis of interobserver variability used a relatively low number of women and intraobserver variability was not analyzed . Although each observer performed the measurement twice, it was performed without appropriate time intervals between two measurements, and thus intraobserver variability could not be analyzed . In summary, we have provided reference values of the fetal gall bladder throughout gestation in the korean population . As shown in our study, the reference values of the fetal gall bladder in the korean population are not statistically different from those of the caucasian and african - american populations, and non - visualization of the fetal gall bladder is a relatively common finding with little clinical significance as a sign of fetal abnormality.
In 1982 chang and der, two postdoctoral fellows working in geoffrey cooper's laboratory, discovered kristen rat sarcoma virus and murine sarcoma virus; retroviral oncogenes related to rodent sarcoma virus genes . The human kras gene is a homolog of these two oncogenes . A normal form of human c - ras has been called kras or kras2 (kristen rat sarcoma viral oncogene homolog or alternatively kristen murine sarcoma virus2 homolog). In 1983, der described an abnormal form of the p21 protein expressed by colon and lung carcinoma cell lines and showed that the gene encoding this protein is able to transform nih3t3 cells . This finding was later confirmed by parada and weinberg, who described the transformation of nih3t3 cells by an activated kras oncogene . Aberrant p21 proteins were encoded by the altered kras gene and their expression in carcinoma tissue was causally linked to an abnormal state of activation . Since then, it has been accepted that kras is one of front - line sensors that initiate the activation of an array of signalling molecules allowing the transmission of transducting signals from the cell surface to the nucleus, thus affecting cell differentiation, growth, chemotaxis, and apoptosis . A signal transduction cascade initiated by the activated form of kras kras elicits changes in the cytoskeleton and consequently affects cell shape, adhesion and migration [4, 5]. In the following paragraphs, kras protein, gene, oncogenesis, and cancer therapy is reviewed kras belongs to a group of small gtp - binding proteins, known as the ras superfamily or ras - like gtpases . The entire ras superfamily is characterised by the presence of a catalytic g domain, but includes members with distinct evolutionary specializations with respect to different cellular process . The ras subfamily (ras, rho, rab, arf, rac, and ran) includes the most frequently studied proteins, such as harvey - ras (h - ras), neuroblastoma - ras (n - ras), and two variants of kristen - ras (k - ras)one, known as kras4a, which is weakly expressed in human cells and the dominant form, known as kras4b, which is much more highly expressed . The kras gene product, kras protein, contains 188 amino acid residues with a molecular mass of 21.6 kd and participates in intracellular signal transduction . As mentioned above, the kras protein remains inactive until it binds to gtp, as depicted in figure 2 . Once the gtp is bound to the kras protein, kras undergoes conformational changes that involve two regions of the protein, thus activating it . These two important regions are known as switch 1 (aminoacids 3038) and switch 2 (aminoacids 5967), which form an effector loop, controlling the specificity of the binding of this gtpase to its effector molecules . This conformational change in the kras protein affects its interactions with multiple downstream transducers gtpase - activating proteins (gaps)which amplify the gtpase activity of the ras protein 100,000-fold . The change also affects interactions with guanine - exchanging / releasing factors (gefs / grfs) promoting the release of gtp . The kras protein also has intrinsic gtpase activity, stimulated by gaps, which acts as a timer associated with direct interactions with the effectors . Mutations found in an oncogenic form of the ras p21 protein impair gtpase activity and make the kras protein unresponsive to gap proteins . Mutated forms of p21 rapidly exchange gdp for gtp, which it prefers as a substrate, thus inducing the active state . Such aberrant forms of kras protein deregulate many effectors, thus affecting several important cellular pathways . Many gtp derivatives targeting ras or raf effectors have been developed to repair the defective gtpase activity that influences the aberrant ras signalling . However, little is known about the specificity and transport of compounds modified by gtps through the plasma membrane . The first domain includes 85 amino acids at the n - terminus and is identical in the three forms of ras (kras, nras, and hras). The second domain contains 80 amino acids, with lower sequence identity (7080%) among the three forms of ras protein . These regions are important for the signalling function of the kras protein and jointly form the g - domain (amino acids 1165, figure 3). The g - domain of the kras protein includes the gtp - binding pocket, where p - loop - phosphate binding loops (aminoacids 1016 and 5659) interact with the b - phosphate and c - phosphate of gtp . The region between amino acids 32 and 40 (the core effector region) is essential for the interactions between the putative downstream effectors and gaps . Ras protein also contains a hypervariable region (hvr) at the c - terminus (amino acids 165188/189; the third domain), which guides posttranslational modification and determines plasma membrane anchoring . This region plays an important role in the regulation of the biological activity of ras protein . Switch regions i and ii play important roles in the binding of regulators and effectors . The phosphate binding pocket - p loop permits temporary binding of gtp to the ras protein . This is also the region of gtpase activity, which negatively regulates the ras protein via a ras - gtp hydrolysis reaction and binding of guanosine diphosphate . The kras protein acts like a plasma membrane - localized molecular switch, regulating multiple signal transduction pathways . It is synthesized in the cytosol, where it is farnesylated by farnesyl transferase at the cysteine residue of the carboxy - terminal motif caax (where c represents cysteine, a is an aliphatic amino acid, and x is any amino acid). The aax amino acid motif is cleaved by proteases, whereas the c - terminal carboxyl residue of the kras protein is methylated . Cleavage of the axx peptide motif and methylation occur at the cytosolic surface of the endoplasmatic reticulum and are mediated by the ras - converting enzyme rce1 . C - terminal farnesylation plays an important role in membrane localization . In the splice variant kras4a, the axx motif undergoes additional palmitoylation by palmitoyl transferase, resulting in proper targeting of kras4a to the membrane . However, there is no detectable palmitoylation of the predominant splice variant kras4b, which probably reaches the plasma membrane via a microtubule - dependent mechanism, thus avoiding the golgi apparatus [13, 15]. Posttranslational farnesylation and carboxymethylation are believed to be important for the oncogenicity of the ras protein . Treatment with farnesyl transferase inhibitors has been shown to inhibit anchorage - independent growth of both kras - transformed mouse fibroblasts and human tumour cells containing kras and nras mutations . Activation of downstream signalling pathways by kras can also be triggered by signals from subcellular compartments, such as the endoplasmatic reticulum and the golgi apparatus [16, 17]. While wild - type kras usually promotes cell cycle progression, it can also induce growth arrest, apoptosis, and replicative senescence when increased to abnormal levels . This can be triggered by cellular stress, ultraviolet or ionizing irradiation, heat shock, and some cytokines . In these circumstances, triggering of growth arrest can represent a defence mechanism against inappropriate activation of ras . It has been demonstrated that the wild - type kras gene is a tumour suppressor that is frequently lost during tumour progression in many types of cancer . Once the kras gene mutates, it acquires oncogenic properties (table 1) and seems to be causally involved in the development of various human cancers [19, 20]. Loss of the wild - type kras allele has been observed in both human and mouse tumours, indicating that absence of the normal allele may facilitate transformation by one copy of the oncogenic kras allele . Oncogenic mutations in the kras gene prevent the hydrolysis of gtp, thus permanently activating the ras molecules . Expression of a mutated kras gene in fibroblasts has been shown to augment metalloproteinase 2 (mmp2) expression in the matrix and enhance the invasion of cancer cells . Overexpression of this mutated form of kras also inhibits glycosylation of the integrin 1-chain, resulting in altered polarisation and increased adhesiveness of colon cancer cells . In addition, expression of this oncogenic form of kras protein has been shown to be associated with upregulated carcinoembryonic antigen (cea) expression and disturbance of epithelial cell polarization . There are two copies of the kras gene in the human genome, designated kras1 and kras2 . The mrna encoded by the main kras2 is 5.5 kb long, and differs from transcripts of the transforming kristen murine viral gene by only six codons . Analysis of human placental and embryonic cdna libraries has revealed that 900 bp of the kras1 gene is homologous to the corresponding sequence of the kristen murine sarcoma virus2 homolog, with one intervening sequence, and 300 bp of the kras2 is fully homologous to the viral counterpart . The kras1 gene is a pseudogene derived from kras2 by alternative mrna splicing . Mcbride and colleagues found that the protooncogenes kras1 and kras2 are localized at human chromosomes 6 and 12, respectively . Later, kras1 and kras2 were mapped by in situ hybridization to chromosome positions 6p11 - 12 and 12p11.1 - 12.1, respectively . Alternative splicing of exon 4 produces two mrna forms, known as 4a and 4b . Exon 5 can be skipped during alternative splicing, giving rise to isoforms krasa and krasb . The 6th exon encodes the c - terminal region in krasb and is not translated (the 3untranslated region, 3utr) in krasa . Krasb is the predominant splice variant of kras2, and is referred to, briefly, as kras . There are indications that allelic losses of chromosome region 12p commonly occur in human cancers, and a frequently deleted region is near the kras gene at position 12p12 - 13 . Further, recent studies on lung adenocarcinoma suggest there is an association between the incidence of allelic losses in the 12p12 - 13 region and kras gene mutation . Diagnostics of kras gene mutations in clinical setting is limited by two factors: first, in the time of testing, kras mutated tumour cells may be in minority, outbalanced by wild type tumour cells and wild type non - tumour cells present in the sample . Second, analytically preferable snap - frozen tumour samples are rarely available for kras mutation testing . Instead, formalin fixed paraffin - embedded (ffpe) tissue is used . There, integrity of dna may be severely compromised by procedure of formalin fixation (especially by its long duration and low ph). All the known principles of dna polymorphism detection are applicable to kras mutation detection and demand a dedicated review outside of the scope of this paper . More than 60 methods described can be divided into sequencing methods [3037], methods based on specific interaction with oligonucleotide, methods based on specific interaction with enzyme [3840], and conformational methods [4147]. While many specificity and/or sensitivity enhancement of methods were described as well [4853], analytical validation, systematic comparison, and assessment of methods side by side is lacking . To authors best knowledge, only communaut europene (ce) marked kras mutation detection kits are supplied by dxs (mutations in codons 12 a 13 are tested using principle of arms - pcr and scorpion primers, vienna - lab (reverse dot blot assay format), tib molbiol (kras lightmix clamped hybridization probes for codon 12), and invigene (qpcr with sequence suppressor agent stopprimer for the unwanted excess component, applicable for first two nucleotide positions in codons 12 and 13). Kras expression is regulated both during the initiation of transcription by the binding of proteins to its promoter and during transcriptional elongation by micrornas affecting kras mrna stability . Both human and murine kras gene promoters contain a nuclease hypersensitive polypurine - polypyrimidine element (nhppe). The g - rich strand of nhppe located in the proximal promoter sequence is able to form an intramolecular parallel g - quadruplex, consisting of three g - tetrads and three loops, which recognizes and binds nuclear proteins that are involved in transcriptional repression of kras expression . Accordingly, it has been reported that sequestration of nuclear proteins that bind to nhpp by an oligonucleotide mimicking the kras g - quadruplex resulted in 40% inhibition of kras transcription, compared to controls . The transcription of kras is regulated, in part, by an interaction between the promoter region and the 65 kda esxr1 protein and, in part, by micrornas (mirnas). Esxr1 is a human protein with an n - terminal homeodomain in the nucleus and a c - terminal proline - rich repeat region i in the cytoplasm . The n - terminal fragment of esxr1 binds to the taatgttatta consensus sequence in exon-1 of the kras gene, thus inhibiting its mrna expression . Mirnas contain a 21 - 22 nucleotide long noncoding sequence that is able to regulate gene expression . In 2005 it was estimated that there are more than 500 mirnas, which collectively regulate approximately 30% of all human genes, including the ras gene family . Regulation of gene expression by mirnas probably occurs as a result of imperfect hybridization of the mirna to the complementary sequences located in the 3untranslated region (3utr) of target messenger rna (mrna) species . This interaction between mirna and mrna both decreases mrna stability and represses protein synthesis by preventing access to ribosomes . Interestingly, many altered mirnas have been identified in human cancers [6163], including some of the most thoroughly analyzed mirnas members of one group, the oncomirs, are upregulated in cancer and can act like oncogenes . The second group, the anti - oncomirs, probably act as tumour suppressors by targeting oncogenes, repressing the cell cycle and division of cancer cells . For example, mirna - let7 is an oncogene - antioncomir pair that negatively regulates ras protein levels and decreases cell proliferation rates [68, 69]. Kras, nras, and hras harbour multiple let-7 mirna complementary sites (lcss) in their 3utrs . Zhang et al . Found that reducing the activity of let-7 in hela cells resulted in a 70% increase in ras protein levels, while takamizawa et al . Found that let-7 expression was 80% lower in 60% of lung cancer adenocarcinoma and squamous cell carcinoma lines than in normal lung tissue . Moreover, a correlation between low levels of let-7 mirna and significantly higher ras protein expression has been found in lung squamous cell carcinomas . These results suggest that let-7 is able to downregulate the expression of ras in human carcinomas . These molecular findings provide a strong rationale for developing novel therapeutic treatments aimed at decreasing kras protein expression in cancer cells . In many cases kras protein expression is dramatically increased due to mutations in the kras gene sequence, thus making cells refractory to current therapies, such as those involving use of epidermal growth factor receptor inhibitors . Such oncogenic forms of the kras gene are prevalent in pancreatic carcinomas (> 80%), colon carcinomas (4050%), and lung carcinomas (3050%), but are also present in biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia [73, 74] and breast cancer . Mutations in the kras gene have important effects on the process of carcinogenesis, which depend on the cells and tissues involved . The mutations found most frequently in the kras gene of cancer cells are located at positions 12 and 13 in exon 1, and less frequently in codons 61, 63, 117, 119, and 146 [77, 78]. Allelic mutations result in amino acid changes, namely gly to asp, ala, arg, ser, val, or cys in codon 12 and gly to asp in codon 13 . Somatic missense mutations at positions 12, 13, 61, and 63 enable perturbation of the intrinsic gtpase activity of the kras protein, resulting in reductions in gtp hydrolysis capacity . Mutations in codons 12 (figure 4) or 13 are known to lead to conformational changes in the kras protein . Mutation in codon 12 of the kras gene causes the encoded kras protein to freeze in its active state for a much longer duration than its nonmutated counterpart . Mutations resulting in the substitution of amino acids 116, 117, 119, and 146 reduce the nucleotide affinity of the kras protein, thereby affecting the rate of gdp / gtp exchange . The oncogenic forms of the ras protein have a profound effect on the downstream effector pathways, resulting in much higher proliferation rates of cancer cells expressing such forms . The transforming ability of the kras oncogene may result from overexpression of the mutant kras allele or from deletion of the wild - type allele . Overexpression of kras can also be induced by the loss of p16ink4 (cdkn2a), p19ink4 (cdkn2d), or p53 . (2001) have shown that the wild - type kras allele can suppress the oncogenic function of the mutated allele . In addition, the radiosensitivity of tumour cells is altered by oncogenic ras expression, probably as a result of the effect of the kras mutation on several intercommunicating pathways . The prevalence of mutations in the kras gene at the time of diagnosis is highest in pancreatic cancers (> 80% of cases), notably pancreatic adenocarcinomas predominantly harbour kras forms with a guanine to thymine transversion in codon 12 . Wei and colleagues examined samples collected from 30 patients with pancreatic cancer and found that 24 of them showed mutations at codon 12 and only one at codon 13 . However, concurrent kras mutations frequently occur in patients with pancreatic cancer . A positive association has been found in patients with pancreatic cancer between tobacco exposure and mutations in the kras gene . Similar associations have also been reported for coffee drinking, and milk, butter, and alcohol consumption [88, 89]. However, no direct evidence of a causal relationship between these dietary components and mutations in the kras gene has been presented . The second highest incidence (about 50% of cases) of mutations in the kras gene is found in colon cancers [90, 91]. The first stage is characterized by the development of a small, benign tubular type of adenoma or polyp with sporadically detectable kras mutation(s). The second stage is more aggressive and is usually associated with patches of definitive carcinoma cells, which may grow into invasive cancers characterising the third stage . Mutations of the kras gene have been identified in tissues from both adenoma and carcinoma cases, but at much lower frequencies in colon adenoma tissues than in carcinoma tissues [93, 94]. The incidence of mutation in the kras gene has been found to be low and to occur mainly in the small adenomas of patients with familial adenomatous polyposis, who have a predisposition to colon cancer in the kras gene associated with colon cancer appear most often in codons 12 (28%) and 13 (8%) of exon 1 and less frequently in codon 61 . In colorectal cancer, the main substitution (gly to asp) has been found to occur in codon 12 . Mutation from ggt (gly) to gtt (val) in codon 12 has been observed more frequently in primary metastatic carcinoma, suggesting that this mutation may confer a more aggressive phenotype in colorectal carcinoma . A mutation in codon 13, resulting in the substitution of gly with asp, observed in colon cancer has been shown to be associated with reduced survival rates . This kind of kras gene mutation has also been shown to occur more frequently in unstable, than in stable, colon tumours [97, 98]. Losses of kras wild type alleles in both mouse and human lung adenocarcinomas and squamous carcinomas have been found in many studies, notably in 67% to 100% of chemically induced murine lung adenocarcinoma cases harbouring a mutant kras . In humans, kras mutations appear in 1030% of lung carcinoma cases, demonstrating strong associations with a history of smoking and poor prognosis [100, 101]. Among both current and former smokers, further, although some researchers have found sporadic kras mutations in non - smokers with early onset of cancer, smoking history is an important factor and is correlated with increased occurrence of mutations in the kras gene in lung cancer cases . Mutations in the kras gene in codons 12 and 13 were detected in 21% of nsclc (non - small cell lung cancer) tumour samples examined in the tribute iii trial . Nsclc patients have a tendency to accumulate activating mutations in either the egfr or kras genes . However, a clinical study has shown that mutations of these two genes are, in general, mutually exclusive . Although higher kras mutational frequency is primarily found in cancers of the pancreas, colon and lung, possible links between kras hyperactivity and human breast cancer have been explored recently . Hollestelle at al . Found mutations in 12.5% of cases but the sanger cosmic database version 28 (http://www.sanger.ac.uk/genetics/cgp/cosmic/) records only a 5% incidence . The lower frequency of kras mutations in breast cancer cell lines suggests that the gene mutation may be less important in breast cancer carcinogenesis than in other forms of cancer, although mutations at a hotspot in the kras gene have been found in a small subset of breast cancers . Many clinical trials have shown that a poorer response to chemotherapy, a shorter time - to - progression, and worse overall survival are consistently associated with specific mutations in oncogenes . Kras is one of the most frequently mutated oncogenes in many cancers, and it is also one of the most important predictors of resistance to targeted therapy using egfr1 tyrosine kinase inhibitors (egfr - tkis). Two of the most important egfr - specific tkis are gefitinib (iressa, zd1839) and erlotinib (tarceva). The first indications of the predictive strength of the association between the kras gene and therapeutic responses to the egfr - tki gefitinib were originally observed in nsclc patients with tumours bearing the wild - type form of the kras gene and constitutively activated egfr1 gene, due to activating mutations in exons 18 to 21 or high copy number / amplification of the egfr1 gene . Clinically, better responses to tyrosine kinase inhibitor treatment were observed in patients with adenocarcinomas and well - differentiated tumours, female patients, non - smokers, and people of asiatic origin [107109]. Clinical research data show that gefitinib monotherapy is well tolerated and active against a wide range of tumour types, including colon, head, neck, breast, prostate, and lung cancers, especially nsclcs . Egfr - tkis are usually used as the second line therapy in patients after failure of chemotherapy . However, gefitinib did not pass the registration procedure in the european union because insufficient clinical benefit was demonstrated, probably because european clinical trials did not include sufficient good responders clinical data also suggest that the drug represents a new therapeutic option for nsclc patients with brain metastases . After the successful tribute and talent clinical trials, erlotinib (tarceva) was also approved by the us fda in 2002 as a second or third line treatment for nsclc after failure of standard chemotherapy . However, molecular analysis revealed that patients who have activating mutations in the kras gene (exon 1: codons 12, 13, or 61) with or without increases in egfr copy numbers did not derive benefit from this therapy and had about a 96% chance of disease progression . Similarly, eberhard et al . First observed the relationship between kras mutations and the outcome of erlotinib therapy in a randomized phase iii clinical trial in which the drug was used, in combination with first line gold standard chemotherapy (carboplatin and paclitaxel), to treat advanced nsclc patients . Patients with the kras mutation exhibited a shorter time to progression (three months) and a shorter overall survival (four months) when treated with a combination of erlotinib and first line chemotherapy, such as treatment with cisplatin, compared to the group with wild - type kras, for whom the time - to - progression was 12 months . Most nsclc patients in the erlotinib treatment study had expressed wild - type kras, and their kras status had greater prognostic than predictive value as a biomarker . However, in colorectal cancer, mutations in the kras gene are important predictive (as well as prognostic) biomarkers, since the effectiveness of treatment with cetuximab and panitumumab is impaired in tumours with the activating mutation . Information regarding the status of the kras gene allows the selection of appropriate therapies for patients who do not display activating mutations and the selection of alternative therapies for patients with mutations . Although results pertaining to the role of kras in the prognosis of clinical outcome or prediction of therapeutic responses to egfr1 tyrosine kinase inhibitors are interesting, they need to be validated in larger and prospective trials, using standardized and sensitive mutation detection techniques . If the associations are confirmed, knowledge of the mutation status of kras in nsclc tumours could help physicians decide which patients should receive gefitinib and/or erlotinib . Interestingly, kras gene mutations also seem to provide strong predictive indication of therapeutic responses to other classes of tyrosine kinase inhibitors, as recently demonstrated for the imatinib mesylate (glivec). Imatinib is the standard drug for patients with chronic myeloid leukaemia (cml) and patients with gastrointestinal stromal tumours (gists), expressing the bcr - abl fusion protein and tyrosine kinase receptor c - kit, respectively . Further, drug resistance to imatinib is usually attributed to mutation of the imatinib - binding sites of these proteins, although amplification of the bcr - abl fusion gene or overexpression of multidrug resistance proteins may be involved in some cases . However, a recent study by agarwal et al . Colorectal cancer is another frequent neoplasia that is associated with activation of the egfr1 pathway, so it is not surprising that novel and successful therapeutic strategies for this condition involve egfr1 protein kinase inhibition . In contrast to nsclc, two monoclonal antibodies against egfr1, rather than small molecular inhibitors of egfr1, are generally used for treating colorectal cancer: cetuximab (erbitux) and panitumumab (vectibix). In accordance with the effect of small molecular egfr1 inhibitors in nsclc, kras alterations play a critical role in the response of colorectal cancer patients to such therapeutic monoclonal antibodies . Indeed, kras mutation status is the most important predictor of resistance to cetuximab or panitumumab; both the median progression - free survival of cetuximab - treated patients and overall survival was recently found to be superior in a kras wild - type group than in a kras mutant group (31 versus 10 weeks, and 16 versus 7 months, respectively). On september 27, 2006, the us fda approved the completely humanized monoclonal anti - egfr1 igg2 antibody panitumumab (vectibix) for clinical use in the third line treatment of patients with metastatic colorectal carcinoma who had progressed after standard chemotherapy . Kras panitumumab therapy was tested in a randomized study involving 463 patients, and the results showed that the wild - type kras gene is essential for its therapeutic activity . Progression - free survival of patients with wild - type versus mutant kras gene tumours was 12 versus seven weeks, while response rates obtained in another study were 17% versus 0% . These findings strongly indicate that kras gene status should be routinely tested as a critically important diagnostic biomarker to determine which patients will derive therapeutic benefit from egfr1 inhibition . Indeed, analysis of the kras gene status in colorectal cancer cases has become conditio sinequa non for deciding whether or not to apply cetuximab or panitumumab therapy in routine clinical practice and fda has updated vectibix and erbitux labels in 2009 to include this information . Surprisingly, the effects of kras gene mutations on tumour sensitivity to cytotoxic chemotherapies and radiation have only been explored in a few studies . However, expression of a 12 val mutated form of kras has been shown to increase the resistance of cancer cells to radiation therapy . Similarly, the presence of oncogenic kras has been found to significantly increase the sensitivity of cells to a novel class of anticancer agents, cucurbitacins, in a p53- or p21-dependent manner . In contrast, an ovarian cancer cell line tov-21 g bearing a mutant allele of kras is reportedly significantly more sensitive to cisplatin and radiation, but not to paclitaxel or campthotecin, than the corresponding kras wild type line . However, results of clinical studies by rosell and colleagues (1995) showed that patients with a mutation in the kras gene had poorer clinical responses to paclitaxel monotherapy than wild type controls, suggesting that kras gene status is a predictive marker of paclitaxel resistance . In a phase iii retrospective study on nsclc patients (tribute), randomly treated with carboplatin and paclitaxel with erlotinib or placebo, patients with kras mutant tumours showed poorer clinical outcomes when treated with erlotinib plus chemotherapy compared to chemotherapy alone . An updated clinical trial (crystal) involving 540 metastatic colorectal cancer patients demonstrated that cetuximab in combination with folfiri (folic acid, fluorouracil, and irinotecan) in first line therapy is highly effective against kras wild type, but not mutant, tumours . However, further analysis of the data showed that neither the response nor the progression - free survival of patients treated with chemotherapy alone were significantly affected by kras gene status, although the overall survival of patients with kras mutant tumours was significantly shorter than that of patients with kras wild type tumours . Recently we have also shown that egfr may represent a predictive molecular marker for poor response to preoperative chemoradiotherapy in locally advanced gastric carcinoma . Responses to chemoradiotherapy were found in 60% of egfr - negative patients, but only 13% of egfr - positive patients (p = .044), and pathologic complete responses were observed in 29% of patients with egfr - negative staining, but none (of eight) egfr - positive patients (p = .16). The above findings regarding the role of the kras gene in tumour responses to cytotoxic therapies appear to conflict somewhat . The predictive and prognostic significance of oncogenic kras seems to have been mixed in many studies, and the contributions of variations in the gene to clinical outcome appear to differ according to tumour types and therapeutic interventions . Clearly, further studies are urgently needed to confirm and clarify the findings in large prospective biomarker - oriented clinical trials . Other clinical trials have also demonstrated that activating mutations in the kras gene can contribute to tumour progression by affecting the expression of vascular endothelial growth factor (vegf), which plays a critical role in tumour angiogenesis . Inhibition of kras expression by selected kras antisense oligonucleotides has been shown to be associated with significantly reduced secretion of vegf - a165 into the medium of colorectal cancer cell cultures . In addition, in a cohort of patients with pancreatic tumours, 25/33 (76%) with kras mutations showed higher vegf expression, and their median survival was shorter, than those with tumours expressing the wild - type allele . Similar findings have also been reported from a study of nsclcs, in which higher vegf expression was observed in 50% of tumours bearing a kras gene mutation . Although these studies suggest that kras gene status could play an important role in responses to anti - vegf targeted antiangiogenic therapy, a recent study by hurwitz and saini showed that groups of patients bearing either kras mutant or wild - type tumours derive therapeutic benefit from first line application of the anti - vegf monoclonal antibody bevacizumab (avastin). Furthermore, although both groups of patients (i.e., those with wild - type kras and mutated kras genes) benefited from adding bevacizumab to chemotherapy, both progression - free survival and survival was better for wild - type kras patients, both with chemotherapy alone and with chemotherapy plus bevacizumab . Bevacizumab did not increase the percentage of patients with mutated kras who responded to treatment . An optimal therapeutic drug should be able to specifically target the mutated kras gene or its product, have minimal systemic toxicity and be orally active . Unfortunately, drugs like this remain to be developed and less efficient strategies need to be used in clinical trials . However, in addition to the cancer therapies mentioned above, several therapeutic agents and strategies can directly suppress the activating mutant form of the kras gene, and thus improve the efficiency of chemotherapy and biological therapy . One possible approach for inhibiting kras expression is to use antisense oligonucleotides or viral constructs delivering antisense sequences in order to inactivate the mutant oncogene rna message . In addition, synthesis of mutated kras protein has been repressed by applying a small interfering adenovirus - mediated rna (sirna), and the specifically designed sirna was shown to have prolonged anti - proliferative effects against various tumour cancer cell lines expressing mutated kras proteins . Another, similar strategy to target mutant kras mrna is based on designing an mrna ribozyme that specifically interacts with a mutated form of the kras mrna and encodes catalytic rna molecules that bind to the mrna substrate by base - pair complementation, leading to translation arrest and/or degradation of the specific mrna . The kras - specific ribozyme strategy has also been shown to suppress successfully the proliferation of kras - mutated tumour cells . Recently an interesting novel strategy employing farnesyltransferase inhibitors (ftis) was shown to inhibit the biochemical transactivation initiated by the mutated kras gene . Farnesyl transferase is an enzyme that primarily regulates zinc metabolism by the addition of a farnesyl group to the cysteine residue of a protein . At least 30 proteins (including kras) require posttranslational farnesylation to reach their membrane positions and function properly in cell signalling . Farnesyl inhibitors represent a novel class of biologically active anticancer drugs that inhibit cell growth . After the discovery that ras proteins have to be farnesylated to become functionally active, several farnesyl inhibitors were developed . However, phase ii and phase iii clinical trials conducted to date have found that kras ftis might not be sufficient to inhibit the mutated - overactive forms of kras protein . The reason for this is probably incomplete inhibition of farnesylation, because garnesylation of kras protein by geranyltransferase i leads to suppression of the effects of farnesyltransferase inhibitors [131, 132]. It should be noted that although kras inhibitory strategies have shown promise in preclinical trials and have been partially successful in clinical trials, there are insufficient data on their efficacy in combination with anti - egfr1 strategies to recommend their routine use as yet . The evidence from various studies summarized in this review demonstrates that the kras protein is an important signal transducer involved in the regulation of various cellular responses during cell proliferation, differentiation, and survival . A pivotal function of kras protein in the regulation of the mapk and pi3k / akt pathways is its effect on the proliferation rate of both normal and cancer cells . Activating mutations of the kras protein, which frequently occur in cancer cells, overall, this review summarizes novel approaches allowing the management of cancers with or without kras mutations, and highlights the importance of early identification of somatic mutations in the kras gene in cancer biopsies.
Palisaded encapsulated neuromas clinically manifest as solitary, firm, non - pigmented, dome - shaped nodules on the face of adult patients . Palisaded encapsulated neuroma is known as a benign tumor of the facial skin, and is rarely found in the oral mucosa . Microscopically, the tumors are characterized by moderately cellular, fascicular proliferation of spindle cells that show some areas of parallel nuclei . A bundle of nerves interposed between the schwann cells typically aggregates in palisades and is identified by s-100 protein immunohistochemical stain . An alternate designation i.e. Solitary circumscribed neuroma (scn) was proposed by fletcher in 1989 . Irrespective of the nomenclature, pen / scns are considered as reactive hyperplastic processes . In 2010, koutlas and scheithauer considered pen / scns as relatively common true neuromas of the skin or mucosa . As a peripheral nerve sheath tumor, pen accounts for only 0.04 to 0.05% of oral biopsy specimens . Other peripheral nerve sheath tumors are neurofibroma, schwannoma (neurilemmoma), mucosal neuroma associated with multiple endocrine neoplasia iii, nerve sheath myxoma and granular cell tumor . In the mouth, pen is mostly found on the hard palate and maxillary labial mucosa as a small, superficial and usually painless nodule . The lesion is frequently diagnosed between the 5 and 7 decades of life, with equal sex predilection . A preferred treatment for pen is conservative local surgical excision; although gross total resection has been recently claimed to be the treatment of choice . A 48-year - old man was referred to the oral medicine department of babol dental school, complaining of a tongue mass persisting for one year . Physical examination revealed an exophytic sessile mass measuring 0.3 0.4 cm with rubbery consistency on the anterior one - third of the dorsal surface of the tongue (fig . Clinically, the overlying mucosa was depapillated and had increased vascularity . Under the impression of a pg, excision of the mass was done and no recurrence was reported at 12 months postoperatively . Histopathological sections showed an encapsulated mass within the connective tissue, composed of interlacing fascicles of spindle cells that were consistent with schwann cells . The nuclei, showing a parallel orientation within the fascicles, were characteristically wavy and pointed, with no significant pleomorphism or mitotic activity . The overlying epithelium was atrophic and no rete ridges were seen (figs . 3 and 4). Giemsa special stain revealed no mast cells within the stroma, ruling out the differential diagnosis of neurofibroma . Photomicrograph showing an encapsulated proliferation of neoplastic cells (hematoxylin and eosin staining; original magnification 40). No evidence of malignancy is observed (hematoxylin and eosin staining; original magnification 400). Mucosal neuroma can be distinguished histologically from other neurogenic tumors such as neurofibroma, neurilemmoma and pen . Briefly, mucosal neuroma is not encapsulated and does not have palisading nuclei; whereas neurofibroma and pen are encapsulated . Mucosal neuromas are usually associated with the multiple endocrine neoplasia syndrome iii (men iii), a rare syndrome with potentially fatal consequences such as medullary carcinoma of the thyroid . Other clinical signs of the patients with men iii can be considered in the diagnosis of oral lesions such as a mucosal neuroma . However, when oral lesions are present in absence of other diagnostic signs, histopathological evaluation can be helpful . The microscopic examination of the mucosal neuroma shows nerve bundles in various sizes surrounded by normal connective tissue, which are not usually seen in pen . A traumatic neuroma is not a true tumor, yet it develops as a proliferation of neural tissue that is caused by injury to a peripheral nerve . Traumatic neuromas are usually associated with pain, ranging from pain on palpation to a constant severe pain . These include the presence of perineural cells surrounding individual microfascicles, the greater abundance of interstitial collagen, mucoid matrix and myelin components, and the more orderly parallel arrangement of axons in traumatic neuroma . Antoni a, organized spindle cells in palisaded whorls, and antoni b, haphazardly distributed neoplastic cells, are two common patterns which are often found during histopathological examination of schwannomas . Other microscopic criteria include verocay bodies and the more definite palisading in the nuclei than that in pen . Contrary to the latter, it is extremely difficult to microscopically differentiate neurofibroma from pen, especially when an incisional biopsy has been performed . The absence of marked fibrous capsule and the irregular arrangement of the neoplastic cells are the main differential clues seen in neurofibroma compared with pen . The significant presence of mast cells, usually observed among tumoral cells of the neurofibroma, is also detectable using histochemical or immunohistochemical staining methods . As regezi and colleagues stated, pen / scn may be misdiagnosed clinically once identified somewhere in the mouth other than the palate . This may be an obvious clinical impression since intraoral pen / scns are mostly found on the hard palate . Conversely, the tongue involvement comprises less than 8% of pen / scn cases . As for this case, the pen resembled a pg on the dorsal surface of the tongue (a common site for neurilemmoma and neurofibroma, but not for pen). An erythematous lesion in a less commonly affected site rarely happens to be a pen . Besides, the tongue is a potential site for pg . Have reported that pg is most commonly seen on the attached gingivae, tongue, lower lip and buccal mucosa . The age of patient may be an important clinical parameter when the list of differential diagnoses of a lesion is formulated . Our patient was 48 years old, which was close to the recently reported average age for pg patients (52 years). Pyogenic granuloma usually occurs in patients older than 39 years, with equal gender distribution . Soft tissue enlargements of the oral cavity often present a diagnostic challenge because a diverse group of pathological processes can produce such lesions . Pyogenic granuloma can manifest as a painless smooth or lobulated mass with a surface that bleeds quite easily . Because of their high level of vascularity, young pgs are red, whereas older lesions are more collagenized and appear pink or normal colored . Clinically, oral pg occurs as an exophytic lesion manifesting as small, erythematous papule on a pedunculated or sometimes sessile base . Pyogenic granuloma arises in response to various stimuli such as chronic low - grade irritation, traumatic injury and hormonal factors . However, the effect of female hormones on oral pg was questioned by bhaskar and jacoway since they found lesions both in males and females with no sex predilection . Palisaded encapsulated neuromas may be misdiagnosed clinically once they appear somewhere in the oral cavity other than the palate . A pen arising on the dorsum of the tongue may mimic a pg with similar clinical morphology . Even the patient s age may be misleading . On the other hand, peripheral nerve sheath tumors must therefore be included in the list of differential diagnoses for a pg - like lesion on the tongue.
The syndrome with ectrodactyly, ectodermal dysplasia, cleft lip and cleft palate (eec) is a complex disease with a variety of abnormalities of the ectodermal and mesodermal germinal layer . The first author who described this disease was eckoldt in 1804 . Split hands or feet are characterized by agenesis of the third ray and possible fusion of the remaining fingers or toes . The ectodermal component of this syndrome includes hair with hypotrichosis, hypopigmentation, the teeth with hypodontia, enamel hypoplasia and microdontia and ultimately the nails, which present themselves dystrophic in most cases . A number of associated anomalies are also frequently found . In the 230 published cases, 84% of patients presented with split hands or feet . Dysplasia of the ectoderm arises in 77% of patients and clefts appear in 68% of cases . Important hints may indicate an eec syndrome already in the clinical examination of the patient and in the investigation of family history . Two case reports of our department shall demonstrate the management of eec syndrome affected patients with all - in - one closure of the lip cleft and palate cleft and the treatment regimen in manifest ectodermal dysplasia . A newborn baby with a unilateral complete cleft lip and palate (left side- cleft palate width 23 mm) first child of non - consanguineous marriage was referred to our department . Additionally, the child presented a deletion of the central finger on the right hand and fusion of the second and third finger on the left hand as well as fusion of the second and third toe on both feet . Paper - thin, dry and reddened skin and sparse scalp hair were also visible (figure 1). The father suffered from the eec syndrome, too-, showing similar hand malformation and a bilateral cleft lip and palate, which was treated in multiple surgeries . In our patient, feeding of the child was possible with a palate obturator . For (naso)alveolar molding lip taping with an elastic plaster (dynacleft, barrie on, canada) the cleft lip width was still 12 mm . For improved soft - tissue conditioning, lip adhesion two months later, the one - step procedure of the cleft lip and palate closure was performed . The operative procedure involves firstly in intravelar veloplasty with microscopic view, secondly in restore the nasal floor with two choanes and pedicled flaps without push back and at the end doing only a gingivoperiosteoplasty but without touching the germ area . At the end, a palatal plate was fitted for 7 days . In our protocol, we do a secondary osteoplasty with 9/10 years of age . The operation time was 60 min for the palate . During the closure of the palate to prevent pressure damage on the tongue the mouth gag was often released . Nevertheless, after surgery, the child had to be re - intubated at the intensive care unit due to an exfoliative stomatitis and dramatic swelling of the back of the tongue with impairment of the upper respiratory tract (figure 3). Supportive treatment with an anti - edematous and anti - inflammatory medication (cortisone intravenously) was initiated the further post - operative course of treatment was without complications (figure 4). We suppose that there is a connection with the eec which makes the tissue more sensitive but this is a hypothesis that is not proven . The clinical examinations showed a complete cleft lip and palate (left side) and deletion of the central finger on the right hand, fusion of second and third finger of the left hand and fusion of the second and third toe on both feet . Swelling of the back of the tongue and exfoliation after the all in one closure and before re - intubation . Follow - up 6 months after surgery with harmonious upper lip and no abnormalities in the epidermis . Until now, no treatment concerning hands and feet was performed . This case presents the sister of the above described boy, born 3 years and eight months later: born at 38 week of gestation with a birth weight of 2985 g she showed a bilateral cleft lip, syndactyly on all extremities and a conspicuous dryness of the skin (figure 5). Clinical findings and the family history lead again to the eec syndrome . Similar to the brother an obturator plate with nasoalveolar molding was adapted . At the age of 8 weeks a lip adhesion was performed . With 4.5 months the clinical examination of the patient 3 h after birth shows the typical characteristics for the eec - syndrome . No complications have occurred in wound healing due to the eec - syndrome until now, no treatment concerning hands and feet was performed . . Characterized by ectrodactyly, ectodermal dysplasia, cleft lip and/or cleft palate . Since then, numerous reports in the literature have expanded the clinical appearance . The transmission is usually autosomal - dominant trait with variable expressivity and reduced penetrance [4 - 6]. Some authors believe that the classic case of the eec syndrome is caused by mutation of the p63 gene . The variability in the phenotypic expression is explained by the interaction of ectodermal with the mesodermal germ layer . The eec syndrome must be differentiated from other syndromes, which also show an ectodermal dysplasia and orofacial clefts as the rapp - hodgkin syndrome, the aec syndrome (syndrome with ankyloblepharon filiform adnatum, ectodermal dysplasia and cheilognathopalatoschisis, [10 - 12]). Others have their own characteristics, for example the rapp - hodgkin syndrome presenting a short stature and special lineaments . The eec syndrome has to be distinguished from other diseases with acral anomalies and oral cleft, including the acrorenal syndrome and from the fetal alcohol syndrome . Minor anomalies of the eec syndrome are renal malformations, deafness, mental retardation and choanal atresia . Other recently reported associated anomalies are the insufficiency of the pituitary gland, anorectale malformations and hypoplasia of the thymus . Until now, three types of eec syndrome and their respective gene loci were molecularly identified . Balanced chromosome changes or interstitials deletions were found: type 1 is linked to gene locus 7q11.21q21.3, type 2 and type 3 with chromosome 19 locus with 3q21 (p63) [21 - 23]. Recently, heterozygous mutations in the p63 gene have been shown to 3q27 by amino acid substitutions in the dna - binding domain, which are considered as the main cause for the formation of the gap in hands and feet [25 - 27]. The ultrasound in prenatal diagnosis plays an important role in the early detection of ectrodactyly and cleft lip and palate [28 - 30]. For a more accurate prenatal diagnosis of this syndrome, a molecular study was introduced . With the use of a dna extraction from fetal chorionic villi, a prenatal dna analysis can be carried out in a pregnancy at risk for the eec syndrome ., eec children will be treated like other non - syndromic cleft patients . In the first 24 h after birth, an obturator plate and if necessary and due to dry skin possible, upper lip taping is adapted for alveolar molding . The gastric tube see in figure 4 was removed after the obturator plate was inserted . If the cleft lip remains more than 15 mm a lip adhesion is an option . Normally, this will be performed at the age of 8 weeks, in the first case it was delayed due to surrounding circumstances . Approximately 8 weeks later, a closure of all layers in a single operational step will be performed . One - stage procedures with 34 months can lead to disturbance in growth of the maxilla, but the extend was similar to the mean values of multistage procedures assessed in the eurocleft study . Children and their parents have no further psycho - social stress due to multiple surgical interventions at an early age . The main therapeutic approach depends on the expression of the ectodermal dysplasia and should be evaluated individually for each patient . It is mandatory that the patient has a close follow - up with, if necessary, functional physiotherapy, speech therapy and early functional orthodontic care . Whenever cases of eec syndrome occur, it is important, according to their phenotypic characteristics, to follow an interdisciplinary approach to reduce complications, to minimize undesirable sequelae and provide the best possible medical care . Ethics and consent: a written consent from the parents for the publication of photos is available.
Paclitaxel is an antineoplastic drug isolated from a bark extract of taxus brevifolia (taxaceae) the governments of the us and canada approved paclitaxel for sale in 1992, and a parenteral solution of paclitaxel subsequently became commercially available in japan in 1997 . Paclitaxel is used clinically in the treatment of ovarian, breast, endometrial, stomach, and non - small cell lung cancers in japan . The main adverse drug reactions to paclitaxel include gastrointestinal symptoms, peripheral neuropathy, arthralgia, muscular pain, nausea and vomiting, epilation, and pyrexia . Paclitaxel tends to be soluble in n, n - dimethylacetamide, acetonitrile, methanol, and ethanol but is relatively insoluble in water . Because 50% ethanol is used as the solvent for clinical paclitaxel injections, we hypothesized that impairment of specific central nervous system (cns) functions by ethanol or its cleavage product, acetaldehyde, as well as adverse reactions related to intoxication, may occur following treatment with this preparation . Thus, the possibility of adverse reactions following intake of ethanol accompanying paclitaxel administration should not be overlooked . Since many hospitals in japan are located in rural areas and are not conveniently accessible by public transportation, most patients drive to the hospital . Thus, it is important to consider the possible cns depressant actions of ethanol contained in injectable drug formulations, in order to reduce the risk of serious car accidents . Furthermore, in the road traffic act in japan, the breath ethanol concentration that constitutes drunk driving is 0.15 mg / l this threshold is lower than those in the uk, usa, and canada (0.40 mg / l), and those in australia, germany, and france (0.25 mg / l). It is important to ensure that patients who receive paclitaxel injections containing ethanol do not have breath ethanol concentrations exceeding the legal threshold . Although research on plasma ethanol concentrations following paclitaxel administration has been published previously, only a few reports have evaluated the correlation between ethanol intake during chemotherapy and the ethanol concentration in exhaled breath . Here, we investigated the concentration of ethanol in exhaled breath after chemotherapy with an intravenous paclitaxel infusion . Thirty japanese outpatients (mean age 55 8.6 years [range 3574]; 2 male and 28 female) who received treatment with paclitaxel (80330 mg / day) for breast, ovarian, or gastric cancer were eligible subjects for this research . This clinical study was approved by the institutional review board for clinical trials at gunma university hospital (maebashi, japan). Written consent was obtained from all patients after they were informed of the study procedure . The volume of ethanol administered and the infusion rate of ethanol were calculated from the volume of the paclitaxel infusion and the administration time . Immediately after administration of the intravenous infusion to a subject, a balloon - type gas detector tube (kitagawa gas detector tube system; komyo rikagaku kogyo kk, kanagawa, japan) was used to measure the concentration of ethanol in exhaled breath . The levels of aspartic acid aminotransferase (ast) and alanine aminotransferase (alt) were noted from the medical records, and the alcohol drinking history was taken from each patient . Correlations between the total amount of ethanol administered and the ethanol concentration in exhaled breath, and between the intravenous infusion speed and the ethanol concentration in exhaled breath, were calculated using pearson s correlation coefficient . Thirty japanese outpatients (mean age 55 8.6 years [range 3574]; 2 male and 28 female) who received treatment with paclitaxel (80330 mg / day) for breast, ovarian, or gastric cancer were eligible subjects for this research . This clinical study was approved by the institutional review board for clinical trials at gunma university hospital (maebashi, japan). Written consent was obtained from all patients after they were informed of the study procedure . The volume of ethanol administered and the infusion rate of ethanol were calculated from the volume of the paclitaxel infusion and the administration time . Immediately after administration of the intravenous infusion to a subject, a balloon - type gas detector tube (kitagawa gas detector tube system; komyo rikagaku kogyo kk, kanagawa, japan) was used to measure the concentration of ethanol in exhaled breath . The levels of aspartic acid aminotransferase (ast) and alanine aminotransferase (alt) were noted from the medical records, and the alcohol drinking history was taken from each patient . Correlations between the total amount of ethanol administered and the ethanol concentration in exhaled breath, and between the intravenous infusion speed and the ethanol concentration in exhaled breath, were calculated using pearson s correlation coefficient . The patient characteristics, the amount of paclitaxel administered, the speed of the intravenous infusion, and the concentration of ethanol in exhaled breath are summarized in table i. the average ethanol concentration in exhaled breath immediately after the intravenous infusion of paclitaxel was 0.028 0.015 mg / l (range 0.000.06). Ethanol concentrations in exhaled breath of individual patients hepatic function in all patients was assessed to be within the normal range, as indicated by ast and alt values of 1233 u / l and 1262 u / l, respectively . The correlation coefficient between the total amount of ethanol administered via the intravenous infusion and the ethanol concentration in exhaled breath was weak (r = 0.25; p = 0.055) [figure 1a]. In contrast, the intravenous infusion speed had a relatively stronger positive correlation with the concentration of exhaled ethanol (r = 0.49; p = 0.11) [figure 1b]. Relationship between the ethanol concentration in exhaled breath and (a) the total amount of ethanol administered via the intravenous paclitaxel infusion; and (b) the speed of the paclitaxel infusion . The patient characteristics, the amount of paclitaxel administered, the speed of the intravenous infusion, and the concentration of ethanol in exhaled breath are summarized in table i. the average ethanol concentration in exhaled breath immediately after the intravenous infusion of paclitaxel was 0.028 0.015 mg / l (range 0.000.06). Ethanol concentrations in exhaled breath of individual patients hepatic function in all patients was assessed to be within the normal range, as indicated by ast and alt values of 1233 u / l and 1262 u / l, respectively . The correlation coefficient between the total amount of ethanol administered via the intravenous infusion and the ethanol concentration in exhaled breath was weak (r = 0.25; p = 0.055) [figure 1a]. In contrast, the intravenous infusion speed had a relatively stronger positive correlation with the concentration of exhaled ethanol (r = 0.49; p = 0.11) [figure 1b]. Relationship between the ethanol concentration in exhaled breath and (a) the total amount of ethanol administered via the intravenous paclitaxel infusion; and (b) the speed of the paclitaxel infusion . More than 90% of ethanol is metabolized by alcohol dehydrogenase (adh) and aldehyde dehydrogenase 2 (aldh2) in the liver it has been reported that people with low aldh2 activity show hereditary sensitivity to the effects of alcohol, and approximately 50% of japanese people are poor alcohol metabolizers thus, the percentage of japanese people who experience facial flush and heart palpitations in association with elevated blood aldehyde concentrations after drinking alcohol is larger than that of europeans and americans . Therefore, there is a greater risk of intoxication leading to a car accident in people who have poor ethanol metabolism, because the blood ethanol concentration remains high even after consumption of a relatively small amount of alcohol . Ethanol is eliminated primarily by a saturable (michaelis - menten) process hence, the half - life of ethanol changes according to the dose or the rate of administration . Paclitaxel injections contain 50% (v / v) ethanol; thus, if 300 mg of paclitaxel is injected, 25 ml ethanol is also administered . Furthermore, because the first - pass effect does not apply to intravenous infusions, the effects of ethanol will be greater than with oral administration . In this study, an ethanol concentration in exhaled breath that exceeded the threshold for drunk driving, as specified in the road traffic act, was not detected in any patient, but there was one case that reached more than 40% of the threshold . Moreover, a previous report described several cases that exceeded the threshold defined by the law the relationship between the ethanol concentration in breath and that in blood has been investigated, and a method of deducing the blood concentration from the concentration in breath has been established . Moreover, when considering the cns effects, the ethanol concentration in breath (which reflects the arterial blood ethanol concentration) is considered to be a more suitable indicator than the venous blood ethanol concentration . The ratio of venous blood ethanol concentrations to exhaled breath ethanol concentrations is approximately 2000: 1 the average blood ethanol concentration estimated from our findings was 0.06 0.03 mg / ml . Webster et al . Reported that the average plasma ethanol concentration after administration of paclitaxel in caucasian patients was 0.07 0.10 mg / ml when the average doses of paclitaxel in both studies (155 76 and 293 35 mg, respectively) are taken into consideration, the estimated blood ethanol concentrations may have been a little higher in our study . The difference in the body size between japanese and caucasian subjects may have affected this . Because ethanol has a fast elimination rate, its concentrations steady state rapidly, and this is why the plasma ethanol concentration at the end of administration depends on the infusion speed . Thus, the ethanol concentration in exhaled breath after administration of paclitaxel is considered to be affected by the infusion speed but not by the total amount of ethanol administered . There were several subjects who complained of facial flush or light - headedness after the end of the intravenous infusion, which may have been a response to the ethanol metabolite, acetaldehyde in these cases, markers other than the breath ethanol concentration should be considered, in order to assess the degree of intoxication . In general, patients with high sensitivity to ethanol tend to present with symptoms of alcohol impairment and also have impaired decision - making ability . The gender bias of the patients should be mentioned as a limitation of this study . Because most patients in the study were outpatients with breast cancer or ovarian cancer, the majority of the patients were female . It has previously been shown that when the same dose of ethanol is administered to male and female subjects, higher blood concentrations are reached in females than in males, and this may have affected our results . We have shown that the ethanol concentration in exhaled breath after administration of paclitaxel is affected by the infusion speed rather than by the total amount of ethanol administered . However, it is difficult to predict from this information which patients will show a high breath ethanol concentration . Hence, all outpatients receiving paclitaxel should avoid driving from hospital when possible and, if driving is unavoidable, they should drive only after taking a sufficient break . The possible effects of the ethanol additive should be considered carefully when administering drugs, such as paclitaxel, with a high volume of ethanol additive.
Chronic myelogenous leukemia (cml) represents about 1520% of all cases of adult leukemia in western populations . Cml is a clonal myeloproliferative disease characterized by the presence in> 95% of patients of the t(9;22)(q34;11) translocation known as the philadelphia chromosome . This translocation causes expression of the bcr - abl fusion protein, a tyrosine kinase with constitutively increased kinase activity which is thought to be necessary and sufficient for the initiation of cml . The development of small molecule tyrosine kinase inhibitors (tkis), such as imatinib mesylate, which inhibits the increased bcr - abl tyrosine kinase activity, have dramatically improved the prognosis of patients with cml [5, 6]. Imatinib mesylate induces complete hematologic remissions and cytogenetic responses in the majority of patients in chronic phase cml, but the response in more advanced stages is usually only partial and less durable . About 4 - 5% of patients per year develop resistance to imatinib either because of bcr - abl gene amplification or more commonly point mutations in bcr - abl [7, 8]. If imatinib is discontinued because of toxicity or other reasons, the majority of patients relapse fairly promptly . One hypothesis that could explain the customary relapses after stopping therapy is that tkis and conventional cytotoxic drugs therapies are unable to eliminate all bcr - abl positive cells, presumably sparing a relatively small number of leukemic early progenitors and stem cells that are quiescent and are not killed by the inhibitors or other drugs at clinically tolerable concentrations . These cells constitute a reservoir of bcr / abl positive cells capable of functioning as leukemic stem cells or limited stem cells . If for any reason a patient has to stop therapy, these cells have sufficient self - renewal ability to recreate the disease by reconstituting the stem cell population and also enhancing the probability that the leukemic cells will develop resistance to the drugs, leaving bone marrow transplantation as the only option for survival . In order to search for critical differences in biochemical pathways and regulatory networks between cml and normal quiescent progenitors and stem cells that might ultimately serve as selective targets, we decided to compare the gene expression profiles of the normal and cml quiescent cell fractions . As a strategy to enrich for these quiescent cells, we isolated cd34 + cells by positive selection from the mononuclear cells of normal bone marrow and cml blood samples and then separated the cd34 + cells into g0 and g1/s / g2/m fractions by flow cytometry . We found 1, 204 genes significantly upregulated and 1, 133 downregulated in cml - g0 cells compared to normal g0 cells (resp ., 3.1% and 2.9% of the genes represented on the affymetrix chip), thereby permitting us to compare gene expression profiles in highly enriched normal and leukemic quiescent progenitors and stem cells . A total number of eight patients diagnosed with ph+ cml (three in accelerated phase and five in chronic phase) were used in this study . The cml samples were all obtained from patients hospitalized at the memorial hospital during the period 19902006 . The bone marrow samples were purchased from cambrex (cambrex bio science rockland, inc ., mononuclear cells were isolated on a ficoll gradient (ficoll - paque plus, ge healthcare, cat #17 - 1440 - 03) from total nucleated cells of bone marrows of four healthy donors and peripheral blood of eight patients with untreated ph+ cml (five in chronic and three in early accelerated phase with 8.518% blasts in the peripheral blood). In the case of the patient samples, total cells after ficoll were frozen in liquid nitrogen in rpmi plus 10% dimethyl sulfoxide and 10% fetal calf serum and stored until selection . Cd34 + cells were positively selected using a midimacs immunomagnetic separation kit (miltenyi biotec, bergisch gladbach, germany, cd34 progenitor cell isolation kit, cat #130 - 0460701) after one round of purification, the recovered cells were passed through another round of purification using a second column . This way the purity of the cd34 + recovered cells was 96100% as assessed by flow cytometry . For the statistics about the starting number of cells from each sample and the cd34 recovered fraction, cd34 + cells were resuspended at a concentration of two million per 0.5 ml of staining buffer (sb: hbss without nahco3, 10 mm hepes, 1% bsa, 2% fetal calf serum) plus hoechst 33342 (bisbenzimide h 33342, cat #b2261, sigma, st . Louis, missouri, usa) at a final concentration of 20 nm / ml and incubated for 45 in a water bath at 37c . After washing once with sb plus 10% hoechst, the cells were resuspended in 0.5 ml of sb plus pyronin y (cat #p9172, sigma) at a final concentration of 1 g / ml and kept in a water bath at 37c for 20, washed once and resuspended in 1 ml of sb . After being stained, the cells were sorted using a moflo flow cytometer (dakocytomation, dako colorado, inc . Fort collins, colorado, usa) applying a gate in the region of logarithmic fluorescence intensity of 1000 + /100 for both pyronin y (fl3) and hoechst (fl7). Approximately 50,000 cells from the cml / g0 and cd34+/g1/s / g2/m fractions were analyzed for brdu incorporation using a roche kit (cat #11 296 736 001). Cells were pulsed with brdu for an hour in complete growth medium, fixed with ethanol, stained with a mouse anti - brdu monoclonal antibody, and then incubated with an anti - mouse - ig fluorescein conjugated antibody . Total rna was isolated from each group of cells sorted from the g0 control fraction (bm) or cml sample using trizol reagent (cat #15596 - 026, invitrogen corp ., carlsbad, ca, usa). Quality of rna was ensured before labeling by analyzing 5 pg of each individual sample using the rna 6000 picoassay and a bioanalyzer 2100 (agilent technologies, inc ., samples with a rna integrity number (rin) greater than 7.0 were considered suitable for labeling . For each sample meeting this standard, 20 ng of total its rna were labeled using the genechip two - cycle target labeling kit (affymetrix, inc ., santa clara, ca, usa). Ten micrograms of labeled and fragmented crna were then individually hybridized to the human genome u133 plus 2.0 array (affymetrix) at 45c for 16 h. automated washing and staining were performed using the affymetrix fluidics station 400 according to the manufacturer's protocols . Finally, chips were scanned with a high - numerical aperture and flying objective (fol) lens in the gs3000 scanner (affymetrix). In summary, from each sample (cml or bone marrow), we extracted rna and performed a separate gene expression profile and the changes in gene expression were derived from the statistical analysis in which we compared cml samples (eight independent gene expression data) versus bone marrow (five independent gene expression data). The microarray data were quantile - normalized, and the gene expression values were estimated using the rma method . A linear regression model was used to model the gene expression values, in which a batch factor was added to the model to account for potential batch effect since arrays were run in two distinct batches two years apart . Differences between the g0 gene expression values of cml and normal samples were tested using the moderated t - statistics . Storey's q - value that control false discovery rate was used to correct for multiple hypothesis testing . The microarray data have been deposited on the geo public repository (http://www.ncbi.nlm.nih.gov/geo/) under accession no . Rt - qpcr assay was used to determine the level of expression of the genes we found up and downregulated on the microarray . We used the iscript one - step rt - pcr kit with sybr green (cat #170 - 8892, biorad, hercules, ca, usa) with the following conditions: 10 @ 50c, 5 @ 95c, 10 @ 95c, and 30 @ 60c using 1 ng of rna for each reaction as a template . All samples were run in quadruplicate on an abi 9700 platform (perkin elmer / applied biosystems, foster city, ca, usa). The relative expression of each gene was calculated using the ct method using gapdh as a reference gene . Forward and reverse primers were designed in different exons, in order to avoid dna contribution to our final pcr product, using primer 3 software (available on line at: http://frodo.wi.mit.edu/). For the sequence of the primers used, see table 1 in supplementary material available online at doi:10.1155/2011/798592 . Three or four hundred total cd34 + cells or cd34 + g0 and g1/s / g2/m enriched cells per plate were assayed for colony growth in 1.3% methylcellulose as described in detail previously .the cloning efficiency (c.e .) Values are the average of 4 plates counted for each bar shown . Unless otherwise stated, either single (100 ng / ml) or three early acting cytokines: kl, fl, tpo (50 ng / ml each) with or without additional cytokines as noted: g - csf, gm - csf (10 ng / ml each), il-3, il-6 (20 ng / ml each), and epo 1 iu / ml were added the drugs shown to the cultures to stimulate or inhibit colony growth . Unless otherwise noted, 3 cytokines refer to kl+fl+tpo and 5, 6, 7, or 8 cytokines refer to kl + fl + tpo + g - csf + gm - csf il-3 quadruplicate plates of each sample were counted at days 14 or 15 using an inverted microscope including estimates of colony lineage and size . A standardized scale and colorcode for estimating colony size has long been used in our laboratory which has been verified by plucking single large and multiple smaller (pooled) colonies and hemocytometer counts of the numbers of cells contained in the different sized colonies . Gfu - gm: tiny <40 cells, small 40100, medium 100010,000, large 10,00040,000, x - large 40,000100,000, xx - large> 100,000 cells; cfu - e and bfu - e and mixed: tiny <505000, medium 500050,000, large 50,00010, x - large 105 10, xx - large> 5 10 cells . G - csf, gm - csf, and il-3 were obtained as gifts from kirin brewery co., gunma, japan and kl (kit ligand or scf, stem cell factor), fl (flt3 ligand), tpo (thrombopoietin), il-6, and epo (erythropoietin) were purchased from r & d systems, inc . Cd 133/2 apc (293 c3) was purchased from miltenyi biotech, gladback, germany . Cord blood samples were obtained from the new york blood center as samples judged too small for clinical use; three or 4 samples were pooled for our studies . Defrosted mononuclear peripheral blood cells from eight cml patient samples or five fresh normal bone marrows were individually used to isolate the cd34 + fraction using the midimacs immunomagnetic separation kit from miltenyi (see material and methods) with a percentage of purity of recovered cells varying between 95100% . Cd34 + cells from each samples were subsequently stained with hoechst 33342 and pyronin y and sorted individually according to their dye content (figure 1(a)). The region chosen for sorting the quiescent fraction (g0) allowed us to avoid collecting dead cells and cross - contamination with cycling cells . We also sorted the proliferating fraction (g1/s / g2/m), but due to its high heterogeneity, we did not conduct a detailed analysis on this fraction for comparison with the cd34+/g0 cells . Table 1 summarizes the numbers and percentage of cd34+/g0 cells and cd34+/g1/s / g2/m recovered from each patient and normal bone marrow . After percoll and ficol separation of total blood cells from normal bone marrow or cml blood on average 22% of normal and 11% of cml mononuclear cells were recovered . After passing the mncs twice on miltenyi columns for positive selection of cd34 + cells, 3.06% of normal and 3.15% of cml highly enriched cd34 + cells were obtained from the mnc fractions . Due to the low quality of rna recovered, one bm sample (#2) was not used to generate microarray data . The cd34 + cells were further separated into proliferating (g1/s / g2/m) and quiescent (g0) fractions by flow cytometer using hoechst and pyronin y staining . After sorting, the mean and range of recoveries of normal and cml cd34+/g1/s / g2/m were 18% and 14.7%, respectively, while the recoveries of normal and cml cd34+/g0 cells were 4.3% and 3.05%, respectively, or an average of 0.016 and 0.013%, respectively, of the total normal and cml starting cell populations . An average of 27% (2232%) of unstimulated cml cd34 + g1/s / g2/m cells incorporated brdu after an incubation period of one hour while less than 1% of cml or normal cd34 + g0 cells incorporated brdu without cytokine stimulation immediately after separation . Figure 1(b) shows representative pictures of cml cd34 + g1/s / g2/m and g0 cells immediately after separation after 1-hour incubation with brdu . The few g0 cells that incorporated brdu all showed punctated nuclear labeling indicating an early stage of dna synthesis while the g1/s / g2/m cells had varied nuclear staining patterns indicating different stages of dna synthesis . Without cytokines, the viability of both normal and cml g0 and g1/s / g2/m cells declines rapidly and few viable labeled or unlabeled cells remain after 2 - 3 days and almost none after 4 - 5 days . However in the presence of 6 or 7 cytokines (kl + fl + tpo, each 50 ng / ml, + g - csf + gm - csf + il-3 il-6, each 10 ng (ml), viability remains excellent (95100%) and the majority of both normal and cml total cd34 + cells and g0 and g1/s / g2/m cells are induced to proliferate rapidly in liquid culture with average doubling times of ~30 hours . Continuous exposure to brdu (5 m) is toxic to both proliferating normal and cml cells, and viability declines rapidly after 2 days so results comparing incorporation of brdu during continuous exposure of cytokine stimulated g0 cells is limited to the first 48 hours . With stimulation by 7 cytokines, 92% and 94% of cml g0 cells incorporated brdu during continuous exposure for 24 and 48 hours, respectively, (average of 3 experiments) while the corresponding 24 and 48 values for normal g0 cells were 8% and 72%, respectively . These experiments suggest that the majority of both normal and cml cd34+g0 and g1/s / g2/m cells remain viable and can be stimulated to proliferate with 7 cytokines, but that the cml / g0 cells are more poised than the normal g0 cells to begin proliferating . To validate the robustness of our microarray data, we performed quantitative real - time pcr (qpcr) independently on each of four cml cd34+/g0 and two normal bone marrow cd34+/g0 samples that were different from the ones used to generate the microarray data . On each sample, we tested the expression of all the genes represented in figure 1(c), averaged the results obtained either from the four cml samples or two bm and then calculated the resulting fold change in expression for each gene between the two groups . Since the amount of rna recovered after the sorting was very limited, we performed a single - step rt - qpcr using the ct method (the primers used are listed in table 1, in supplementary material available online at doi:10.1155/2011/798592). This way, we were able to use as little as 0.1 ng per reaction enabling us to do four replicates per gene tested . We found that out of fourteen genes tested, which were differentially expressed between the normal and cml / g0 by the microarray, thirteen were confirmed by qpcr (figure 1(c)). Using a q value of less than 0.05 as a threshold, we found 1,204 genes significantly upregulated and 1,133 downregulated in cml / g0 compared to normal bm / g0 cells (for a complete list of the genes see supplementary material available online at doi:10.1155/2011/798592). As an additional criterion for selecting the genes significantly differentially expressed, we considered in the analysis only those with differences in expression higher than three folds . This resulted in 292 down- and 192 upregulated genes to be considered . Using this as a starting point, we later extended the analysis to look for genes that could corroborate our initial tentative conclusions, but in this second step, we considered also genes that had at least a two - fold difference and more than one set of probes changed . We have grouped the genes according to their reported functions and discussed the possible significance of the most relevant findings regarding differences between normal and cml cd34+/g0 cells . In the genes linked to cell cycle regulation, we found an almost equal number of them differentially expressed characterizing cml / g0 cells as nonproliferative when compared to normal g0 cells, (e.g., upregulation of mtss and downregulation of cdc14b), and as proliferative via upregulation of cdc6 and cyclin b2 . The most striking difference is in the number of genes upregulated in the cml / g0 cells that are either involved in dna replication (topo2a, rrm2, gins1 and 2) or are part of the mitotic spindle machinery (map9, cetn3, anln, dlg7). This subset of cml cells seems to be in a nonproliferative state but essentially ready to enter into the cell cycle upon stimulation, having the machinery for cell division and dna replication expressed and ready to work . This conclusion is compatible with findings reached independently by another group, showing that cml cells are much more easily triggered into cell cycle than their normal counterparts . Among the most significantly differentially expressed genes that we found between the cml and normal quiescent cells, many of these genes the first three genes belonging to this group are prominin-1 (cd133), id1, and flt3 . A second group includes genes that have been found overexpressed in both of two independent studies that have analyzed hematopoietic stem / progenitor cell transcriptomes, hlf, rbpms (hermes), gata3, tnfsf10 (trail), and crhbp [20, 21]. The third group includes: cd110/mpl, gbp2, sptbn1, arg2, birc3, crhbp, hla - e, hoxa3, hoxb6, spink2, nrip1, prkch, rapgef2, and tloc1, all genes that are overexpressed in hsc - enriched populations of bone marrow / cord blood and mobilized peripheral blood cells . Last but not least, we found as differentially expressed msi2 (musashi-2), another well - known stem cell marker, hes-1 a hematopoietic stem cell marker and il7, that is, expressed in human adipose - derived stem cells . All these genes are overexpressed in hematopoietic stem / progenitor cells compared to more differentiated ones, but in our microarray, all of them are downregulated in the cml / g0 fraction compared to the normal / g0 fraction . This provides additional evidence that with the methods employed, we are indeed enriching for quiescent hematopoietic stem cells and early progenitors in normal bone marrow but when we apply the same technique to enriching cml quiescent stem and progenitor cells, the latter are in a more advanced stage of differentiation . Because the cml g0 cells were enriched from the blood of patients with highly elevated wbc counts while the normal g0 cells were enriched from the bone marrow, this might offer a partial explanation . However, since previous labeling studies conducted in vivo in cml patients with massive myeloid expansion have shown there is continuous trafficking and exchange of early progenitors as well as maturing cells between the bone marrow, spleen, and blood and in whom the differential counts and proliferative kinetics of the bone marrow and circulating cells were very similar, it is more likely that the gene expression results accurately reflect the average results of the entire cml / g0 subpopulation . Another element that supports our conclusion that the cml / g0 cells are more differentiated than the normal g0 cells is that six genes that belong to the polycomb repressive complex 1 (prc1), respectively, scml1, phf1, pcgf3, cbx7, l3mbt1, and 4, and one (epc2) belonging to the prc2 group, are downregulated in the cml / g0 fraction (all of them apart from scml1, with a difference in terms of relative gene expression under the twofold level, so they are not listed in table 1). The prc1 and prc2 complexes belong to the groups of epigenetic regulators and act as gene expression repressors . Downregulation of genes belonging to these two groups in the cml / g0 fraction reinforce the idea that the bulk of quiescent cml cells belong to a more differentiated state than the normal counterpart, as also proposed for blastic phase cml cells in a previous paper by jamieson . Another series of genes differentially expressed in the cml / g0 fraction enabled us to localize more precisely where the predominant myeloid expansion takes place.cml g0 cells express a series of megakaryocytic (nfe2, tesc and cd41) and erythrocyte markers (cd36, klf1, tfr2, ank1, and xk and four different hemoglobin chains (hbb hbq1 hbd, and hbg1) plus gata1, a gene whose expression is linked to hematopoietic cell differentiation . The overexpression of gata-1 is of particular interest since graf has shown that if gata-1 is expressed in myeloid cell lines, the cells' phenotype is completely changed to erythroid, probably due to inactivation of the myeloid regulator pu-1 by gata-1 . An unexpected finding, in view of the more prominent hyperproliferation of megakaryocytic than erythroid cells in cml, is that smad7, whose expression promotes megakaryocytic over erythroid differentiation, is downregulated in cml / g0 cells . On the other hand, two key genes promoting lymphoid differentiation (bcl6 and gata3) are downregulated and a gene expressed in neutrophils (ncf4) is upregulated, as might be expected . These data might be explained by the fact that cml g0 cells are neoplastic cells and therefore exhibit a common cancer - associated characteristic variously termed lineage infidelity, promiscuity, or ambiguity, a well - known phenomenon occurring in leukemia . It appears that the cells retain the phenotype of the original cells but at the same time deregulate many other pathways characteristic of other hematopoietic lineages, which could also explain why there's expression of fetal hemoglobin chain (hbg1) together with the adult hemoglobins . In conclusion, it appears that at least the majority of cml / g0 cells overexpress genes usually associated with erythro - megakaryocytic development which is probably correlated with the thrombocytosis frequently seen in patients with cml and also with the spontaneous growth of erythroid colonies in vitro by cml progenitors in the absence of erythropoietin, whereas normal progenitors always require epo . Figure 2(a) shows the average results of multiple cloning experiments comparing the cloning efficiencies (c.e.s) of normal and cml total cd34 + cells and the g0 and g1/s / g2/m subsets . No epo was added in any of the experiments, but the cml cells consistently produced erythroid colonies without epo, often including large or x - large bfu - e, and sometimes comprising over half of the total colonies, whereas in the absence of epo, the normal cells rarely produced any erythroid colonies and then only tiny or very small ones . In almost all experiments, the cml g0 cells generated more and larger erythroid colonies than the g1/s / g2/m cells . In other experiments not included in figure 2(a), the addition of epo to other cytokine combinations greatly augmented further growth of cml erythroid colonies as well as stimulating normal ones . It is also evident in figure 2(a) that cml total cd34 + and cd34 + g0 cells produced more total colonies than the corresponding normal cells when stimulated by the three early acting cytokines kl, fl, and tpo and to a lesser extend by kl+g and gm - csf, again showing the cml progenitors are more easily triggered into cycle than the normal cells . However, there was little difference or the normal g0 cells had higher total c.e.s when the cells were near maximally stimulated with 57 cytokines . Figures 2(b) and 2(c) show typical experiments comparing the cloning results of two normal and two cml g0 cells in more detail . Except for a few tiny or very small cfu - e, normal g0 cells shown in figure 2(b) produced only cfu - gm colonies without epo and had the greatest incremental growth of large and extra - large colonies with 57 cytokines; with addition of epo, about 1/4 to 1/3 of the normal colonies were erythroid or mixed, some very large . In marked contrast, the cml g0 cells shown in figure 2(c), when stimulated in the absence of epo by all the single cytokine and combinations shown (except g - csf alone and g - csf+gm - csf), produced a mixture of cfu - gm and cfu - e / bfu - e with about a third to one half of the colonies being erythroid or mixed and often very large . The maximum total cloning efficiencies of these two g0 samples were ~1424% after stimulation with 37 cytokines, but in other cml and also normal g0 samples total c.e.s of up to 4042% were observed after stimulation with multiple cytokines . No consistent differences were noted between the maximum total c.e.s between normal and cml cd34 + cells or g0 or g1/s / g2/m subsets, although there was more variability in the quality of the cml samples . Class i homeobox (hox) genes comprise a family of 39 transcription factors that share a highly conserved dna binding domain . Since several of them have been shown to play a role in hematopoiesis, we looked at their expression profile in our microarray . We found three members of this family under expressed in cml / g0 cells: hoxb3, hoxa5, and hoxa3 . Hoxb3 is expressed in the primitive cd34 + population, that is, highly enriched for human hematopoietic stem cells (hscs), and it is downregulated as the cells differentiate into committed progenitors, and together with hoxb4 it is required for normal hsc function . Hoxa5 is another gene involved in hematopoietic lineage commitment and maturation, and it seems to act as a repressor of the generation or proliferation of erythroid progenitor cells . Regarding hoxa3, there is not much information about its function in the hematopoietic system . Taken together, the pattern of expression of the hoxa genes in our microarray suggests, again, that the cml cd34+/g0 stem progenitor cells are more mature than the normal counterpart . Another two transcription factors are differentially expressed in the normal and cml / g0 fractions: nmyc is under expressed and wt1 overexpressed in cml / g0 cells . Nmyc is a well - known oncogene found to be expressed in neuroblastomas and retinoblastomas and also in myeloid and lymphoid leukemias, but it has not previously been reported to be dysregulated in cml . This gene plays a role in preventing differentiation so its expression is downregulated in cells that progress through more mature stages in order to acquire their final phenotype . Nmyc is under expressed in the cml / g0 progenitors so its level of expression may simply reflect their more advanced differentiation and more rapid maturation as previously reported . Wt1 pattern of expression in normal hscs is biphasic: high in quiescent cd34+/cd38, low in committed progenitors, and high again in differentiated cells (cd34). So, how to explain the higher expression of wt1 in cml / g0 cells? It has been reported that the oncogenic signaling from bcr / abl can induce wt1 expression and that while in normal mice only a few immature cells in the bone marrow express wt1, when cml is induced the percentage of bone marrow cells expressing wt1 rises considerably . The effect of this gene if expressed in progenitors cells is to keep them in a state in which they are not responsive to differentiation inducing signals . So, it could be that while wt1 tends to keep the cml / g0 cells in an aberrant quiescent state other programs for differentiation are still turned on, such as those inducing differentiation in the m / e lineages, another manifestation of lineage infidelity . As a strategy to identify novel anticancer treatments specific for the cml quiescent population, we looked for genes that were upregulated in the cml fraction and that in the literature had been previously found to have a potential role in other type of cancers . Following these criteria, we found three genes: pvt1, anxa2, and marcks . Marcks is a gene important for cell proliferation: it has been reported that while its expression is low in cell lines that are actively proliferating its expression increases when they stop dividing and enter the g0 phase . Additionally, the over expression of marcks inhibits proliferation of human tumor - derived choroidal melanoma cells . So its role in cml / g0 cells could be to keep them in an artificial quiescent state and as a consequence protect from the action of cytotoxic drugs . Blocking marcks activity would be one step necessary to make these cells respond again to cytokine stimuli and make them reenter the cell cycle . Marcks is a prominent intracellular substrate of protein kinase c: since different pkc inhibitors have been developed and available (like enzastaurin, ly317615.hcl), this hypothesis could be tested . Anxa2 is a lipid- ca - actin binding protein that has been reported to be upregulated in different human tumors like hepatocellular and pancreatic carcinoma and acute promyelocytic leukemia . But while in these type of tumors it seems to have a positive role in promoting cancerogenesis and metastasis in other tumors, it seems to slow down their aggressiveness or tumor cell migration, like in osteosarcoma and prostate cancer . The different functional roles of anxa2 in different malignancies probably reflect its tissue specificity, so without further evidence, it would be premature to suggest it may have a specific role in the cml / g0 population . Nonetheless, it remains a potential target candidate gene . The pvt1 gene encodes a number of alternative transcripts, but no protein or regulatory rna products have been found so far; recently, it has been suggested that this region might encodes for different mirnas where one at least seems to be oncogenic . The amplification of this locus has been shown to contribute to the pathophysiology of ovarian and breast cancer, and over expression of pvt1 has been detected in a subset of cases of aml and in other myeloid malignancies . Pvt1 role in cancer seems to be that of an inhibitor of apoptosis, so this is another potential target gene that is worth consideration . These three genes were not identified as overexpressed in a previous work that did gene expression profile comparing total cd34 + cml and normal cells, so it is plausible that the different expression levels of these three protein is due to the fact that they belong to the specific quiescent subpopulation of cd34 + cml cells rather than the total cd34+/cml cells . The gene most downregulated in cml cd34+/g0 cells compared to normal bone marrow cd34+/g0 cells is prominin-1 or cd133 (19.6 fold). Prominin-1 is a pentaspan transmembrane glycoprotein which was first isolated in 1997 from plasma membrane protrusions on murine neuroepithelial stem cells and was so named because of its prominent localization in these protrusions (from latin, prominere). There are two isoforms of the gene, ac133 - 1 and -2 (which is 27 nucleotides shorter): it was demonstrated that ac133 - 2 rather than ac133 - 1 is the predominant transcript expressed in hematopoietic stem cells (hscs) derived from fetal liver, bone marrow, and peripheral blood as well as in epidermal stem cells, a wide variety of fetal and adult tissues and several poorly differentiated human carcinomas, but not in more differentiated tumors . Based on these findings it was postulated that ac133 - 2 might serve as a good marker of undifferentiated cells, including stem and progenitor cells present in stem cell niches in multiple fetal and adult tissues . In contrast, the undeleted ac133 - 1 transcript originally found in the retinoblastoma cell line was not detectable in fetal liver or kidney or in adult pancreas, kidney, placenta, or brain, but was strongly expressed in fetal brain, suggesting distinct roles for the two isoforms in development and homeostasis of different fetal and mature organs rather than redundancy . Both cd133 isoforms localize to the plasma membrane and have been extensively used alone or in combination with other markers for the identification of stem and progenitor cells from many adult normal tissues and organs including leukemic stem / progenitor cells . Most studies have reported cd133 expression is reduced or lost during later stages of differentiation, but in a recent report, using a knock - in lacz reporter mouse model (il10/cd133) and immunostaining, cd133 expression was observed in the full spectrum of undifferentiated and differentiated colonic epithelial cells in both mice and humans . Both cd133 + and cd133 metastatic cancer cells formed colonospheres and tumors in nod / scid mice that could be serially xenotransplanted, and it was noted the cd133 cells formed more aggressive tumors and were more enriched for phenotypic markers thought to be more typical of cancer initiating cells (cd44+cd24) than cd133 + cells . In view of our finding that cd133 is downregulated in cml cd34 + g0 cells compared to normal cd34 + g0 cells, it is of particular interest that the authors suggested that its downregulation in aggressive colonic cancer may indicate transformation of primary cd133 + cancer cells into more malignant cd133 metastatic tumors . The cd34 antigen has been widely used as a marker of human stem cells and progenitor cells for both clinical stem cell transplantation and laboratory studies characterizing and comparing normal and leukemic stem and progenitor cells, although it is recognized that a small subset of cd34 cells also have repopulating ability and can give rise to cd34 + cells . Coexpression of cd34 and cd133 has been observed, and the cells expressing both antigens were found to have a higher cloning potential than those only expressing cd34 . Numerous studies have led to the recognition that hscs are concentrated in the lin - cd34+cd38/lo fraction . Of particular interest, wagner et al . Observed that the more primitive cd34+cd38 slowly dividing cells expressed higher levels of cd133 than the fast dividing, presumably more mature, cd34 + cd38 + cells . Unlike normal bone marrowenriched progenitors which form almost no erythroid colonies or only tiny ones in the absence of epo as shown in the examples in figures 2(a), 2(b), and 2(c), cml progenitors often produce erythroid colonies of varying sizes, some very large, after stimulation with various single cytokines or combinations of cytokines without epo . The production of erythroid colonies by cml progenitors in the absence of epo is consistent with the previous finding that bcr - abl tyrosine kinase supports normal erythroid development in erythropoietin - deficient murine progenitor cells . Under epo deprived conditions, we observed marked inhibition of cml erythroid colony growth by several potent inhibitors of bcr - abl, thus providing evidence that bcr - abl increased tyrosine kinase activity cooperates with kl and other cytokine activated pathways in early cml progenitors to reprogram and distort their direction of lineage commitment, which in normal bone marrow and mobilized peripheral blood progenitors, and to a lesser extent in cord blood cells, is more dependent on epo for production of erythroid cells . The production of erythroid colonies by cml progenitors without epo at first appears counterintuitive because the major expansion in cml clearly occurs in the granulocyte lineage . However, it is quite compatible with the upregulation of the genes noted earlier that are involved in early erythroid development in cml cd34 + g0 cells compared to normal cd34 + g0 cells . As already noted, prominin-1 (cd133) is the most downregulated gene in cml cd34 + g0 cells compared to normal g0 cells and expression of the cd133/2 antigen is also low in both cml cd34 + g0 and g1/s / g2/m and bcr - abl driven all-3 cells compared to normal bone marrow or cord blood cd34 + g0 and g1/s / g2/m cells (figure 3). Unlike normal bone marrow or mobilized peripheral blood cd34 + g0 or g1/s / g2/m cells which form no or only tiny erythroid clusters on stimulation with 3 to 7 cytokines without epo (figures 2 and 4), we observed that pooled cord blood g0 and g1/s / g2/m cells often produced small, medium, and even large erythroid colonies in the absence of epo (figures 4(b) and 4(c)). Figure 4 summarizes a number of representative experiments comparing the total cloning efficiencies (c.e.s) of cd34 + g0 and g1/s / g2 m cells from normal bone marrow, normal mobilized peripheral blood, pooled cord blood samples, and chronic phase cml peripheral blood samples . In all except some of the cord blood samples, the g0 cells had higher total c.e.s and almost always also produced larger gm and erythroid colonies (not shown) than the g1/s / g2/m cells (figure 4(a)), providing additional evidence that they are more primitive . To further examine the effect of cd133 on direction of lineage differentiation, we compared the formation of gm and erythroid colonies by cord blood cd34 + quiescent and proliferating cd133 + and cd133- cells . We observed that cord blood cd34 + g0 133/2 + cells form numerous myeloid colonies, some very large, but no erythroid colonies when stimulated by either 3 gfs or 7 gfs w / o epo (figure 5(a)). In contrast, while cord blood cd34 + g0 cd133/2 negative cells had similar total c.e.s with 7 gfs w / o epo, they formed a mixture of myeloid and erythroid colonies with about a third of the latter being large or extra large . In a similar experiment shown in figure 5(b) with pooled cord blood g0 and g1/s / g2/m cells, again the cd133 + cells produced only myeloid colonies and cd133+/2 negative cells from both the quiescent and proliferating fractions formed many erythroid colonies . The cells derived from the pooled cordblood samples shown in figure 5(b) had lower total c.e.s than those in figure 5(a), but both the g0 and g1/s / g2/m cd133/2 negative cells had higher percentages of erythroid and mixed colonies, some very large . Whereas the results clearly demonstrate that cd133/2 + cord blood - derived g0 and g1/s / g2/m cells in the absence of epo only form gm colonies and the cd133/2 cells form a mixture of erythroid and gm colonies, the distinction is less consistent and the results are more variable in comparable cml cells . Figure 6(a) illustrates colony formation by cml g0 and g1/s / g2/m cells with relatively low c.e.s enriched from pooled samples of two chronic phase cml patients . Both g0 cd133/2 positive and negative cells formed almost entirely tiny or small erythroid colonies when stimulated with 7 cytokines while only the cd133/2 negative g1/s / g2/m cells formed any colonies, again mostly tiny or small erythroid ones, but with some gm colonies . The three cytokines, kl+fl+tpo, stimulated growth of very few or no colonies . Figure 6(b) shows an example of colony formation by cd34 + g0 and g1/s / g2/m cells enriched from another chronic phase cml patient in which the cells formed mostly gm colonies, but with further separation of g0 cd133/2 + cells, the total c.e . Increased by about a third and there were higher proportions of large and x - large gm colonies and almost no even very small erythroid clusters . An example of a third cml cloning experiment is shown in figure 6(c) in which chronic phase cml g0 cells had very high total c.e.s when epo was added to the 7gfs, especially the cd133 + cells . As usually observed, the g0 cells had higher c.e.s than the g1/s / g2/m cells . Both the quiescent and proliferating cd133 negative cells formed mostly erythroid colonies while the cd133 positive cells formed predominantly gm colonies even in the presence of epo, although about 17% of the colonies were large or x - large bfu - e and mixed colonies . In summary, based on our observations so far, it appears that prominin-1, the product of the gene most downregulated in cml cd34 + g0 cells, is also underexpressed on the surface of both cml cd34 + g0 and g1/s / g2/m compared to normal bone marrow or cord blood cd34 + g0 and g1/s / g2/m cells (figure 3). Cord blood cd34 + g0 133/2 positive cells form numerous myeloid colonies, some very large, but no erythroid colonies when stimulated by either 3 gfs or 7 gfs without epo (figures 5(a) and 5(b)). In contrast, under the same conditions, cord blood cd34 + g0 cd133/2 negative cells form a mixture of myeloid and erythroid colonies with some of the latter being large or x - large . The role of (downregulated) cd133 in cml cd34 + g0 and g1/s / g2/m cells is presently less clear and appears to be more complex . The cml g0 cells in figure 6(a) had low c.e.s and both the cd133 positive and negative fractions produced almost entirely tiny and small erythroid colonies even when stimulated by 7 cytokines, while only the g1/s / g2/m cd133 cells produced a substantial number of colonies, mostly erythroid . In the experiment shown in figure 6(b), the cml g0 cd133 + fraction when stimulated with the same 7 gfs without epo increased the total c.e . By about a third compared to the total g0 cells, but the colonies were almost entirely gm, whereas the total g0 cells produced about 10% small- and medium - sized erythroid colonies . Finally as shown in figure 6(c), the cml cd133 + g0 cells when stimulated by 7 gfs + epo had one of the highest c.e.s (47.25%) we have observed with mostly large and x - large gm colonies, but also about 17% large and x - large bfu - e and mixed colonies, while the cd133 cells formed mostly erythroid colonies, mostly large or x - large . The above observations suggest that expression of cd133 in both normal and cml progenitors favors gm differentiation while cd133 negative cells produce a mixture of erythroid and gm colonies . However, the precise function of cd133 is still very uncertain and confounded by the necessity to use multiple cytokines to stimulate growth . Cml and cord blood progenitors are less dependent on epo in producing erythroid cells than normal bone marrow progenitors, but addition of epo markedly shifts the cells toward erythroid differentiation . Overall, the results suggest that prominin-1 may have an important role in determining the direction of lineage commitment, especially in cord blood and cml progenitors . We found no consistent difference in the numbers or sizes of the colonies produced by either normal or cml quiescent or proliferating cd133 + or cd133 cells, although some experiments suggested enrichment of one or the other might further enhance their overall proliferative potential . An attempt was made to see if it was possible to correlate changes in gene and surface antigen expression as measured by flow cytometry in subpopulations of normal and cml cells, but because of the small number of cells usually recovered it was only possible to do limited surface phenotyping in a minority of enriched samples . Examples of representative experiments showing similarities and differences in expression of surface antigens are shown in figures 7 and 8, and a summary of the overall results of multiple surface phenotyping experiments is given in figure 9 . In these and other studies, there were insufficient cd34 + total cells or subsets to compare additional surface antigens other than those shown . As illustrated in figure 1(a) and table 1, the cd34 + g1/s / g2/m cells are usually much more numerous than the cd34 + g0 cells so their phenotype is very similar to that of the total cd34 + cells (not shown). Figure 7(a) shows a typical study comparing surface markers of normal mononuclear cells from mobilized peripheral blood and the enriched total cd34 + cells from the same sample showing marked enrichment of cd34+cells and reduced expression of antigens expressed on more differentiated cells, including cd14, cd45ra, cd90, cd3, and cd19 . Figures 8(a) and 8(b) show comparisons of cell surface antigen expression of normal and cml total cd34 + and cd34+/g0 cells in several typical experiments . As would be expected, in most but not all normal and cml samples, expression of cd33, cd38, and cd45ra were reduced in the cd34 + g0 cells compared to the total cd34 + cells . Figure 7(b) shows the changes that occur in surface antigens during 12 days in liquid culture when normal cd34 + cells are stimulated to proliferate by two combinations of 4 cytokines . In the latter experiment, following stimulation with both cytokine combinations, cd34, cd38, glycophorin a, cd90, and cd45 ra expression rapidly declined and this usually occurred even faster in stimulated cml cd34 + cells (not shown). Most surface markers associated with granulocyte / macrophage, erythrocyte or megakaryocyte differentiation increased both in normal and cml cd34 + cells (e.g., cd 14, 15, 36, 41), although cd61 declined slightly . As is evident from comparing individual experiments, there was considerable variation in both normal and cml individual samples, but we have nevertheless attempted to provide an overall summary in figure 9 of the most consistent findings in multiple experiments of the similarities and differences in surface antigen expression between normal and cml total cd34 + cells and cd34 + g0 cells and when the latter are stimulated to proliferate . Overall, we did not detect any consistent differences between normal and cml total cd34 + cells or in their g0 and g1/s / g2/m subsets except that the cml g0 cells usually had lower percentages of cells expressing cd38 and sometimes of hla - dr than the normal g0 cells, but the results were not consistent enough to draw any firm conclusions . This study was initiated based on the assumption that quiescent cml cells and early progenitor cells may differ in their pattern of gene expression from comparable normal cells and that if critical differences could be identified, they would help to reveal the underlying molecular mechanisms responsible for the excessive overproduction of the cml population . It was further hoped that some vulnerable differences would be discovered that would be susceptible to targeting by highly selective drugs, since it was assumed that the quiescent cells would be largely unaffected by existing bcr - abl inhibitors such as imatinib and dasatinib as well as other drugs active against proliferating cells . So far, there has been only one other study attempting this, which was performed using the same methods but using fewer samples (five cml and two normal) and an older microarray platform with considerably less genes represented (14,500 versus 38,500). Because at least 10 cells were required for each sample for microarray analysis and the enriched cd34 + g0 cells comprised less than 0.02% of the starting cell populations, it was difficult to obtain sufficient cells, especially patient samples, to carry out the study . We initially collected 10 cml samples, but 2 had insufficient rna so only 8 are included . We also performed microarray analyses on cd34 + g0 and g1/s / g2/m cells enriched from cord blood and normal g - csf mobilized peripheral blood samples, but excluded them from the final analysis because some of the results differed from cells enriched from normal bone marrow samples and we considered the latter to be more appropriate normal controls . Both the normal and cml quiescent and proliferating fractions consisted of 96100% cd34 + primitive blast cells and less than 1% of cd34 + g0 cells incorporated brdu, so almost all of the latter were either in g0 or early g1 . Because there are no definitive markers to distinguish stem cells from early progenitor cells, it is unknown what percentage of each was present in the cd34 + g0 fractions, but we presume the majority of cells in both the normal and cml fractions were progenitors in various stages of development . Ideally, we would have preferred to also compare normal and cml stem cells, but were unable to do this because of a lack of clear stem cell markers and insufficient cell numbers . Some of the genes that were differentially expressed in normal and cml / g0 cells revealed a number of interesting findings which correlate nicely with some of the biological and functional abnormalities previously observed in cml cells . (i) in keeping with the gene expression data, normal and cml quiescent g0 cells are more highly enriched in primitive cells than the proliferating g1/s / g2/m cells . (ii) the cml g0 cells are in a slightly more advanced stage of development than the normal g0 cells, and as previously reported by graham et al ., the cml cd34 + g0 cells are more similar to the cd34 + proliferating cells than are the normal g0 and g1/s / g2/m fractions . (iii) in keeping with their more advanced stage of development and their upregulation of genes involved in dna replication or part of the mitotic spindle machinery, cml / g0 cells are more poised to begin proliferating than normal g0 cells and are more sensitive to growth stimulation by single cytokines or combinations known to act on early progenitors and stem cells . While normal and cml / g0 cells are almost equally responsive to stimulation by multiple cytokines, the cml cells are triggered into cycle more rapidly . (iv) once cml quiescent progenitors are stimulated by cytokines to begin proliferating, they undergo further differentiation and maturation more rapidly than normal quiescent progenitors, but both granulopoiesis and erythropoiesis are usually less efficient than in normal hematopoiesis as shown in cloning experiments in which the cml cells form many more small cfu - gm, cfu - e, and bfu - e compared to normal progenitors [14, 32]. (v) whereas normal cd34 + cells form almost entirely granulocyte / monocyte clusters and colonies in clonogenic experiments when stimulated by cytokines in the absence of erythropoietin, cml cd34 + g0 cells consistently spontaneously form a combination of gm and erythroid colonies in the absence of epo . The gene expression data clearly shows that cml / g0 cells have marked overexpression of genes associated with development of the erythrocyte and megakaryocyte lineages, and graham et al . (vi) prominin-1 (cd133) is the gene most downregulated in cml g0 cells, and there is lower expression of cd133 on the surface of these cells . Cord blood g0 cd 133 + cells form only gm colonies without epo while cd133 cells form a mixture of gm and erythroid colonies . The downregulation of cd133 appears to be associated with the spontaneous formation of erythroid colonies by cml progenitors in the absence of epo, but its precise role remains to be better clarified . It has been known for many years that both normal and neoplastic cell populations contain significant numbers of resting or quiescent cells that are considerably less sensitive to the damaging effects of irradiation, cytotoxic drugs, and other injurious agents than are proliferating cells [59, 60]. The dormant state is a protective mechanism that is of crucial importance in enhancing a population's probability of survival under adverse conditions, and early on the concept that dormant cancer cells are important obstacles to curability was widely recognized by both basic and clinical scientists [15, 25, 61]. If one accepts the premises that almost all lethal cancers originate in adult stem cells or early progenitors functioning as stem cells, that these cells are essential for initiation, maintenance, and expansion of the cancers, and that a large fraction of these cells, like normal stem cells, reside in a quiescent state in which they are resistant to most therapies, then it is obviously important to better understand their derivation and properties, to determine how normal and cancer quiescent stem cells may differ, and to look for ways to develop specific targeted therapies based on these differences . Activation of quiescent cells following a mitogenic stimulus by serum, cytokines, or other factors is highly complex and involves the coordinated and selective induction of expression and repression of hundreds of genes including specific cyclins, cyclin - dependent kinases (cdks), and protein kinase inhibitors (pkis). Many cell cycle genes are transcriptionally silent in quiescent cells, and they express only a limited number of cytokine receptors, and recent studies have shown that sirnas and micrornas are also involved in repressing gene transcription and translation in quiescent cells . When one considers the staggering complexity of all of the cytokines and other regulatory factors and cellular interactions that determine whether a quiescent stem cell residing in its protected niche is going to divide while simultaneously deciding what kinds of cells to produce, it is hardly surprising that our present understanding of the mechanisms regulating stem cell behavior is very incomplete . In our analysis, it appears that in many cases, clusters of presumably related genes that are differentially expressed are all associated with a particular stage of development or function, suggesting that their common dysfunction in cml g0 cells may involve aberrant co - regulation . Most authorities agree that the hsc population is heterogeneous, but it is still uncertain at what level of development the system permits changes to occur in phenotype and functionality, or when the differentiation hierarchy becomes fixed . Circumstantial evidence for both normal and leukemic stem cells favors a heterogenic model in which there is a continuum of stem and early progenitor cells with gradually declining potentials for self - replication, pluripotency, and other stem cell properties, but that some cells may also exhibit flexibility in responding to different stochastic influences in their developmental milieus . Some degree of reversibility may also exist whereby early progenitors can retain or reassume more primitive stem cell properties if needed, such as the ability for more extensive self - replication in order to replace or supplement damaged stem cells . However, it is likely that significant reversibility is restricted to early progenitors and that once they have become committed to differentiate along a particular lineage, it is doubtful that they can revert to functioning as stem cells . With regard to cancer stem cells, some years ago we postulated that most leukemias originate in limited stem cells which have more limited pluripotency than normal primitive hscs, but retain sufficient self - replicating potential to initiate a lethal leukemia . Many current researchers now agree, although some prefer to call them leukemia or cancer initiating cells to distinguish them from true hscs . While cells undergoing differentiation and maturation can become temporarily arrested or slowed in their progression through other phases of the cell cycle under certain conditions (e.g., hypoxia, increased cell density, exposure to toxins, cytotoxic drugs, irradiation, or other damaging agents), it is usually only stem cells and primitive progenitors that remain in a quiescent state for extended periods under normal steady - state conditions . Once progenitors become firmly committed to differentiation and maturation, serial cloning studies conducted in vitro and cytokinetic labeling studies with h - thymidine conducted in vivo [24, 62] have shown that both normal and leukemic cells usually proceed to undergo a variable but limited number of maturation divisions to produce terminally differentiated cells (which may be highly abnormal in leukemia and other malignancies), and which are incapable of reverting to regain significant self - renewal or other essential stem cell properties . While our in vivo h - thymidine labeling studies were less extensive in patients with lymphomas or solid tumors growing in ascetic form [63, 64], in cases in which it was possible to distinguish neoplastic cells in differing states of maturity, it appears that once the neoplasticcells become committed to maturation, if the environment is suitable, they usually continue to divide but are only capable of a limited number of divisions before dying spontaneously and are incapable of reproducing the disease . Overall, our in vivo labeling studies strongly suggest that the number of dormant cancer stem and progenitor cells continue to increase as the population of cancer cells expands and that they, thus, constitute a progressively greater obstacle to curative therapy in many types of cancer [11, 25, 65]. The constitutive tyrosine kinase activity of the p210 protein causes abnormal phosphorylation of regulatory proteins in numerous interacting signaling pathways [3, 25, 66, 67]. The overall signaling networks altered by bcr - abl are highly complex, indeed reaching a level of complexity that some observers have likened to heisenberg's uncertainty principle in quantum mechanics . Nevertheless, although the specific signaling changes responsible for each of the biological abnormalities that have been described are still incompletely defined, it is highly likely that the faulty signaling disrupts multiple interactive networks that normally tightly regulate the orderly well - coordinated processes of proliferation, differentiation, and maturation in normal hematopoiesis . This misregulation can probably explain all the abnormalities observed in early - stage cml including the initial overproduction of gm progenitors, the imbalanced lineage apportionment, the inefficiency of production of both granulocytes and erythrocytes, and all the other more subtle dysplastic morphological, biochemical, and functional changes that have been described . Gleevec and some of the newer bcr - abl inhibitors are highly effective in globally inhibiting the increased tyrosine phosphorylation of multiple proteins involved in these signaling pathways [25, 6971] and since bcr - abl is usually the sole mutation in early stage cml, the progenitors are restored to near normal behavior when the kinase is adequately inhibited . At higher concentrations, the abl tki inhibitors are lethal to both fresh primary cml progenitors and bcr - abl - driven cell lines while at still higher concentrations, they also kill normal progenitors and cell lines not driven by bcr - abl, the exact normal: cml lethal concentration ratios depending on the particular cells and inhibitors . However, as suggested by the usual relatively slow induction of remissions in cml patients over the course of weeks or months, it is doubtful if the bcr - abl inhibitors when administered in clinically tolerable doses actually kill many of the proliferating cml progenitors and precursors . Rather, by inhibiting bcr - abl's constitutive tyrosine kinase activity, they at least transiently restore the cells to functioning more normally, and in so doing the cml progenitors cease excessive cell production, presumably by reacquiring the ability to respond properly to quorum sensing signals that ensure maintenance of normal homeostatic cell density limits . Meanwhile, the later cml progenitors which are already committed to differentiation continue to proceed through a limited number of maturation divisions and then die as terminally differentiated cells, just as do normal cells . After the body burden of leukemic cells has been sufficiently reduced, the residual normal stem cells are released from the cml cells' (poorly understood) inhibitory effects and resume production, usually resulting in a complete hematologic or cytogenetic remission . The bcr - abl inhibitors are clearly a very important advance since they are able to induce durable remissions in the majority of cml patients in chronic phase, but they are not usually curative since most patients relapse if therapy is discontinued, probably because quiescent cml stem / progenitor cells are not killed by the drugs and are able to reproduce the disease [9, 72]. Several mechanisms of resistance have also been well described as noted earlier [7, 8] thus, while gleevec and other bcr - abl tkis are very effective in the early stage of cml in largely eliminating the majority of proliferating ph+ progenitor and precursor cells, more attention should be given to seeking ways to selectively kill the quiescent leukemia stem and progenitor cells . While our own studies so far have not revealed any specific vulnerable targets, it is important that the search be continued . The alterations in gene expression described here must be confirmed in a larger number of patients, and if possible with further technological advances, in still more highly enriched populations of early progenitors and stem cells . As the search proceeds, the significance of some of the differences in gene expression reported here may become clearer and eventually lead to discovery of new ways to selectively kill the quiescent cml stem and progenitor cells . For a number of reasons, it has become increasingly difficult to obtain large enough samples of cml cells to carry out the rigorous procedures required to isolate sufficient numbers of highly enriched stem and progenitor cells for further studies, so this is another important issue that must be addressed . In a broader sense, it is perhaps even more important to design similar therapeutic strategies for other types of cancer . Much of the recent development of anticancer drugs has been directed towards producing different classes of drugs that block segments of one or more signaling pathways that are known to be dysregulated by the particular initiating mutation(s) commonly found in different types of cancer . However in advanced malignancies it is often difficult to distinguish the importance of the primary causative mutation(s) compared to that of secondary or still later (passenger) mutations and the situation may become further complicated by multiple epigenetic changes . A huge number of new drugs of different classes are now available, but few cause complete or durable remissions and they are almost never curative . Greater emphasis should therefore be placed on more clearly identifying and whenever possible selectively targeting the primary driving mutations in the cancer stem or early progenitor cells in early stage disease, and also in developing new strategies to selectively kill the quiescent stem or progenitor cells that escape most current therapies.
Herpes simplex virus (hsv) is a neurotropic virus that has a large linear, double - stranded dna genome protected by a capsid with icosahedral symmetry surrounded by an envelope consisting of a lipid bilayer with embedded glycoproteins, having yet a proteinaceous region between the capsid and envelope called tegument . The hsv belongs to the family of herpesviridae, subfamily alphaherpesvirinae, and genus simplex virus [2, 3]. It is a virus that has a very complex life cycle and stands out as one of the most common pathogens in the etiology of sexually transmitted diseases worldwide . Hsv infects the mucosa of the mouth, eyes, and the human anogenital tract . After primary infection, the virus replicates productively within mucosal epithelial cells and enters sensory neurons via nerve termini . The virus is then transported to neuronal cell bodies where latency is established . The virus can remain in this latent state indefinitely but can be reactivated at any time during the lifetime of the host [4, 5]. During latent infection, no infectious virus is produced from infected cells, symptoms are absent in the host, and the transmission does not occur . However, reactivation can occur only in some cells, in the absence of symptoms, enabling the transmission of the virus . This seemingly benign strategy has critical importance to the survival of the virus, where the infected cell continues throughout the life of the host, providing a reservoir for periodic reactivation . Hsv is presented in the form of two distinct serotypes: herpes simplex type 1 (hsv-1) and type 2 (hsv-2), which are closely related but contain sufficient differences to enable type identification . Hsv-1 is usually transmitted during childhood by nonsexual contact, typically found in orofacial lesions, with latency in the trigeminal ganglia . Hsv-2 is the cause of most genital herpes and is almost always sexually transmitted, with latency in the lumbosacral ganglia . However, in the last decades, hsv-1 has been pointed out by several studies as a main causative agent of genital herpes, especially in college students [7, 8]. Regardless of which serotype of hsv is acquired, the infections remain intermittent throughout the host's lifetime, in symptomatic or subclinical form with or without periodic reactivations . The extent and frequency of recurrent genital herpes are highly type - specific, with hsv-2 reactivation outpacing hsv-1 by three to one . Thus, although an increase in genital infections attributable to hsv-1 has been well recognized in recent years, the situation regarding recurrence has apparently remained unaltered, with hsv-2 being the dominant agent . When there is a reactivation, the virus is excreted on the mucosal surfaces, allowing its transmission to susceptible individuals . Most hsv-1 and hsv-2 infections are subclinical . However, when the infection is symptomatic, the clinical manifestations of hsv-2 are typically characterized by recurrent, painful, vesicular, and ulcerative lesions, located in the genital and anal areas . In contrast, symptomatic hsv-1 infections are usually manifested as recurrent orolabial and facial lesions . Both hsv-1 and hsv-2 can cause genital herpes with greatest incidence among women of reproductive age, with risk of maternal transmission of the virus to the fetus and neonate . The acquisition of genital herpes during pregnancy has been associated with spontaneous abortion, intrauterine growth restriction, prematurity, and congenital and neonatal herpes . Vertical transmission from an infected mother to her baby can cause severe disease resulting in sequelae or death of the infant . Most neonatal infections result from exposure to hsv in the genital tract during passage through the birth canal, although they can also be transmitted to the fetus during the intrauterine phase . The risk of disease in the newborn is significantly higher when the mother acquires genital infection for the first time with hsv-1 or hsv-2 during pregnancy . Recurrent infections are rarely associated with disseminated neonatal disease in the newborn of immune - competent mothers . In fact, the pregnant women who acquire genital herpes as a primary infection in the latter half of pregnancy, rather than prior to pregnancy, are at the greatest risk of transmitting the virus to their newborn [2, 3]. Neonatal herpes has three major categories: skin, eye, and mouth disease (sem), central nervous system infection (cns), and disseminated infection (di). It has been observed that sem in itself is rarely fatal, but, without antiviral therapy, most cases progress to cns or di . Clinically, cns disease typically presents with nonspecific symptoms such as lethargy, poor feeding, irritability, vomiting, and seizures . The disseminated infection is manifested in various combinations such as hepatitis, acute adrenal insufficiency, myocarditis, and consumption coagulopathy . In brazil, there is a major lack of studies on the prevalence of genital infection by herpes simplex virus . Furthermore, the diagnosis of genital infection with these pathogens is not included among the mandatory prenatal examinations . Thus, there is no official data available on infection during pregnancy nor on the likely consequences of such infections in newborns . In this study we evaluated the prevalence for herpes simplex virus types 1 and 2 genital infection among pregnant and nonpregnant women, its association with the presence of cervical abnormalities detected by colposcopy and cytology, sociodemographic characteristics, and reproductive activity . This study included 236 sexually active women, 106 (44.9%) of whom were pregnant and 130 (55.1%) nonpregnant, enrolled among those who attended the health service by spontaneous demand for cervical screening programme or to prenatal care, and agreed to participate in the study . The participants were residents in natal, rio grande do norte state, brazil, who sought gynecological care at two primary health care units from may 2010 until september 2011 and who agreed to participate in the research . Study subjects were asked to fill in an anonymous questionnaire in order to identify different demographic and behavioral risk factors that may have implied their exposure to hsv-1 and hsv-2 . The patient's ethnicity was identified based on self - reports according to the criterion of the brazilian institute of geography and statistics (ibge), which classifies ethnicity into five categories: white, black, mulatto, asian, and native . In this study, the black, mulatto, asian, and native categories were combined into a nonwhite category . Data on sexual behavior included the age at the first sexual intercourse and the number of sexual partners during the lifetime . The study was approved by the ethics committee for medical research of the federal university of rio grande do norte (protocol 030/10cep / ufrn) and the written informed consent has been obtained from each subject . All women participating in this study underwent a clinical examination followed by a visual inspection of the vulva, vaginal walls, and cervix by colposcopy, with only two gynecologists, to detect possible abnormalities of these structures . During the examination, a solution of 5% acetic acid was applied initially and the first visual analysis was performed . After washing to remove the acetic acid, lugol solution of i2 (1%) in equilibrium with ki (2%) in distilled water was applied, and the second inspection was done . The colposcopy was considered with alteration, when at least one of the abnormal features (acetowhite lesions, punctuation, mosaics, inflammatory punctuation, congestion, and ulceration) in a localized area in the transformation zone was found . Two specimens containing exfoliated uterine cervical cells were collected from each patient using a cervical brush . One of these specimens was used to obtain a smear on a slide that was stained by the papanicolaou method and analyzed by cytopathological examination, based on the bethesda system . We considered as normal cytology, or cytology without alterations, the samples that presented no pathological alteration or only inflammatory changes . Cytology with alterations were the samples in which we detected the presence of atypical squamous cells of undetermined significance (asc - us), squamous intraepithelial lesions of low grade (lsil) or squamous intraepithelial lesions of high - grade (hsil). The other cervical specimen was conditioned in a tube containing a preserving solution (pbs + vancomycin + nystatin) and sent to a laboratory where it was processed for dna extraction . The tubes containing the cervical specimens were submitted to rigorous agitation before removal of the brush and were centrifuged at 300 g per 10 min . The supernatant was removed and the resulting pellet was processed for dna extraction using qiamp dna mini kit (qiagen, hilden, germany) following the manufacturer's recommendations . Around 30 ng of the dna samples was submitted to a polymerase chain reaction (pcr) to amplify a 110 bp fragment of the human -globin gene, using the primers pco3+/pco4 + to analyze the quality of target dna and the absence of pcr inhibitors . The products of pcr were submitted to electrophoresis on 8% polyacrylamide gel, followed by silver staining . The positive samples for -globin were analyzed for the presence of dna from hsv-1 and hsv-2 in separated reactions by pcrs specific for each type . Each reaction was composed of a master mix (promega, madison, wi, usa), 10 mm of each primer, and 2.5 mm of dna sample, to a final volume of 25 l . The pair primers hsv-1a (5-ccctgtctcgcgcgagccac-3) and hsv-1b (5-tcagccacccatacgcgtaa-3), which amplify a fragment of 142 bp, were used to detect the presence of hsv-1, while the primers hsv-2 (a) (5-ggacgaggcgccaaagcacacg-3) and hsv-2 (b) (5-tccgtccagtcgtttatcttcac-3), which generate a product 270 bp, were used to detect the presence of hsv-2 . The conditions for both reactions were as follows: incubation at 50c for 2 min and denaturation of dna at 95c for 5 min, followed by 40 cycles of 94c for 1 min, 45 s at 58c for annealing, an extension step at 72c for 30 s, and a final extension step at 72c for 10 min . The products of pcrs underwent vertical electrophoresis on polyacrylamide gel at 8% and subsequent visualization by silver staining . Statistical analysis of the results was performed using the chi - square test, while difference in proportion and associations between risk factors and hsv infection were analyzed by calculating the odds ratio (or) and their respective confidence intervals (95% ci). The analysis of individual questionnaires revealed that the profile of the segment of the population studied was characterized by being composed of women aged 1472 years, mean 30.3 10.8 years . Most of them were less than 30 years of age, were of nonwhite ethnicity, were married or living in a stable relationship with her partner, had elementary education or less, had family income (monthly minimum wage) up to one minimum wage (approximately us$300.00), had their first sexual intercourse and the first pregnancy at age 18 or less, had more than one sexual partner in their lifetime, was not pregnant at the time of the survey, and had at least one pregnancy . The presence of any of the serotypes of hsv alone or both together was detected in 95 women, revealing an overall prevalence of 40.3% of genital infection in the studied population (23.7% hsv-1 alone, 11.9% hsv-2 alone, and 4.7% the two serotypes together). The presence of at least one of the serotypes of hsv was detected in 36/106 (34.0%) pregnant women and in 59/130 (45.4%) nonpregnant women . We observed a significant higher prevalence rate of hsv-1 infection among non - pregnant women (30.0%), when compared to pregnant women (16.0%) (p = 0.0001). Hsv-2 alone was found in 13/106 (12.3%) pregnant women and in 15/130 (11.5%) nonpregnant women . The coinfection by the two serotypes of hsv, in the same patient, was detected in 5.7% of pregnant and 4.7% of nonpregnant women (table 1). Most of the participants in this study underwent morphological examinations by colposcopy (92.8%) and cytology (96.6%) to detect the presence of changes of the uterine cervix . The colposcopic examination was carried out on 90/106 (84.9%) pregnant women and in 129/130 (99.2%) nonpregnant women . In the group of pregnant women, the colposcopic examination showed that 38/90 (42.2%) had visible abnormalities of the uterine cervix . Among nonpregnant women, the colposcopic examination showed that 45/129 (34.9%) had cervical changes (table 2). Cytological examination was performed on 100/106 (94.3%) pregnant women and in 128/130 (98.5%) nonpregnant women (table 3). The cytological alterations were more prevalent in pregnant (41/100) than nonpregnant (22/128) women (p = 0.0001). We also analyzed the correlation between the presence of genital infection by any of the two serotypes of hsv and morphological findings detected by colposcopy and cytological examinations . There was not any significant difference in the prevalence rates for hsv-1 between the groups with or without colposcopic abnormalities, in both pregnant and nonpregnant women . However, the prevalences of hsv-2 were significantly higher in women with colposcopy abnormalities in both pregnant and nonpregnant women, compared to those with normal colposcopy (table 2). Similar results were found for women with or without cytological abnormalities (table 3). Analysis of the association between genital infection by hsv-1 and hsv-2, each individually, and the considered variables revealed the absence of association between genital infection with hsv-1 and all variables tested (table 4). The genital tract infection by hsv-2 was found to be associated with morphological alterations detected by colposcopy or cytology, ethnicity, marital status, and number of lifetime sexual partners . The nonwhite and single women and those who had multiple partners presented a greater risk of acquiring genital hsv-2 infection (table 5). It has been shown that the prevalence of genital tract infection by hsv-1 and hsv-2 varies substantially in the different geographic regions, including those within the same country . In this study, we found an overall prevalence rate of 40.3% . The found prevalence rate was almost three times higher than that reported for united states women (14.0%). It was also higher than that described in a similar previous study involving women of natal, brazil (28.4%). Considering each serotype of the hsv individually, hsv-1 was more prevalent than hsv-2, in the segment of the population studied, presenting coherence with the results reported in other studies, such as those reported for women of the united states, for women of israel, and for women of natal . This could be explained by changes in attitudes, behavior, and the sexual practices among adolescents, including oral - genital sexual contact . Furthermore, the use of condoms for vaginal intercourse could have reduced the exposure to genital infection by hsv-2, contributing to an increasing proportion of cases of hsv-1, compared with hsv-2 as cause of genital herpes infection . Hsv-1 was detected in 23.7% of women surveyed, presenting a prevalence rate almost equal to that found in a previous study for natal residents women (23.0%), but slightly smaller than the prevalence rate reported for women of the united states (32.0%). Hsv-2 was detected in 11.9% of the study participants, representing double the prevalence rate of 5.4% reported by pereira et al . In 2012, but close to that described for american women (16.2%). Still the prevalence rate described in this study was very similar to that reported for women of israel (13.3%), while it was less than the estimated prevalence for women of south america (varying between 20.0% and 40.0%). Coinfection by the two serotypes of hsv was detected in 4.7% of the women surveyed, which was above that in previous research conducted by pereira et al . Hsv-1 was more prevalent among nonpregnant women, whereas hsv-2 presented similar prevalence among pregnant and nonpregnant women . The prevalence of genital hsv-1 infection found in the nonpregnant women was higher than that described for women of the united states (14.0%). Regarding pregnant women, hsv-1 was found with a prevalence rate very similar to that reported in a previous study in natal's women (23.0%). The prevalence rate of hsv-2 was practically equal to that described for women in korea (17.0%) but was less than that reported for united states' women, approximately 22.0% . The prevalence rates found in nonpregnant women participating in this study for both hsv-1 and hsv-2 were higher than those described for women in the united states, which were 4.4% and 9.4%, respectively . Pregnant women had higher percentages of abnormal results in both colposcopic and cytological examinations, when compared to nonpregnant women . We observed in this study a higher proportion of women infected with hsv-2 among those who had abnormal colposcopy and/or cytology, in both groups of pregnant and nonpregnant women . This suggests that hsv-2 has a higher ability to cause lesions in the genital tract . This result is consistent with the literature data reporting that the extent and frequency of symptomatic recurrent genital herpes is highly type - specific, with hsv-2 reactivations being much more frequent, outpacing hsv-1 by three to one . Furthermore, it was shown that genital hsv-1 recurs infrequently and the rate of recurrences decreases rapidly over time, with a median recurrence rate declining by about 50% from the first to the second year of infection . When we evaluated the existence or lack of association between genital hsv-1 infection and sociodemographic variables and sexual activity, it was found that there was no association between the variables tested and genital hsv-1 infection among women of this study, corroborating with the results obtained in a previous study conducted in natal . However, a significant association was observed between the variables of ethnicity, marital status, and number of sexual partners over a lifetime and the occurrence of genital infection by hsv-2 . No association was observed with chronological age, education, and age at first sexual intercourse . These results are consistent with those obtained for israel women, regarding education, age of first intercourse, and number of sexual partners . With regard to ethnicity,, the morphological alterations may not have been caused exclusively by hsv, once that there are other pathogens that cause cervical lesions, such as hpv, that were not analyzed in this study . Besides, the studied population is limited to women attended in two primary health care units and may not be representative of the female population of natal . The analysis of a larger number of patients would allow obtaining more conclusive results . In conclusion, results of the current study show a high prevalence rate of both hsv-1 and hsv-2 in the studied population . The significant proportion of pregnant women infected by hsv-2 that presented morphological alterations in the uterine cervix suggests the importance of including the colposcopy or cytology exams as part of routine prenatal care.
Extragonadal germ cell tumors (eggcts) have the same histological components as their gonadal counterparts, but a gonadal mass is not detectable by palpation or high - resolution ultrasonography) here, we report a case of primary mediastinal seminoma with complete obstruction of the superior vena cava and no recurrence of the residual mass after intensive chemotherapy . His bilateral jugular veins were distended, and swollen lymph nodes were palpable in the right subclavicular region . A chest x - ray showed a mediastinal mass . Computed tomography (ct) of the chest revealed a solid soft tissue mass (95 75 112 mm) [in diameter] in the upper mediastinum, as well as a leftward displacement of the trachea and complete obstruction of the superior vena cava (figure 1figure 1pretreatment ct shows a solid soft tissue mass (95 75 112 mm) in the upper mediastinum, with leftward displacement of the trachea and complete obstruction of the superior vena cava . ). Based on the suspicion of a mediastinal tumor with lymph node metastasis, mediastinal radiotherapy and methylprednisolone were immediately started . Biopsy of the right subclavicular lymph nodes revealed metastatic seminoma (figure 2 figure 2biopsy specimen from a right subclavicular lymph node . Well - defined tumor cells with large nuclei and clear nucleoli and a large clear reticulum are formed as map - like alveolar tumor cells . Cobblestone growth can be seen with lymph node involvement surrounding the alveolar tissue . ), and the patient was referred for chemotherapy . Pretreatment ct shows a solid soft tissue mass (95 75 112 mm) in the upper mediastinum, with leftward displacement of the trachea and complete obstruction of the superior vena cava . Well - defined tumor cells with large nuclei and clear nucleoli and a large clear reticulum are formed as map - like alveolar tumor cells . Lactate dehydrogenase, -human chorionic gonadotropin and alpha - fetoprotein were all in the normal ranges . Ultrasound revealed a uniform, echogenic structure of the bilateral testicles, without any findings suggestive of malignancy . Radiotherapy and methylprednisolone were discontinued, and systemic chemotherapy was simultaneously started with a combination of cisplatin, bleomycin and etoposide (bep). Since lung function test values deteriorated after one cycle of bep, bleomycin was excluded from subsequent chemotherapy, and the regimen continued with etoposide plus cisplatin (ep). A partial response was observed after completing 3 cycles, but no further shrinkage was observed on subsequent examinations . Salvage surgery was then considered for the residual mass because the superior vena cava was still completely obstructed by the tumor . Although implantation of a prosthetic vessel was required, the procedure was not performed because it was considered to be a high risk surgery . Instead, the patient received 2 courses of vinblastine, ifosfamide and cisplatin (veip) as salvage chemotherapy . On completion of the chemotherapy, the evaluable lesions were considered to represent a " stable disease " (figure 3figure 3posttreatment ct shows less enhancement of a smaller soft tissue mass (30 18 36 mm).). As of december 2010, there is no evidence of residual tumor growth, new masses or metastatic foci . The patient is being closely followed up on an outpatient basis and has been free of recurrence for 32 months after intensive treatment . Posttreatment ct shows less enhancement of a smaller soft tissue mass (30 18 36 mm). Our patient was first diagnosed as having an egct arising from the mediastinum when he presented with chronic cough and complete obstruction of the superior vena cava . Since few patients with early germ cell tumors arising in the mediastinum have any symptoms, these tumors are often quite large by the time they are diagnosed and the mean maximum diameter of such tumors is reportedly 4.6 cm) in 2008, kesler et al . Reported that 10 surgical deaths occurred among 158 patients who underwent additional resection of residual primary mediastinal nonseminomatous tumors) like primary testicular seminoma, primary mediastinal seminoma has a good prognosis, and the rate of necrosis of the residual mass is as high as 92% when partial remission is obtained after chemotherapy)
Acute respiratory infections (aris) are important under - five morbidities causing lots of discomfort, frequent visits to a healthcare provider, admission to an indoor facility, and even mortality [1, 2]. India's reproductive and child health programme has components for the prevention and control of ari since 1992 . Aris, however, still continue to be the single largest contributor of childhood morbidity and mortality with estimated 35 episodes every year, 4 million pneumonias, and 1 million deaths [46]. Estimates also indicate that 3050% of opd attendance and 2040% of hospital admissions may be attributed to ari and pneumonia . For a disease of such magnitude and severity, it is important that all aspects of its epidemiology have to be known . Yet unfortunately, there are few studies reporting the detailed epidemiology of aris . Of the few studies that exist, very few studies have been conducted in peri - urban communities and fewer involved active surveillance done prospectively by qualified and trained health practitioners . Besides, most studies are 1 to 2 decades old [817]. In view of above, a prospective study to find out the current epidemiology of aris among infants and toddlers, the most affected age group, was planned . We report here the incidence, prevalence, clinical pattern, severity, seasonal pattern, and gender differences . This community - based follow - up study was conducted in 2011 - 12 at mehrauli, a peri - urban field practice area attached to the department of community medicine of a tertiary care teaching hospital of new delhi . Optimum sample size was worked out to be 106 children or 5512 child - weeks of exposure . Owing to logistics needed for the fortnightly follow - up, the study was restricted to one of the wards selected randomly (ward 4). Cut - off age for enrollment of children was kept as 24 months so as to ensure a follow - up of 12 months before child ceased to be toddler . The only inclusion criterion was parents' willingness to let their children participate, while exclusion criteria were two - fold: (i) child suffering from any chronic / severe illness and (ii) family of child not a permanent resident of ward 4 . Our sampling frame was constituted of 264 under - two children identified through a house - to - house survey one month prior to the study . Of these, 23 were not eligible for being included in the study (1 had thalassemia, 1 had nephrotic syndrome, 1 had very low birth weight, and 20 were temporary residents). Three parents refused to let their wards participate and were replaced by another eligible child residing closest to the refusal . History regarding presence of ari currently or in the past fortnight was elicited from a responsible caregiver and recorded on mics proforma of unicef . All children, whether or not their parents reported a current / past bout of ari, were assessed clinically by a qualified trained medical practitioner on the designated day . Additional information on type of ari, its severity, other accompanying symptoms, and so forth was assessed and recorded in a separate interview schedule validated by a faculty from paediatrics department . Background information on the physical, biological, and sociodemographic environment as well as the general health status of the child was noted . In context of physical environment, overcrowding, indoor air pollution, environmental tobacco smoke, and so forth history of breast feeding, immunization, and exposure to ari from siblings was assessed as part of the biological environment . Education, occupation, income, and social class formed the main stay of our sociodemographic assessment . Information collected on interview schedules was cross - checked for completeness and correctness by supervisors and a database was created using microsoft excel software . Incidence density of all aris has been described in terms of mean number of episodes per 100 child - weeks of exposure . Chi - square test has been used to detect statistical significance at 5% level of significance . A total of 2752 contacts were made with 67 boys and 39 girls at their residences . One enrolled subject went out of town for 2 months, thus reducing our observation period by 8 child - weeks to 5503 child - weeks of exposure . Four - fifth of study subjects were from middle income group and the remaining from high income families . A little over 5% of the mothers were illiterate and 32.1% had less than five years of schooling . Close to half of the subjects lived in overcrowded (47%) or ill - ventilated (21%) houses, and 14% of households did not have a separate kitchen and 18% were using biomass fuels in addition to liquefied petroleum gas (lpg). Besides, 45% of study subjects were exposed to environmental tobacco smoke (ets). One - fourth of subjects were having mild - to - moderate protein energy malnutrition (pem). Health assessment based on history as well as physical examination revealed that ari was the commonest illness (437 episodes in 105 children) followed by diarrhea (253 episodes in 85 individuals) and fever (69 episodes in 48 individuals). Prevalence of ari, diarrhea, and fever was thus found to be 34.5, 19.9, and 5.4 percent, respectively . Only 1 child did not seem to suffer from any type of ari during the study period, 105 (99.1%) children suffered from no pneumonia, cough, and cold (alone or in combination with other types of ari), 38 (35.8%) had pneumonia, and 5 (4.7%) had otitis media . Of the 437 ari episodes, 390 (89.2%) affected the upper respiratory tract only, 47 (10.7%) were pneumonias, and 5 (1.1%) were otitis media . Out of those children suffering from ari, proportions of children suffering from 1 to 3, 4 to 5, and 6 to 7 episodes of ari were 35.2, 45.7, and 19.1 percent, respectively . Of the 38 children who suffered from pneumonia, 9 (23.7%) had two episodes . The mean duration of uri was 6.16 days (range: 414 days) as compared to 11.65 days for lri (range: 719 days). The overall incidence of ari (all types) was found to be 7.9 episodes/100 child - weeks of exposure; incidence was higher in infancy (9.4 episodes/100 child - weeks) as compared to toddlers (7.0 episodes / per 100 child - weeks). Incidence of pneumonia was found to be 0.8 episodes/100 child - weeks (1.1 in infancy and 0.7 in toddlers). Highest incidence of pneumonia was seen in children less than 2 months of age (1.7 episodes/100 child - weeks). Incidence of no pneumonia, cough, and cold was nearly constant throughout infancy, decreasing in the second and third years of life (lowest incidence of 3.3 episodes/100 child - weeks seen in children of 3036 months). The incidence of pneumonia was one - tenth that of ari and that of severe pneumonia or otitis media was roughly one - tenth that of pneumonia . No case of otitis media was seen in infants less than 2 months of age and none of the toddlers were found to suffer from severe pneumonia . Incidence of pneumonia was, however, noted to be higher in boys (0.9 episodes/100 child - weeks) as compared to girls (0.6 episodes/100 child - weeks). Similar pattern was seen for severe pneumonias also (0.1 for boys as compared to 0.05 for girls). In contrast, the incidence of no pneumonia, cold, and cough as well as otitis media was found to be higher in girls . Figure 1 shows the monthly incidence of ari (all types including otitis media), no pneumonia, cough, and cold, pneumonia, and otitis media among infants and toddlers . The monthly incidence of aris ranged from a low of 5.2 episodes/100 child - weeks (in may) to a high of 15.8 (in february). Two peaks were seen with the more prominent peak falling in the month of february which coincided with spring season . Incidence of pneumonia also showed a fluctuation, ranging from a low of 0.2 episodes/100 child - weeks in may to a high of 1.5 episodes/100 child - weeks in march (visible as a peak) and november (visible as a less prominent plateau). This community - based prospective study conducted in delhi provides information on risk of developing ari in tropical peri - urban settings of developing countries . Our study had a very low attrition rate (1.6%) all of which was attributable to social migrations . We believe that the child - weeks lost to attrition were not systematically different from those included for analysis . Besides, though living standards of delhi are better than those in other areas of india as a whole, owing to the lack of primary health care system and higher levels of pollution (as is the case in most urban areas), there is likely to be a balancing effect, thereby making our estimates representative of similar peri - urban areas . Prevalence of ari in this study stood at 34.3% as compared to 19.9% and 5.4% for diarrhea and fever, respectively . The relative importance of ari as a cause of disease, not just mortality, in young children was, hence, reestablished . Nfhs 3 reported that 5.8% of children under 5 years suffered from ari (cough plus short, rapid breathing) during the 2-week - period preceding the survey . The difference is due to variations in methodology (nfhs used a one - time survey technique conducted by nonmedical personnel and no examination was involved). Have also documented ari to be a major cause of morbidity among rural children with prevalence rates of 35.4 per hundred days of observation . The bangladesh study, however, reports that nearly 9% of their study subjects did not suffer from any ari episode during the 12-month study period which is a highly desirable situation compared to only 0.9% in our case . Our findings are also in concurrence with those reported by koch et al . In their greenland study . Annual incidence of ari in this study was 4.1 episodes / child (7.9 epi/100 child - wks). Acharya et al . In 2003 found ari incidence of 6.4 episodes per child per year . Reported 3.7 episodes of ari among under - five children of wardha, maharashtra in 1996, while awasthi and pande from lucknow have reported the lowest ari incidence of 1.67 episodes . Studies from neighbouring bangladesh too have reported annual ari incidence to be 5.5 episodes per child . Although our observations fall within the range of what others have reported, still it would be the best not to compare our findings with that of others for the following three reasons: (i) while most workers have studied children up to 5 years of age, we focused on infants and toddlers exclusively, the age which is most affected by aris; (ii) some studies were conducted in part of the year only and not a full calendar year; and (iii) the questions used to estimate ari might have been different in different studies . We found annual pneumonia incidence to be 0.44 episodes / child (0.85 episodes/100 child - weeks) which is much higher than that reported by most workers and agencies . For example, the joint unicef - who document on pneumonia of 2006 reports an incidence of 0.30 episodes / child / year for india as a whole and rudan et al . Singh and nayar have reported an annual incidence of pneumonia among under - five children of wardha (maharashtra) in 1996 to be 0.07 episodes / child, while awasthi and pande found pneumonia incidence of 0.09 per child per year in lucknow, up ., however, report a higher pneumonia incidence of 0.52 episodes per child per year from rural udupi in karnataka . In bangladesh much lower incidence of acute lower respiratory infections (i.e., pneumonia) was reported (0.23 per child per year). The reason for the high prevalence of aris and higher incidence of pneumonia in the present study could be (i) higher levels of air pollution, (ii) overcrowded and ill - ventilated living conditions, and (iii) lack of a primary health care mechanism . The latter has also been documented in pakistan by singh and nayar which records that 13% of no pneumonia is likely to turn into pneumonia, and a significant proportion of these would be prevented if appropriate care is available . We found 10.1% of all ari episodes to be pneumonia and 0.7% to be severe pneumonia . Awasthi and pande found 10% of respiratory disease to be pneumonia in lucknow, while acharya et al . Reported 8.7% to be pneumonia and 0.5% to be severe pneumonia in udupi, karnataka . In contrast, 4% of ari episodes were reported to be pneumonia in bangladesh . On the other hand, incidence of otitis media in the current study (0.01%) is much lower than the 30% estimated incidence among ari cases as reported by simoes et al . For developing countries . The difference could be due to difficulty in making an assessment of otitis media which requires otoscopy, a procedure we could not conduct in field conditions . The mean duration of no pneumonia, cough, and cold was 6.16 days as compared to 11.65 days for pneumonia in our study . The bangladesh study reported that 46 percent of uri and 65 percent of alri episodes lasted 15 days or more without giving any more details . Median duration of uri and lri episodes has been reported to be 14 days (range: 725 days) and 19 days (range: 939 days) in the greenland study . Incidence of uri was the highest in the 1217-month - age cohort, while that for pneumonia was highest in 0 - 1-month - age cohort . The bangladesh study found the highest uri incidence among children 1823 months old, followed by infants 611 months old, while the highest pneumonia incidence was reported in infants 06 months old, followed by children 1218 months old . The greenland study also reported the highest prevalence of respiratory symptoms in the 611 months age group with a steep rise occurring from less than 5 months to 611 months and a decrease in incidence thereafter . Thus, the risk of ari generally increases in the later part of life as a toddler, which could be due to the fact that children become more mobile at this age and interact with multiple caregivers and individuals (in contrast to mother only during early infancy) and they are therefore exposed to more numbers of ari cases / carriers . Gender . Our finding of slightly higher incidence of no pneumonia, cough, and cold in girls did not match the findings of nfhs which reported slightly higher prevalence among boys . The reasons for higher reporting for boys could be explained by the gender bias that is prevalent in our society with its attendant preferential treatment for boy - child, especially when history is the only method of assessment (nfhs methodology). The greenland study did not find any gender differential as far as uris were concerned, but that for pneumonia was higher in boys . The presentstudydemonstrated that uri peaks in spring season and to a lesser extent in autumn, while pneumonia peaks a little later . The bangladesh study noted a higher incidence of uri as well as pneumonia (alri) in monsoon and autumn (before winter). On the other hand, no seasonal pattern in the incidence of ari, uri, or lri was observed in the greenland study . The differences are possibly due to differences in the climatic conditions as well as housing conditions of the areas of study . The annual incidence of aris was 4.1 episodes / child, while that for pneumonia was 0.44 episodes / child . Pneumonia incidence was higher than the estimates for india as well as for south asia as a whole . Incidence of pneumonia was roughly one - tenth that of ari and that of severe pneumonia or otitis media was one - tenth that of pneumonia . Since incidence of aris and pneumonia generally decreased with increasing age, targeting infants specifically for prevention and control efforts may be a more effective strategy . Two fortnight - long intensive mass - media campaigns (pneumonia awareness and prevention fortnight) prior to holi (a festival of colors celebrated in the month of march commencing the spring season) and dusshera (indian festival celebrated in autumn during month of october / november), on the lines of breast feeding awareness week and malaria month, just before onset of the peak season, are also likely to be helpful in generation awareness and disease control . More of such studies with larger sample sizes and including rural as well as urban populations need to be conducted . (i) with the study being conducted in a small area, its findings may not be generalizable to other peri - urban settings, within and outside this country . (ii) period of recall of one fortnight, though accepted as standard for this type of study, may have led to missing out on many episodes (those starting after a particular visit and ending before the next), particularly when primary caregiver was not the respondent . (iii) primary researcher being a doctor was ethically bound to provide health education as well as medical guidance to children and their caregivers, thereby altering the natural history and may be even the incidence of aris.
Aplasia cutis congenita (acc) is a rare congenital malformation characterized by noninflammatory, well - demarcated defects of all skin layers, subcutaneous tissue, with possible co - defects in muscles, periosteum, bone and dura . It manifests usually as a solitary defect on the scalp, but sometimes may occur on the face, trunk or limbs . Acc is most often a benign isolated defect, but can be associated with other physical anomalies or malformation syndromes . On the other hand, a split cord malformation (scm), also called a diastematomyelia, is a rare spinal anomaly and refers to a sagittal division of the spinal cord into two symmetrical or asymmetrical hemicords . Variants of this malformation associated with a split of the spinal column, spinal bony spurs, myeloceles, myelomeningoceles, lipomas and dermal sinuses has been previously reported in the literature . However, the association of acc with scm is considered very rare variant and only two reports of this condition are available . A full - term girl, the first child of a 24-year - old mother was born with large skin, muscle, bone and dural defect in the lumbo - sacral area . The patient weighed 2800 g. the patient was evaluated by the pediatric clinic and no other congenital malformations were found . The lesion showed 8 5 cm skin defect, covered with transparent arachnoid membrane . Underneath nerve tissue of the spinal cord was split by a perpendicular bony spur and connected from its tip to the upper lamina [figure 1a and b]. Dura matter was seen on both sides of the lesion extending laterally over the paraspinal muscles . Brain computed tomography (ct) scan showed mild ventriculomegaly without signs of increased intracranial pressure . Spinal x - rays showed a bony spur on the l2 vertebral column and laminar defect in the lumbo - sacral area [figure 1c]. Under general anesthesia and prone position, the arachnoid layer was removed and the split spinal cord was dissected from the bony spur . There was no neural placode and the spinal cord was found with full neurulation . The spur was seen extending from the posterior border of the vertebral body and penetrating the anterior dura [figure 2a]. However, dural folds around the base of the spur invaginating between the two hemicords were seen . Fibrous attachment between the bony spur and the upper lamina was resected [figure 2b]. The bony spur is then resected from its base to expose the opened anterior dura of the spine [figure 3a]. The anterior dura was sutured [figure 3b], and the spinal roots were demonstrated [figure 3c]. Posterior dura was dissected from both the sides from the subcutaneous tissue and reverted to midline and closure was done in a water tight fashion . Skin defect was large and direct approximation of the lips of the skin was not possible . A wide subcutaneous undermining was done to allow a z - plasty - type flap closure . However, the post - operative follow - up showed necrosis in the lips of the flap . Debridement of the necrotic lips was done and wound was allowed to heal by secondary closure with regular wet dressing and boric acid powder . (c) p - a spinal x - ray demonstrating the laminae defects in the lumbo - sacral region (1: bony spur, 2: fibrous band, 3: split spinal cord) (a) split spinal cord is dissected from the bony spur . (b) fibrous band is cut from the attachment site in the upper lamina (a) bony spur is resected from its base to expose the opening of the anterior dura . Although the etiology is still uncertain, a variety of possible causes such as genetic syndromes, teratogens, intrauterine infection with varicella zoster or herpes simplex viruses, fetal exposure to cocaine, heroin, alcohol or antithyroid drugs, infarction of the placenta, and amniotic pathologies are described in the literature . The incidence is 1 to 3/2000 to 10,000 and 25% of the reported cases are familial with a vast majority (69%) showing an autosomal dominant inheritance or part of a syndrome . Lesions can be multiple and on different surfaces of the body, but most of the cases have solitary scalp lesions (70%). It can also be associated with other physical anomalies such as defects of eyes, extremities, limbs, gastrointestinal system, genito - urinary system, and central nervous system . In our case, the defect was seen in the lumbo - sacral area which is besides considered very rare . Scm is an unusual congenital anomaly characterized by sagittal clefting of the spinal cord or filum terminale . Pang et al . Proposed a unified theory of embryogenesis in which all double cord malformations result from a basic ontogenetic error occurring around the time when the primitive neuroenteric canal closes . This basic error is the key step in the formation of an abnormal (accessory) neuroenteric canal (fistula) through the midline embryonic disc that maintains communication between the yolk sac, which is of endodermic origin, and the amnion, which is ectodermic in origin . An endomesenchymal tract condenses around this accessory canal, splitting the developing notochord and causing two hemineural plates to form . Scms are classified as one of 2 types according to the unified theory . In type i scm, the hemicords are always invested with individual dural sacs and the medial walls of the sacs always ensheath a rigid (bony or cartilaginous) midline spur . In type ii scm, hemicords are always within a single dural sac and the midline septum is composed of non - rigid fibrous or fibrovascular tissues . According to this classification, our case can be diagnosed as type i scm with completely separated dural sacs and a bony spur . Cutaneous stigmata are more common in cases of scms than in other spinal dysraphism, with an incidence rate of 20 to 55%; hypertrichosis is the most frequent skin manifestation . The spinal cord was split by a perpendicular bony spur and connected from its tip to the upper lamina . The spur was seen extending from the posterior border of the vertebral body and penetrating the anterior dura . There was no neural placode and the hemicords were found with full neurulation reflected with the fully intact motor power in the lower limbs and excluding the diagnosis of myeloschisis . This association of both pathologies may be explained by an arrest during embryologic development of the skin and subcutaneous tissue formation (as known for acc) and of the regression of the neurenteric canal or the break between the ectoderm and endoderm forming the fistula due to common pathologic process, such as ischemia . In our case, the mother was previously healthy and there was no drug intake history which could be considered as teratogenic . Recent studies demonstrated that magnetic resonance imaging (mri) and 3d ct are useful for the diagnosis of scm . In this case, however, we approached this variant as an open dysraphism without preoperative mri as the lesion was totally exposed and identifiable . Careful intraoperative exploration was sufficient for the identification of the anatomy and the management of this variant.
Smoking cessation is crucial for a better prognosis in patients with coronary heart disease (chd)13 and copd.2,4,5 acute heart attacks are associated with high smoking cessation rates . In patients with a first myocardial infarction (mi) in the 1970s,3 among whom 78% were smoking, the quitting rate was 55% . In a systematic review from 2003, the mean rate of smoking cessation within a year after a new diagnosis of chd was found to be 45%.1 a lower quitting rate was observed in patients with copd, in particular in subjects not participating in smoking cessation programs . Among smoking copd patients in a swedish study, 10% of those receiving usual care had stopped smoking after 3 years, compared to 38% of those who took part in smoking cessation groups.6 among hospitalized patients with respiratory and cardiac diseases who took part in an intensive smoking cessation program in singapore, 60% of the cardiac patients and 40% of the respiratory patients were still abstinent from smoking after 2 months.7 the mortality due to chd has decreased considerably in the western world in the last few decades.8 evidence shows that decreased smoking prevalence in the general population, particularly among patients with heart disease, is an important reason for this decrease.811 the mortality due to copd has decreased less.12 a stronger tendency of persistent smoking among copd patients may partly explain this difference . Copd patients reluctance to quit smoking may lead to a feeling of hopelessness among the doctors who treat them.13 our aim was to determine how the quitting rate in adults is influenced by getting a diagnosis of obstructive lung disease in the population - based troms study . We also wanted to investigate whether influence from this life event differs from being diagnosed with chd . A large proportion of the adult population in troms has participated in health surveys (the troms study) since 1974 . The troms study so far consists of six surveys, and this study deals with subjects who participated in both troms 5 (20012002) and troms 6 (20072008). The attendance rates (of those invited) in troms 5 and troms 6 were 79% and 66%, respectively . Details of the participants have previously been described.14 we included participants who answered the questions on smoking in both surveys . The regional committee for medical and health research ethics and the norwegian data inspectorate approved the troms 5 and troms 6 surveys with a license for further analysis on non - identifiable data, like this study . The invitation included a questionnaire with questions on smoking habits, education, and diseases . We classified subjects reporting daily smoking in troms 5, but not in troms 6, as quitters . We divided length of education into three categories, 12 years, 1316 years, and 17 years . We classified diseases reported in troms 6 and not in troms 5 as new diagnoses . We categorized mi and angina pectoris as chd and asthma and copd as obstructive lung disease . The frequencies of current smoking at baseline (in troms 5) and smoking cessation among smokers were calculated by subject characteristics, and the statistical differences between subgroups were analyzed by chi - square statistics . Significant variables in univariable analysis (p<0.1), as well as age and sex, were entered in the multivariable analysis . For the statistical analyses, we used the ibm spss statistic, version 21 (ibm corporation, armonk, ny, usa). A large proportion of the adult population in troms has participated in health surveys (the troms study) since 1974 . The troms study so far consists of six surveys, and this study deals with subjects who participated in both troms 5 (20012002) and troms 6 (20072008). The attendance rates (of those invited) in troms 5 and troms 6 were 79% and 66%, respectively . Details of the participants have previously been described.14 we included participants who answered the questions on smoking in both surveys . The regional committee for medical and health research ethics and the norwegian data inspectorate approved the troms 5 and troms 6 surveys with a license for further analysis on non - identifiable data, like this study . In both surveys, the invitation included a questionnaire with questions on smoking habits, education, and diseases . We classified subjects reporting daily smoking in troms 5, but not in troms 6, as quitters . We divided length of education into three categories, 12 years, 1316 years, and 17 years . We classified diseases reported in troms 6 and not in troms 5 as new diagnoses . We categorized mi and angina pectoris as chd and asthma and copd as obstructive lung disease . The frequencies of current smoking at baseline (in troms 5) and smoking cessation among smokers were calculated by subject characteristics, and the statistical differences between subgroups were analyzed by chi - square statistics . Significant variables in univariable analysis (p<0.1), as well as age and sex, were entered in the multivariable analysis . For the statistical analyses, we used the ibm spss statistic, version 21 (ibm corporation, armonk, ny, usa). A total of 4,631 subjects participated in both the fifth and the sixth surveys of the troms study, and 4,497 subjects comprising 1,852 men and 2,645 women answered the questions on smoking in both surveys . The mean time difference between attending the two surveys was 6.2 years (standard deviation 0.5 years). The mean age at troms 5 was 61.1 years (range 3081 years); 1,150 (25.6%) reported daily smoking and 1,753 (39.0%) reported former smoking . The highest prevalence of smoking (43.8%) was found in subjects with self - reported copd, whereas the frequency among those with self - reported chd was 19.0% . The characteristics of the population in troms 5 and the respective frequencies of smoking are listed in table 1 . In the sixth survey, 765 subjects (17.0%) reported current smoking, and 428 of the 1,150 who had been smoking in troms 5 had quit (37.2%). A significantly higher quitting rate was found in men compared to women, and the quitting rate increased with the length of education . Self - reported diseases registered in troms 5 had no significant impact on the quitting rate (table 2). The number of days between attending the two surveys had no impact on the quitting rate (odds ratio = 1, p=0.8). Table 3 shows the frequencies of new self - reported diseases reported in troms 6, but not in troms 5, and the association between a new diagnosis and smoking cessation . New diagnoses of asthma / copd (n=79) and chd (n=73) were both associated with increased frequency of quitting, 50.6% (p=0.01) and 52.1% (p=0.008), respectively . Both men and women belonging to any of these diagnostic groups showed increased quitting rates, 54.9% vs 39.8% (p=0.02) in men and 47.1% vs 32.7% (p=0.009) in women . The impact of getting these new diagnoses was particularly strong among the subjects with education length no longer than 12 years (p=0.001; figure 1). Subjects with education length up to 12 years were also more frequently diagnosed with these diseases compared to those with higher education, 16.0% and 9.5% (p=0.01), respectively . Among subjects with higher education, a new diagnosis of both chd and obstructive lung disease was significantly associated with smoking cessation when analyzed by logistic regression and also after adjusting for sex and education level in multivariable logistic regression analyses (table 4). Half of the participants with a new diagnosis of asthma / copd or angina / mi quit smoking during the 6 years of follow - up . However, the association between these new diagnoses and smoking cessation was found only in participants with education length up to 12 years . The much higher smoking rate among those with asthma and, in particular, those with copd at baseline than in those with chd indicates a shift in attitude toward smoking cessation among subjects with copd . The high number of participants and the high attendance rates among those invited are strengths of the study . The prevalences of daily smoking among all the attendees in troms 5 and troms 6 were 31% and 22%, respectively,11 which are close to the national prevalences of 29% and 21%, as registered by statistics norway (https://www.ssb.no)15 in 2002 and 2008, respectively . In our study sample, the prevalence was lower at both points of time, 25.6% and 17.0%, respectively, and a healthy survivor effect may have contributed to the low frequency of daily smoking in our subsample . The study is based on questionnaires and not on objective measurements of smoking, such as cotinine and thiocyanate . In a previous norwegian study, self - reported smoking was strongly related to serum thiocyanate if the question was asked in a neutral setting.16 although the questions on smoking were included in a self - administered questionnaire, underreporting of daily smoking among those with a new diagnosis cannot be ruled out . Shift in diagnosis based on the same illness from troms 5 to troms 6, for instance, between asthma and copd, may have taken place in some subjects . This is no longer a problem when asthma and copd are combined into one category (asthma and/or copd) and subjects with a new diagnosis of any of these diseases are compared with those with neither of the diagnoses at both troms 5 and troms 6 . Although we have found associations between a new diagnosis and smoking cessation, we do not know for sure whether the extra cessations in the subgroups with a new diagnosis were really preceded by a new diagnosis . Subjects in these subgroups could have stopped smoking before the diagnosis was given . In these cases, it is likely that symptoms from a pulmonary or heart disease had raised their awareness of the risk of continued smoking . No previous study has, to our knowledge, described smoking cessation in relation to a new diagnosis of heart or lung disease . Most previous studies evaluated smoking cessation programs, and control groups in such studies can be compared with our participants . However, the quitting rate of 41.7% among subjects with a copd diagnosis reported in troms 5 was more similar to the rate of 38% among the copd patients, who had taken part in the smoking cessation program in a swedish study, than the rate of 10% among the copd patients on usual care.6 in a study from primary care, where smokers received smoking cessation advice and were followed up annually with spirometry, higher rates of abstinence were found among those with copd than among those with normal lung function.17 this result is in line with our findings . We found that the quitting rate increased with increasing length of education, and this association is well known from previous studies.18 qualitative studies have shown that patients with a copd diagnosis may have several reasons for not quitting despite the knowledge of harming themselves,19 and they do not always believe that quitting would give them a better life.20 copd patients often show little interest in receiving help from medication and describe unassisted quitting as the best method to stop smoking, based on willpower, strong motivation, and internal strength.21 in our study, a new diagnosis of copd or chd might have given many participants the motivation they needed to quit without assistance . In another qualitative study, the interviewees who had stopped smoking emphasized that persons close to them had a strong influence on their decision to quit.22 this gives a reason to believe that the decreasing acceptance of smoking in the society also influences copd patients . Half of the participants with a new diagnosis of asthma / copd or angina / mi quit smoking during the 6 years of follow - up . However, the association between these new diagnoses and smoking cessation was found only in participants with education length up to 12 years . The much higher smoking rate among those with asthma and, in particular, those with copd at baseline than in those with chd indicates a shift in attitude toward smoking cessation among subjects with copd . The high number of participants and the high attendance rates among those invited are strengths of the study . The prevalences of daily smoking among all the attendees in troms 5 and troms 6 were 31% and 22%, respectively,11 which are close to the national prevalences of 29% and 21%, as registered by statistics norway (https://www.ssb.no)15 in 2002 and 2008, respectively . In our study sample, the prevalence was lower at both points of time, 25.6% and 17.0%, respectively, and a healthy survivor effect may have contributed to the low frequency of daily smoking in our subsample . The study is based on questionnaires and not on objective measurements of smoking, such as cotinine and thiocyanate . In a previous norwegian study, self - reported smoking was strongly related to serum thiocyanate if the question was asked in a neutral setting.16 although the questions on smoking were included in a self - administered questionnaire, underreporting of daily smoking among those with a new diagnosis cannot be ruled out . All diagnoses were self - reported, and their correctness could not be confirmed . Shift in diagnosis based on the same illness from troms 5 to troms 6, for instance, between asthma and copd, may have taken place in some subjects . This is no longer a problem when asthma and copd are combined into one category (asthma and/or copd) and subjects with a new diagnosis of any of these diseases are compared with those with neither of the diagnoses at both troms 5 and troms 6 . Although we have found associations between a new diagnosis and smoking cessation, we do not know for sure whether the extra cessations in the subgroups with a new diagnosis were really preceded by a new diagnosis . Subjects in these subgroups could have stopped smoking before the diagnosis was given . In these cases, it is likely that symptoms from a pulmonary or heart disease had raised their awareness of the risk of continued smoking . No previous study has, to our knowledge, described smoking cessation in relation to a new diagnosis of heart or lung disease . Most previous studies evaluated smoking cessation programs, and control groups in such studies can be compared with our participants . However, the quitting rate of 41.7% among subjects with a copd diagnosis reported in troms 5 was more similar to the rate of 38% among the copd patients, who had taken part in the smoking cessation program in a swedish study, than the rate of 10% among the copd patients on usual care.6 in a study from primary care, where smokers received smoking cessation advice and were followed up annually with spirometry, higher rates of abstinence were found among those with copd than among those with normal lung function.17 this result is in line with our findings . We found that the quitting rate increased with increasing length of education, and this association is well known from previous studies.18 qualitative studies have shown that patients with a copd diagnosis may have several reasons for not quitting despite the knowledge of harming themselves,19 and they do not always believe that quitting would give them a better life.20 copd patients often show little interest in receiving help from medication and describe unassisted quitting as the best method to stop smoking, based on willpower, strong motivation, and internal strength.21 in our study, a new diagnosis of copd or chd might have given many participants the motivation they needed to quit without assistance . In another qualitative study, the interviewees who had stopped smoking emphasized that persons close to them had a strong influence on their decision to quit.22 this gives a reason to believe that the decreasing acceptance of smoking in the society also influences copd patients . It has been suggested that a hard core of smokers will constitute an increasing proportion of copd patients who still smoke.23 the high quitting rates among subjects with both an established and a new diagnosis of copd in our study contradicts this pessimism . The study supports pursuit of early diagnosis of copd and gives reasons for a more optimistic attitude among health workers when they discuss smoking cessation with their copd patients.
Adenocarcinoma of the pancreas is a devastating disease, and only a small proportion of sufferers achieve an attempt at curative surgical treatment which leads to a median survival of about 12 months . In spite of advances in surgical technology, perioperative care, and adjuvant treatments the improvement in outcome from this disease has been minimal . Improving survival could be the result of an understanding of the biology of the cancer, and its risk factors with a view to detecting it early possibly in high - risk individuals with secondary prevention strategies in selected patients and also devising novel modes of interfering with its initiation and progression . There have been numerous lines of evidence implicating tobacco smoking as a risk factor for pancreatic cancer and this has been well recognised in the field of pancreatology . Realisation of this in the wider healthcare community, however, had been limited until the publication of the 2004 us surgeon general's report on health consequences of smoking . Tobacco usage is the single largest preventable cause of disability disease and death in the developed world and probably also plays a great role in morbidity and mortality amongst people within the developing world . A recent meta - analysis demonstrated the significant strength of the association between cigarette smoking and pancreas cancer and calculated that the population attributable risk secondary to tobacco use for the malignancy was about 20% . However, the exact etiological cause for the vast majority of these cancers continues to remain speculative . Tobacco smoking is the strongest risk factor for pancreas cancer, but the mechanism of disease causation has not been elucidated although the various steps in the sequence of development of the malignancy have been described beginning from pan - in 1 to invasive adenocarcinoma . On a background of this recent meta - analysis summarising the evidence linking tobacco use with pancreatic cancer, we have reviewed the available laboratory and experimental data, providing a summary of the current knowledge of tobacco - driven pancreas carcinogenesis . Established medical databases including pubmed, medline, scopus, and index medicus were searched for a variety of different combination of the terms pancreas, pancreatic, cancer, adenocarcinoma, tobacco, use, smoking, and carcinogens to identify peer - reviewed publications exploring the relationship between tobacco smoking and pancreas cancer . After excluding duplicate results, a total of 235 publications were initially identified . They were subjected to careful scrutiny and we excluded case - control, cohort studies and other cross - sectional / longitudinal studies published up until 2008 . This was due to an excellent and comprehensive meta - analysis summarising the epidemiology of tobacco use and pancreas cancer (vide supra) published in 2008 . All publications dedicated to laboratory, experimental and clinical consequences of tobacco carcinogen effect on the pancreas or development of pancreas cancer were identified and the full - text was carefully scrutinised . Nicotine is the major psychoactive substance in tobacco and is responsible for tobacco dependence and addiction . It is thought to induce a euphoric state in users by the activation of the mesolimbic dopaminergic reward system in the nucleus accumbens of the brain, and this may be related to the highly addictive properties of the drug . Individuals have genetically based differences in their ability to metabolize nicotine as well as genetic differences in the psychological reward pathways that may influence individual response to smoking initiation, dependence, addiction and cessation . While nicotine itself has some cancer - promoting abilities which we will discuss later, most of the health consequences of tobacco use are secondary to the other chemicals within it tobacco smoke contains 4000 compounds and 50 carcinogens as compared to 3000 compounds and 30 carcinogens in processed unburnt tobacco . These carcinogens include polynuclear aromatic hydrocarbons (pah), tobacco - specific nitrosamines (tsna), aromatic amines, aza - arenes, and aldehydes, other organic compounds like benzene, inorganic compounds like hydrazine, and various metals (reviewed by hecht and hoffmann). Seven compounds have been identified in the family of tsna's nnn (n-nitrosonornicotine), nnk (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), nnal (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol), nat (n-nitrosoanatabine), nab (n-nitrosoanabasine), iso - nnal [iso 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol] and iso - nnac [iso 4-(n - methylnitrosamino)-4-(3-pyridyl)butyric acid)]. Nitrosation of nicotine during tobacco processing and cigarette smoking leads to the formation of tobacco - specific nitrosamines, of which 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nnk) is the most notorious and is now classified by the international agency for research on cancer as a group 1 carcinogen . In the context of pancreatic cancer tsnas, nnk and nnal tsnas are procarcinogens . Following exposure, they need to undergo a series of steps uptake, metabolic activation, and dna and protein adduct formation which subsequently leads to altered growth kinetics in the target organ and development of neoplasia . These metabolic pathways of nnk and nnn have been demonstrated in vivo in mice, rats, and hamsters and in subcellular fractions, cultured cells and tissues from animals and humans . Nnk can undergo hydroxylation, oxidation and carbonyl reduction to yield numerous metabolites which include diazoxides, formaldehyde, nnk - n - oxide, and nnal . These metabolites react with dna and protein resulting in adducts formation . In mice and rats, nnk - derived electrophiles react with nucleophilic centers in dna to yield a variety of products including 0-methylguanine which causes miscoding of dna during replication . This production of 0-methylguanine and 0-ethylguanine had also been confirmed histochemically in pancreatic duct cells in vitro . Until recently, nicotine had been considered to be responsible for only the addictive property of tobacco only; however, of late it is being increasingly recognised that nicotine might independently possess cancer facilitating properties causing the metastasis of tumours preinitiated by tobacco carcinogens in rat models of lung cancer . This effect is thought to be mediated through nicotine stimulation of nachr (nicotinic acid acetyl choline receptors). Nicotine can also induce epithelial - mesenchymal transition in cultured lung, breast and pancreatic cancer cells . Metastasis sequence and therefore it appears that while nicotine has only limited capacity to initiate tumour formation, it can induce invasion and metastasis in preexistent tumours . Numerous animal models of pancreas ductal cancer have been developed, and these have proved invaluable in elucidating the kinetics of the carcinogen within physiological systems and pathogenesis of ductal cancer of the pancreas . In 1988, the important role of tobacco derived carcinogens in extrapulmonary cancers including the pancreas was highlighted in a commentary by hecht and hoffmann . The same commentary also concluded that two of the nicotine - derived nitrosamines, nnk and nnn, are strong carcinogens in laboratory animals and that they could induce tumors both locally and systemically . Also, the magnitude to the total doses of nnk and nnn required to produce cancer in laboratory animals is similar to the total estimated doses which long - term snuff - dippers or heavy smokers are exposed life time . In f-344 rats, nnk and nnal the doses of carcinogens to body weight needed to cause these neoplasms in rats were similar to lifetime exposure of these chemicals in heavy smokers (40 cigarettes / day). This was the first example of pancreatic tumour induction by a constituent of tobacco smoke . Nnal appeared to be the proximate pancreatic carcinogen of nnk as it induced more tumours than nnk . They are the only carcinogens to induce pancreatic adenocarcinoma in animal models when given systemically [18, 19]. Also, in f-344 rats, there is evidence that following oral nnk administration, there is preferential metabolism of nnk to (s)-nnal followed by its extensive retention in various target tissues including the pancreas of nnk - orally treated animals . Treatment of rats with tsna's also resulted in the induction of pancreatic acinar cell and ductal cell neoplasms . In rodents it has been shown that nnk and nnal are excreted in the bile in significant concentrations . If this is true in humans, it may be the route through which activated carcinogens reach the head of pancreas (carcinogen containing bile refluxing into the pancreatic duct). This theory is attractive given that the pancreatic head is the most frequent site for adenocarcinoma [2325]. A study in rhesus monkeys (n = 4), however, found that biliary excretion of the nnk metabolites was significantly less than predicted from rat experiments . Most animal studies have been extrapolated to humans and the assumption is that the metabolism and physiological distribution kinetics of tobacco derived carcinogens is similar to that in rats and rodents . However, there will be differences in the manner in which our species handles these carcinogens, and a few studies have tried to elucidate this by various means . Essentially, what has been achieved is to show that the pancreas is exposed to these chemicals and that these metabolites bind to pancreatic ductal cell dna and result in mutations there and that various phenotypic changes occur there including cancer . However the exact mechanism by which these carcinogens turn a normal ductal cell into a malignant one has not been described . Tsna's (nnk and nnal) have been detected in the pancreatic juice of smokers in significantly higher quantities as compared to nonsmokers confirming that the pancreas is exposed to these carcinogens . It has been shown that aromatic amines and nitroaromatic hydrocarbons are metabolically activated in the human pancreas . This confirmed that the pancreas is exposed to tsna's and that these carcinogens may play a role in carcinogenesis at this site . Although a strong correlation had been suggested between cigarette smoking and pancreatic cancer, studies on pathological changes in the pancreas of smokers are infrequent . Performed a comparative autopsy study on 73 pancreases obtained by autopsy from 42 heavy cigarette smokers and 31 nonsmoker patients . Although the incidence of pancreatic cancer in smokers was higher than in nonsmokers, the difference was statistically not significant . Ductal changes, including mucinous or squamous cell metaplasia and papillary hyperplasia, were found with equal frequencies in both groups of patients and the authors concluded that the type and the incidence of these ductal alterations were not related to smoking but to the age . There were significant limitations of this autopsy study including limited number of the sections of the pancreas examined, as well as exclusion of other important variables, such as alcohol, diet and diabetes weaken the value of this study . Another autopsy study obtained purified dna from human lung, liver, bladder, pancreas, breast and cervix of 13 men and 6 women and analysed it for dna adducts using the nuclease p1 modification of the 32p postlabelling technique . All tissues examined except the breast had detectable adducts . In lung, bladder and pancreatic tissue a characteristic pattern of adducts was seen which had previously been reported as typical of cigarette - smoke - induced damage; diagonal reactive zone . Smokers and former smokers tended to have higher adduct levels than nonsmokers in the tissues examined but this was only significant for the lung . These results confirmed the finding that cigarette smoking is associated with dna damage in the lung and suggested that similar damage may be related to tobacco - induced neoplasms of other tissues . Tsna's adducts have been found in the pancreas and the levels have correlated with dose and time related to exposure . Over the past 1015 years extensive work into the development of pancreatic cancer has been carried out . A model of stepwise progression from normal to malignant cells, and the molecular alterations involved in these has been described . Very similar to the adenoma - carcinoma sequence in the colon, there is neoplastic progression in the pancreatic ducts [3336]. This progression from a normal pancreatic ductal cell to an infiltrating carcinoma involves sequential multiple genetic alterations . The genetic changes include activating point mutations in k - ras, overexpression of her-2neu, and inactivation of p16, p53 dpc4, and brca2 . A gate - keeper gene for the initiation of pancreatic neoplasia has not been indentified and the cause of the ignition of change in a normal ductal cell towards neoplasia is under investigation . Several mechanisms have been proposed in regard to how tobacco smoking might be involved in pancreatic carcinogenesis such as tobacco - carcinogen - induced adducts resulting in the mutation of the k - ras oncogene and initiating tumorigenesis and subsequent promotion occurring by other factors, . However, this is not yet clear, and other mechanisms such as the autonomic nervous system and its various pathways, lipid peroxidation, and synergistic molecular damage by other environmental, occupational, and other factors may play an important role . A number of animal studies have been performed examining the hypothesis that tobacco smoking - induced k - ras mutations and that these were the beginnings of the malignancy [15, 37, 38]. In one of these models, an in vitro transformation of immortal hamster pancreatic duct cells was studied after exposure to nnk for varying lengths of time . Analysis of pancreatic dna for k - ras mutation at codons 12, 13, and 61 showed g a transition at codon 12 of the k - ras oncogene in tumour cells after 1 and 3 days of nnk treatment . However, no mutation was detected in tumour cells developing 5 and 7 days after nnk treatment . One and 3 day nnk - treated cells were able to grow in the absence of growth factors and serum immediately after the treatment . Also, the tumorigenicity of these transformed cells was determined in nude mice, and the cells treated for 1 and 3 days produced well - differentiated neoplasms, while those that were treated for longer durations did not . Thus, it appeared that although k - ras mutation at codon 12 of pancreatic adenocarcinoma was an early event, it was not necessarily required for the development and/or progression of the tumours in nude mice . Numerous studies have been performed in humans to ascertain the role of k - ras in pancreas development with conflicting results . Nagata et al . Observed a negative association, whilst hruban et al . Found a positive association . In a population - based case - control study, where information on smoking and other life - style factors was obtained at direct patient interview, a meta - analysis of 8 studies investigating k - ras mutations in various cancers including pancreas adenocarcinoma, explored the hypothesised association lifetime history of tobacco consumption . No significant association between the frequency of k - ras mutations (or = 1.26; 95% ci = 0.821.94) and tobacco usage was identified . Subsequently, the pankras 2 study group authors reported a case - case study, where they did not find any association between acquisition of k - ras mutations and tobacco smoking . In combination, these studies have contributed to the conclusion that while both smoking and k - ras mutations are important in the development of pancreas cancer, they are independent of one another . Recent studies have demonstrated over - expression of cyclooxygenase and 5-lipoxygenase receptors in exocrine pancreatic carcinomas . Following on from this, weddle et al . Demonstrated high basal levels of arachidonic acid release and expression of m - rna for adrenergic receptors (1 and 2) in two human cell lines derived from exocrine ductal pancreatic carcinomas . This study therefore underlined the importance of -adrenergic mechanisms in pancreas carcinogenesis (as had previously been reported for lung cancer [46, 47]. Nnk reportedly binds with high affinity to adrenergic and nicotinic receptors and activates downstream signalling pathways, inducing cellular proliferation cancer cell lines derived from human pancreas, breast and lung malignancies . In a hamster model of exocrine pancreatic cancer induced by transplacental exposure to ethanol and the tobacco - carcinogen nnk, schuller et al . Reported a reduction in the development of pancreatic cancer in offspring who had been given the arachidonic acid pathway inhibitors, ibuprofen or mk886 . The reduction was not a result of an interaction with the drugs on nnk metabolism or cancer initiation events, as the nnk was administered as a single dose on day 15 of gestation, while the preventive treatments started 4 weeks later, when the offspring were weaned from their mothers . Of note, no mutations in the ki-, n-, or h - ras or p53 genes were found in theses tumours . There is also recent evidence indicating that nnk is an agonist for nicotinic acetylcholine receptors (nachrs), and in hamsters, nnk - induced alterations in regulatory nachrs may contribute to the development of smoking - associated pac and pdac by disturbing the balance between cancer - stimulating and inhibiting neurotransmitters . In combination -adrenergic, aa - dependent regulatory pathways in pancreatic cancer are a possible novel target - dietary or pharmacologic for cancer intervention strategies in an effort towards the prevention and clinical management of pancreatic cancer . While we are beginning to understand some of the key factors involved in pancreatic carcinogenesis, of which smoking is one, the primary etiology of the disease remains poorly understood . Other epidemiological factors may also be important and a genetic predisposition to the disease, supported by reported familial occurrences of the disease [5254] has long been suspected . As reviewed here, investigators have struggled to show that tobacco contributes directly to the development of acquired mutations in the commonly reported oncogenes and tumour suppressor genes . However, there may well be a more subtle association, with innate or inherited genetic variation, which increases an individuals exposure to damaging agents, or possibly that individuals ability to repair induced dna damage . There may also be interplay here, as with all polygenic diseases, with other environmental factors . Certainly, there is a lot of individual variation in the metabolism of the known tobacco - derived carcinogens . Metabolically activated nnn and nnk bind not only to dna but also to the protein moiety of haemoglobin resulting in globin - carcinogen adducts . Mass - spectroscopic studies have detected a significant overlap of the globin - carcinogen adduct levels between smokers and nonsmokers, suggesting that individuals vary in their ability to activate tsnas . As previously mentioned, there is also considerable variation in adduct levels in the pancreas at autopsy among smokers, which could not be accounted for by duration or daily consumption level . While the evidence for a synergistic role between smoking and other factors is limited, the key suspects are mentioned here . (a) dietary factorsvarious groups of researchers have explored the interaction of tobacco smoke with dietary and other factors in animal models in an attempt to replicate the scenarios found in humans, and there is evidence that some dietary factors act synergistically with these carcinogens . Coadministration of sinigrin (a glucoside found in some plants of the brassica family such as brussels sprouts, broccoli, and seeds of black mustard) with nnk, for example, resulted in a significant incidence of pancreatic tumors in rats . In a long - term bioassay (24 months) exploring the interaction of dietary fat and tobacco carcinogens, f344 rats were given nnk, in the drinking water . In addition, one group of rats was given a high - corn oil diet and the second group received a low - corn oil diet . The nnk + high corn oil group gained more weight and developed more number of pancreas tumours as compared to the nnk + low corn oil group . This elegant and simple bioassay provided supplementary evidence to corroborate epidemiological studies which had suggested a link between daily fat intake and pancreas cancers in smokers . Various groups of researchers have explored the interaction of tobacco smoke with dietary and other factors in animal models in an attempt to replicate the scenarios found in humans, and there is evidence that some dietary factors act synergistically with these carcinogens . Coadministration of sinigrin (a glucoside found in some plants of the brassica family such as brussels sprouts, broccoli, and seeds of black mustard) with nnk, for example, resulted in a significant incidence of pancreatic tumors in rats . In a long - term bioassay (24 months) exploring the interaction of dietary fat and tobacco carcinogens, f344 rats were given nnk, in the drinking water . In addition, one group of rats was given a high - corn oil diet and the second group received a low - corn oil diet . The nnk + high corn oil group gained more weight and developed more number of pancreas tumours as compared to the nnk + low corn oil group . This elegant and simple bioassay provided supplementary evidence to corroborate epidemiological studies which had suggested a link between daily fat intake and pancreas cancers in smokers . (b) obesityobesity is now recognised as a risk factor in development of all cancers, but this is particularly so for pancreatic cancer . Wang et al . Reported a 2.6 fold increase in relative risk of a pancreatic cancer associated death in individuals with a body mass index greater than or equal to 35 . The animal study above, in which the fatter nnk group fed corn oil developed more pancreatic cancers, supports this although the actual cause is as yet unclear . A further study demonstrated that body mass index was positively correlated to the levels of dna, lipid peroxidation related adducts and the total aromatic adducts in pancreatic tumour tissues . These observations support the hypothesis that dna damage related to a combination of carcinogen exposure and lipid peroxidation may be involved in human pancreatic carcinogenesis . Obesity is now recognised as a risk factor in development of all cancers, but this is particularly so for pancreatic cancer . Wang et al . Reported a 2.6 fold increase in relative risk of a pancreatic cancer associated death in individuals with a body mass index greater than or equal to 35 . The animal study above, in which the fatter nnk group fed corn oil developed more pancreatic cancers, supports this although the actual cause is as yet unclear . A further study demonstrated that body mass index was positively correlated to the levels of dna, lipid peroxidation related adducts and the total aromatic adducts in pancreatic tumour tissues . These observations support the hypothesis that dna damage related to a combination of carcinogen exposure and lipid peroxidation may be involved in human pancreatic carcinogenesis . Alcohol has long been suspected of contributing to pancreatic cancer development [59, 60], but a synergistic role with tobacco exposure has also been considered . In pregnant animal models, nnk can cross the placenta following tracheal administration and cause its carcinogenic effect - pancreas cancer on the foetus . Concomitant administration of ethanol can greatly potentiated this effect, but other than recommending that pregnant women neither drink nor smoke, the human relevance of these findings is unclear . The pankras 1 study investigated the presence of k - ras mutations in patients who either smoked, or drank alcohol, or did both . The information in this study was taken from patient notes rather than a questionnaire, but reported a higher risk only in those who did one or the other, rather than both . As discussed previously, the frequency of k - ras mutations is probably not the best tool to assess the impact of suspected carcinogens, and the true impact of smoking in combination with alcohol on pancreatic carcinogenesis has yet to be determined . A key dietary factor worthy of independent mention is alcohol . Alcohol has long been suspected of contributing to pancreatic cancer development [59, 60], but a synergistic role with tobacco exposure has also been considered . In pregnant animal models, nnk can cross the placenta following tracheal administration and cause its carcinogenic effect - pancreas cancer on the foetus . Concomitant administration of ethanol can greatly potentiated this effect, but other than recommending that pregnant women neither drink nor smoke, the human relevance of these findings is unclear . The pankras 1 study investigated the presence of k - ras mutations in patients who either smoked, or drank alcohol, or did both . The information in this study was taken from patient notes rather than a questionnaire, but reported a higher risk only in those who did one or the other, rather than both . As discussed previously, the frequency of k - ras mutations is probably not the best tool to assess the impact of suspected carcinogens, and the true impact of smoking in combination with alcohol on pancreatic carcinogenesis has yet to be determined . (d) genetic factorsa genetic predisposition is frequently alluded to in reviews of the aetiology of pancreatic cancers [63, 64] and certainly there are genetic disorders, such as hereditary pancreatitis [65, 66], the liefraumeni syndrome, peutz - jeghers syndrome and hereditary nonpolyposis colon cancer syndrome . Furthermore, familial pancreatic cancer is a well recognised entity [7173]. A recent retrospective cross - sectional cases - only study suggested an increased risk for pancreas cancer when multiple environmental risk factors (including tobacco smoke exposure) were present on a background of a family history of pancreas cancer . This study has again emphasised the role of environmental factors in modulating the genetic risk for development of pancreas cancer.when considering potential synergy between innate susceptibility and tobacco derived carcinogens, we are beginning to look toward genetic variability in the ways in which tobacco derived compounds or metabolites interact with targets, are metabolised, or even the ways in which the damage incurred is repaired . Polymorphisms in genes coding for dopamine receptors and transporters, nicotinic receptors and serotonin receptors, neurotransmitters and transporters may be relevant, and certainly understanding the genetic factors involved in addiction may ultimately result in more effective tobacco cessation programs reducing the incidence of tobacco related diseases, including pancreatic cancer . In addition, however, we are beginning to understand individual variability of susceptibility to damage at a more molecular level.both the quantity and duration of exposure to carcinogens may determine adduct formation or the capacity to cause damage . The carcinogens themselves may be metabolised at different rates in individuals . In human pancreatic tissues, there is individual variability in the capacity for, and stereoselectivity of, carbonyl reduction of nnk . In individuals whose microsomes metabolize nnk at a lower rate but form predominantly (s)-nnal, stereoselective localization of (s)-nnal in the human pancreas might occur, similar to the rat lung, where s - nnal gets retained resulting in higher incidence of cancers there .one family of proteins with an important role in the metabolism of foreign compounds and drugs, and, therefore, a key suspect in promoting cancer predisposition if relatively ineffective in some circumstances, are the cytochrome p450 (cyp) superfamily . There is a vast literature reporting various genetic polymorphism in cyps which increase cancer susceptibility . As yet, this is largely unconfirmed, with the exception of cyp2a6 polymorphism and tobacco - induced cancer [77, 78]. The role of these in tobacco related cancer has been summarised by others [7880] and is not discussed in depth here . In summary, although they have significant effects on tobacco - derived carcinogen metabolism, they are not considered to play an important role in pancreas cancer causation . Phase-2 metabolising enzymes such as uridine - diphospho - glucuronosyl transferase (udpgt), glutathione s - transferase (gst) [8285] have also been studied but are reported from relatively small single institution studies and need validation in large case - control molecular epidemiological studies.as well as variability in the capacity to cause damage, those pathways involved in the repair of damage have been studied, with significant individual variation reported . In particular, the role of polymorphisms of genes coding for enzymes and proteins within key metabolic and dna repair pathways in modulating risk for human pancreas cancer is being investigated and has been the subject of recent reviews [8688]. Numerous small single centre studies exploring the role of gene - environment interaction and interindividual variability in susceptibility to tobacco derived carcinogen damage [77, 8995] have recently been reported and require validation . In addition, genome wide association studies (gwas) in large numbers of individuals with pancreatic cancer are being performed and may ultimately lead us closer to the key genetic factors involved . The first of these confirmed an earlier epidemiologic finding that blood group o results in a decreased risk for pancreas cancer . Another gwas in japanese individuals, who have a particularly high risk of pancreatic cancer, was reported in 2010 and identified genes on chromosomes on 6, 7, and 12 and a further similar but larger study has suggested other specific loci on chromosomes 1, 5, and 13 to be associated with an increased risk for the malignancy; however, the functional significance of these findings is not clear . A genetic predisposition is frequently alluded to in reviews of the aetiology of pancreatic cancers [63, 64] and certainly there are genetic disorders, such as hereditary pancreatitis [65, 66], the liefraumeni syndrome, peutz - jeghers syndrome and hereditary nonpolyposis colon cancer syndrome . Familial atypical melanoma mole syndrome where pancreatic cancer is a major feature . Furthermore, familial pancreatic cancer is a well recognised entity [7173]. A recent retrospective cross - sectional cases - only study suggested an increased risk for pancreas cancer when multiple environmental risk factors (including tobacco smoke exposure) were present on a background of a family history of pancreas cancer . This study has again emphasised the role of environmental factors in modulating the genetic risk for development of pancreas cancer . When considering potential synergy between innate susceptibility and tobacco derived carcinogens, we are beginning to look toward genetic variability in the ways in which tobacco derived compounds or metabolites interact with targets, are metabolised, or even the ways in which the damage incurred is repaired . Polymorphisms in genes coding for dopamine receptors and transporters, nicotinic receptors and serotonin receptors, neurotransmitters and transporters may be relevant, and certainly understanding the genetic factors involved in addiction may ultimately result in more effective tobacco cessation programs reducing the incidence of tobacco related diseases, including pancreatic cancer . In addition, however, we are beginning to understand individual variability of susceptibility to damage at a more molecular level . Both the quantity and duration of exposure to carcinogens may determine adduct formation or the capacity to cause damage . The carcinogens themselves may be metabolised at different rates in individuals . In human pancreatic tissues, there is individual variability in the capacity for, and stereoselectivity of, carbonyl reduction of nnk . In individuals whose microsomes metabolize nnk at a lower rate but form predominantly (s)-nnal, stereoselective localization of (s)-nnal in the human pancreas might occur, similar to the rat lung, where s - nnal gets retained resulting in higher incidence of cancers there . One family of proteins with an important role in the metabolism of foreign compounds and drugs, and, therefore, a key suspect in promoting cancer predisposition if relatively ineffective in some circumstances, are the cytochrome p450 (cyp) superfamily . There is a vast literature reporting various genetic polymorphism in cyps which increase cancer susceptibility . As yet, this is largely unconfirmed, with the exception of cyp2a6 polymorphism and tobacco - induced cancer [77, 78]. The role of these in tobacco related cancer has been summarised by others [7880] and is not discussed in depth here . In summary, although they have significant effects on tobacco - derived carcinogen metabolism, they are not considered to play an important role in pancreas cancer causation . Phase-2 metabolising enzymes such as uridine - diphospho - glucuronosyl transferase (udpgt), glutathione s - transferase (gst) [8285] have also been studied but are reported from relatively small single institution studies and need validation in large case - control molecular epidemiological studies . As well as variability in the capacity to cause damage, those pathways involved in the repair of damage have been studied, with significant individual variation reported . In particular, the role of polymorphisms of genes coding for enzymes and proteins within key metabolic and dna repair pathways in modulating risk for human pancreas cancer is being investigated and has been the subject of recent reviews [8688]. Numerous small single centre studies exploring the role of gene - environment interaction and interindividual variability in susceptibility to tobacco derived carcinogen damage [77, 8995] have recently been reported and require validation . In addition, genome wide association studies (gwas) in large numbers of individuals with pancreatic cancer are being performed and may ultimately lead us closer to the key genetic factors involved . The first of these confirmed an earlier epidemiologic finding that blood group o results in a decreased risk for pancreas cancer . Another gwas in japanese individuals, who have a particularly high risk of pancreatic cancer, was reported in 2010 and identified genes on chromosomes on 6, 7, and 12 and a further similar but larger study has suggested other specific loci on chromosomes 1, 5, and 13 to be associated with an increased risk for the malignancy; however, the functional significance of these findings is not clear . Tobacco, as it is used in its most prevalent form, that is, smoking, is the most important risk factor for the development of pancreas cancer . There is compelling epidemiological evidence for a disease - causation effect for this which is backed up by physiological evidence based on laboratory studies on subcellular fractions, cell lines derived from human and animal models of the disease, whole animal models and human studies . There is also significant evidence for interindividual variation in susceptibility to tobacco - derived carcinogens which is a field of intense current research . Understanding of the role of not only tobacco carcinogens, but also the possible toxic role of nicotine itself in humans needs to be emphasised in public awareness campaigns . Further work is urgently required to explore the complex interactions between genetic, life - style / environmental factors at both laboratory and population levels to understand the disease better and enable improvement in outcome by not only earlier diagnosis, but also innovative and newer modes of intervention.
Air pollution is a health problem which results in several medical conditions in human beings . Old persons, children, patients with cardiovascular problems, pregnant women, and fetuses are more susceptible to these pollutants . Tehran is the capital of iran, a metropolitan area with more than 12 million inhabitants, more than 3 million cars and many factories, which all lead the city to be one of the most air - polluted cities in the world . In a study conducted in 2004 in tehran, the air quality over 262 days was worse than the standard levels specified by the u.s . Comparing with past years, the quality of tehran's air in 2004 was still threatening to people, especially vulnerable populations . Previous studies have indicated that there is a significant association between air pollution and a high rate of cardiovascular disease, including deep vein thrombosis and other atherosclerotic disorders such as myocardial infarction. [79] a study conducted in seoul, south korea, between 1995 and 1998 investigated the delayed effect of air pollution on mortality due to stroke, and the results showed that o3 and pm10 had the highest relationship with mortality due to stroke on the same day . Meanwhile, co, so2, and no2 densities at 48 hours before indicated a higher risk for mortality due to stroke . A study conducted in isfahan, the second most air - polluted city in iran, showed that there is an association between air pollution and the development of atherosclerosis in its first stages in the early life and emphasized the importance of considering the harmful effects of air pollution on children . Another study conducted in isfahan suggested an independent association between air pollution and systemic inflammatory and coagulation responses by studying a genetic polymorphism in a tissue factor in atherosclerotic lesions . Although many studies have discussed the side effects of air pollution on human health, there is not yet a consensus on the vascular side effects of air pollution . In order to provide more comprehensive data on the effects of air pollution on the health, we investigated the association between air pollution in tehran and the number of stroke admissions in the main referral hospitals of tehran . The patients were admitted in eight referral hospitals, located in different areas of tehran . These are the main referral hospitals in tehran which figured out that more than 90% of patients with stroke would be admitted by them . Complementary data including age, sex, risk, and modifying factors on stroke including hyperlipidemia, hypertension, cardiovascular disease, diabetes, smoking status, and type of stroke were obtained from the patient's file . In order to cover stroke in the younger group during analysis, daily information on tehran's air pollution in 2004 was supplied from the centre for control of traffic, transportation and air pollution of tehran . The data included daily levels of co, so2, nox, pm10, ozone (o3), and meteorological variables (temperature and humidity). We used the information from seven air - check stations of the eleven stations in tehran . We used the air quality index (aqi) to describe air quality which consists of six levels, which are shown in table 1 . Categories of pollutant density, necessary for calculating the air quality control index, are also shown in table 1 . In this table, the average 8-hour density was used for co and o3 and the average 24-hour density was used for so2 and pm10 . The density of pm10 was assessed by radiating beta ray, the density of co was assessed by using a non - dispersive infrared analyzer, the densities of o3 and so2 were assessed by using light spectrometry, and temperature and humidity were assessed by the vaisala model mp 113y and were analyzed continuously . Due to the lack of information on no2, nox was used in some stations . According to the standard table of clean air, offered by the u.s . Environmental protection agency, the density of no2 and nox was not mentioned in this classification . To evaluate the association between each pollutant and the rate of hospitalization separately, we used univariate analysis . We also used multi - variable poisson regression in which the number of hospitalizations for each day was the dependent variable and the previously mentioned air pollutants with a p value lower than 0.2 in the univariate analysis were independent variables . Poisson regression was also performed separately for each stratum of the variables of age (cut - off point: 40 years), sex, and underlying diseases (diabetes, hypertension, hypercholesterolemia, cardiovascular diseases, and smoking status). In this analysis, the adjusted relative risk (rr) was defined as the increase in the rate of hospitalization by the increased level of pollutants . Characteristics of studied subjects (n = 1491) the statistical significance of association between the 24 hour average of air pollutants with the number of stroke admissions on the same day and 48 hours before in univariate analysis based on analysis of variance and univariate poisson regression is shown in table 3 . Statistical significance level of association of average air pollutants with number of hospital admission for stroke as shown in table 3, the variables o3, co, so2, temperature, and humidity on the day of stroke and the variables nox, co, so2, temperature, and humidity 48 hours before stroke had p values lower than 0.2 and were therefore entered into multivariate poisson regression . In the multivariate poisson regression, as shown in table 4, the same - day level of the pollutants had no significant effect on the number of admitted stroke patients . As shown in table 5, the level of nox at 48 hours before had a significant effect on the number of hospitalizations (p = 0.02). Association between the same - day level of the pollutants and stroke admission in the different variables strata through multivariate poisson regression association between the 48 h before stroke level of the pollutants and stroke admission in the different variables strata through multivariate poisson regression as shown in tables 4 and 5, multivariate poisson regression was used to assess the association of same - day and 48 hours before stroke pollutant levels in different variables strata . Same - day temperature had a significant reverse association with hemorrhagic stroke admission (p = 0.034), and in patients without a history of heart disease (p = 0.046) or previous stroke (p = 0.043). Also, temperature at 48 hours before had a significant reverse association with hemorrhagic stroke admission (p = 0.017). The humidity level 48 hours before had a direct significant association with the stroke admission of patients with the history of heart disease (p = 0.007). Same - day nitrogen oxide level had a significant direct association with the stroke admission of patients with the history of hypertension (p = 0.031). The nitrogen oxide level 48 hours before stroke had a significant direct association with stroke admission of patients aged over 40 years (p = 0.025), patients with the history of hypertension (p = 0.005), and in patients without history of hyperlipidemia (p = 0.032). The carbon monoxide (co) level 48 hours before stroke had a significant direct association with stroke admission in female cases (p = 0.019), with ischemic stroke admission (p = 0.013), stroke admission in patients with the history of hypertension (p = 0.005), diabetics (p = 0.009), and non - smoking cases (p = 0.008). The sulfur dioxide level 48 hours before had a significant direct association with admission of patients with a history of heart disease (p = 0.004) and past smokers (p = 0.003). Stroke is a multi - factorial disease that is influenced by several genetic and environmental factors including life style and environmental conditions . The level of nox on the same day had a significant association with the occurrence of stroke, and factors including co, so2, and nox, temperature and humidity 48 hours before stroke had a significant association with stroke admission . These associations were different among different subgroups of age, sex, history of underlying diseases, and type of stroke . It could be concluded that the effect of pollutants on stroke 48 hours before stroke was higher than its effect on the same day as stroke which might be due to a requirement for an incubation time of these pollutants at least 48 hours before influencing the brain . In this study, in comparison with other similar studies, more pollutants were used for studying this relationship . In a study conducted by lokken in the u.s.a . Published in 2009, upon examining 1 101 patients with proven stroke, it was shown that observing hospitalized patients and not having a control group may have resulted in an underestimation of the relationship between air pollution and stroke . Therefore, it might be inferred that the true associations are stronger than the associations which were shown in this study . In a study conducted by hu in the u.s.a . In 2008, it was determined that for people who live in polluted areas and areas without any green space, the risk of mortality is higher in cases of stroke . In our study, we found a similar result in that there was a relationship between mortality as a result of stroke with pollutants including carbon monoxide and ozone on the day of stroke and carbon dioxide 48 hours before stroke . In a study that was conducted by oudin in sweden in 2009, upon studying 556 912 persons (contrasting our study and most previous studies), it was shown that there was no association between air pollution, especially between the nox index and hospitalizations due to stroke . In a study conducted by wang in australia, it was found that there was a significant association between temperature and hospitalization due to stroke and even there was association between the type of stroke with temperature, similar to the results of our study . In a study conducted by henrotin in dordjon, france from 1994 - 2004, it was found that there was a positive relationship between increasing amounts of o3 and pm10 and ischemic stroke, but there was no important relationship with hemorrhagic stroke . However, in our study, there was a significant relationship between ischemic stroke and carbon monoxide 48 hours before stroke and a significant relationship between hemorrhagic stroke and temperature on the same day and 48 hours before stroke in which temperature was a protective factor . The hazards of air pollution should be considered in all age groups even in young adults, and the impacts on elderly should be underscored . In terms of limitations, also, it was not a study on the individual level, i.e., it was performed on the ecologic level and the results were analyzed on the group level, so there may be bias in this study . On the other hand, since tehran is a big city and there are few stations for assessing air pollutants, it was not possible to determine accurate amounts of dangerous pollutants in persons with stroke . Also, the time of stroke was not accessible as an exact hour . In this study, potential confounding factors may have been due to risk factors which were dependent on time (time - varying), day, month, or climate conditions in which these confounding factors were considered in the multivariate analysis . Since diagnosis of stroke was performed in this study without having any information of the air pollution status by the physicians, patients, and even the research team, a considerable bias is not expected . Based on our findings and compared to previous studies, we can infer that air pollution and temperature are effective factors in the occurrence of cerebral stroke . However, they do not have any effect on mortality from stroke . Although this study was conducted in 2004, but considering the large sample size and as the findings of this study are not time - dependant, they can be generalized to different population in different times . According to the significant relationship indicated in this study, it can be concluded that policies should be considered for decreasing air pollution and consequently decreasing stroke, complications, and the mortality rate due to stroke . This could be regarded as an effective step on behalf of the organizations responsible for a healthy environment . We suggest conducting more studies at individual level to describe the role of each pollutant in causing stroke in the future.
A 26-year - old female patient was transferred to the samsung medical center from an outside institution due to severe refractory cardiogenic shock . The patient was diagnosed with non - hodgkin s lymphoma 2 years prior and underwent 6 cycles of chemotherapy, which included doxorubicin . After chemotherapy treatments were completed, the patient suffered from orthopnea and dyspnea, and an echocardiography revealed a severely depressed left ventricular (lv) ejection fraction (25%) and dilated lv . Due to complications from cancer treatments, she had an episode of acute decompensated heart failure and was referred to samsung medical center . On initial presentation, vital signs included arterial blood pressure of 79/27 mmhg, heart rate of 133 beats / min, respiratory rate of 28 breaths / min, and body temperature of 35.9c . She had pulmonary edema with bilateral pulmonary congestion (fig . 1) and multiorgan failure with liver and kidney involvement . Despite the use of inotropes and vasopressors, we were unable to achieve stable vital signs . Percutaneous extracorporeal life support (p - ecls) was then placed using the left femoral artery and vein . However, the left leg became ischemic after several hours, and we decided to shift the cannulas to the right groin . The patient was brought to a hybrid operating room, and the groin was opened bilaterally . Then, the arterial and venous cannulas were inserted through the common femoral artery and vein, and the catheter for the superficial femoral artery was inserted at the same time . The following day, transthoracic echocardiography revealed a distended left ventricle, and the chest x - ray showed worsening pulmonary edema . In this case, we determined that an atrial septostomy was not a viable option since both the femoral veins were already cannulated or had been surgically repaired . Instead, we performed an emergency standard median sternotomy and inserted a vent catheter (malleable 20 fr .) With a tip - cut into the right upper pulmonary vein and passed it into the left ventricle . Then, the inserted vent catheter was connected to the venous line of the p - ecls circuit via a y - shaped connector (fig ., the pump flow was maintained at 2.4 l / min / m, obtaining a mean systemic pressure of 60 mmhg and central venous pressure of 8 mmhg . The flow through the vent catheter was measured to be 1 to 2 l / min . The patient was on p - ecls and left heart venting for 5 days . During this time, 3). Transthoracic echocardiography revealed improved right and lv function and decreased chamber size . When the function of the right heart and the lung improved, the drainage from the femoral vein was gradually reduced by progressively clamping the venous line and lowering the r.p.m . In this way, the system was modified from p - ecls to paracorporeal lv assist device (lvad) by the complete clamping of the femoral venous drainage catheter . Thus, the femoral venous cannula and the oxygenator were removed on postoperative day 5 . The reasons for our decision were complete lv support, possible longer - term support due to the absence of the oxygenator and the low level of anticoagulation, and the prevention of recurrent lv distension . On postoperative day 8, we were able to wean the patient from lvad and remove all cannulas from her chest and groin . She was extubated and was finally discharged home on hospital day 28 . During follow - up, the lv ejection fraction worsened from 40% to 20%, and the symptoms of dyspnea became worse than before hospital discharge . Currently, this patient is on the heart transplant list and is waiting for transplantation . However, due to its inability to directly drain the left heart, its effectiveness in assisting the heart is limited . Additionally, several factors including severely reduced lv function, blood from native pulmonary and bronchial circulation, and increased afterload due to retrograde perfusion from the arterial cannula may lead to lv distension . This resultant lv distension can cause pulmonary edema, pulmonary hemorrhage, and myocardial ischemia . Although paracorporeal lvad placed through a median sternotomy provide better hemodynamic support without the risk of lv distension, complications of central cannulation such as bleeding and infection limit the widespread use of this technique . We report a case in which trans - sternal lv drainage was utilized while the patient was receiving p - ecls that was followed by a subsequent switch to paracorporeal lvad without further surgery . This strategy allowed the transformation of peripheral circulatory support into effective myocardial and systemic circulatory assistance and minimized the surgical risk . Although p - ecls offers excellent support to the blood circulation, its effect on the heart is less favorable . The left atrium can receive blood from the right heart and bronchial circulation . In the case of severe lv dysfunction, the lv cannot eject the received blood to overcome the increased afterload due to the retrograde p - ecls flow . The consequences of lv distension include pulmonary edema, pulmonary hemorrhage, right ventricular distension, and increased intraventricular pressure, which can lead to myocardial hypoperfusion and ischemia . The effect of p - ecls on lv distension was investigated previously by some authors . In an animal model of acute heart failure, kawashima et al . Reported that the resolution of ventricular fibrillation was related to lv unloading and reduction in myocardial oxygen consumption . Myocardial perfusion is proportional to the decrease in the lv wall tension and the compression of intra - myocardial coronary vessels . Therefore, lv decompression favorably affects ventricular recovery and increases the possibility of weaning from p - ecls . For this reason, a careful evaluation of the status of the lv and prompt drainage in the case of a pressure increase there are several techniques to decompress the lv, including a percutaneous trans - septal left atrial approach, lv venting through the right upper pulmonary vein, and direct lv apex cannulation . Although percutaneous lv venting does not require a surgical incision, the effectiveness of lv decompression can be limited according to the degree of mitral regurgitation . Thus, surgical venting is a more favorable option than percutaneous septostomy . Reported minimally invasive lv drainage in which an apical cannula is inserted into the lv apex transcutaneously . Although direct drainage of the lv through a trans - sternal approach is technically more complex, it has the advantage of high efficacy with respect to the intraventricular dynamics . Further, our group was more familiar with the trans - sternal approach at that time . Management after an lv venting procedure is still controversial, particularly since there is uncertainty in terms of patient stability after weaning from p - ecls . There are three ways of weaning from venoarterial ecls, namely the one - stage removal of all ecls cannulae, vent catheter removal and subsequent venoarterial ecls weaning, and venous cannula removal and subsequent paracorporeal lvad weaning . In this case, we decided to shift to paracorporeal lvad from p - ecls by progressively clamping the venous line and lowering the flow to the oxygenator until complete exclusion from the circuit . This allows for a heparin- and oxygenator - free trial of extracorporeal circulation, which reduces the chances of bleeding or thromboembolism . The low risk of bleeding and thromboembolism allows longer - term support for heart transplant than venoarterial ecls . The other advantages of our strategy include prevention of recurrent lv distension and pulmonary edema . In summary, p - ecls offers excellent circulatory support in emergency settings and assures rapid and systemic perfusion . However, p - ecls can negatively affect lv physiology and can potentially jeopardize myocardial recovery . Thus, a careful evaluation of the lv status and prompt drainage in the case of pressure increases should be considered . Our strategy assures complete lv venting and allows for a simple conversion of p - ecls to paracorporeal lvad.
Adult surgeons have applied it to all spectra of surgical diseases including inguinal hernia repair . With advancements in anesthesia and refinements in laparoscopic instruments, it has become conceivable that application of laparoscopy in pediatric surgical diseases including hernia repair is the next step . Unresolved debate still exists regarding the benefit of using laparoscopy over conventional open inguinal hernia repair even between laparoscopic surgeons . Although inguinal hernia repair is widely performed in every pediatric surgery department, the use of laparoscopy has not gained wide acceptance due to the benefits of conventional open repair regarding lower morbidity, good cosmesis, and lower rates of recurrence . Different techniques are being used to tighten up the hernia opening, which vary from a single stitch to a purse string procedure using laparoscopy . Several published comparative studies and large laparoscopic series aim to clarify the feasibility of laparoscopy in pediatric inguinal hernia repair and compare laparoscopy versus conventional repair . The aim of the current study was to compare various aspects of a recently reported laparoscopic flip - flap technique with the conventional open inguinal hernia repair in a short - term follow - up . A retrospective cohort study was performed including all children who underwent congenital inguinal hernia repair in the pediatric surgery department of alwasl hospital from july 2004 to october 2004 by either the new laparoscopic flip - flap technique described by yip et al or conventional open repair . Another 2 flank ports were introduced under direct laparoscopic guidance, and insufflations ranged from 8 mm hg to 10 mm hg . An incision over the peritoneum alongside the anterior and lateral edge of the hernia entry side was made; a peritoneal flap big enough to cover the hernia opening was raised . The anterior and lateral half circumference of the sac was detached from the surrounding soft tissue, and the opening of the hernia sac spontaneously collapsed . A 4 0 polyprolene suture was then introduced into the peritoneal cavity through the anterior abdominal wall . The needle was retrieved through the working port, and the wounds were then closed with absorbable sutures . The medical records of these patients were reviewed, and patients were divided into 2 groups according to the type of surgery done: group a comprised those who underwent the new laparoscopic technique, and group b comprised those who underwent conventional open repair . The following data were collected and analyzed: demographics, hernia site, operative time, intraoperative complications, time of postoperative hospital stay, and postoperative recurrence . Another 2 flank ports were introduced under direct laparoscopic guidance, and insufflations ranged from 8 mm hg to 10 mm hg . An incision over the peritoneum alongside the anterior and lateral edge of the hernia entry side was made; a peritoneal flap big enough to cover the hernia opening was raised . The anterior and lateral half circumference of the sac was detached from the surrounding soft tissue, and the opening of the hernia sac spontaneously collapsed . A 4 0 polyprolene suture was then introduced into the peritoneal cavity through the anterior abdominal wall . The needle was retrieved through the working port, and the wounds were then closed with absorbable sutures . The medical records of these patients were reviewed, and patients were divided into 2 groups according to the type of surgery done: group a comprised those who underwent the new laparoscopic technique, and group b comprised those who underwent conventional open repair . The following data were collected and analyzed: demographics, hernia site, operative time, intraoperative complications, time of postoperative hospital stay, and postoperative recurrence . All patients were male, and the age ranged from 4 months to 7 years (mean, 44 months). Group a included 15 patients (mean age, 39 months), while group b comprised 18 patients (mean age, 44 months). Five patients had bilateral hernias in group a versus 7 patients in group b (all of them had bilateral repairs). Operative time for group a ranged from 40 minutes to 55 minutes (mean, 47.5), in group b, operative time ranged from 20 minutes to 35 minutes (mean, 27.5). The calculated time excluded time consumed for any technical problems with the laparoscopic instruments . In group a, intraoperative complications included 1 case (7%) of vas deferens injury during mobilization of the flaps, and in 3 other cases (20%) the flaps were torn during suturing, which necessitated multiple sutures to close the defect . The range of postoperative hospital stay was from 3 hours to 8 hours (mean, 5.5). Postoperative follow - up of 3 months revealed recurrence in 4 cases in group a (27%), and no recurrences were encountered in group b. failure to close the processus vaginalis accounts for nearly all the inguino scrotal abnormalities seen in infancy and childhood . The concurrence rate of recurrent inguinal hernia after uncomplicated inguinal hernia conventional open repairs is generally reported at 0.5% to 1%, increasing for premature infants and complicated inguinal hernia . With advances in minimally invasive surgery, inguinal hernias have been treated routinely by the laparoscopic method in some centers . However, from the technical point of view, the procedure remains in its early evolution . Recurrence for laparoscopic pediatric inguinal hernia repair is approximately 4%, which is higher than recurrence for conventional open repair . Several technical modifications have been reported aiming to reduce the high recurrence rate after laparoscopic repair . Chan and tam advised normal saline injection at the extraperitoneal space to separate the vas deferens and testicular vessels from the peritoneum . Care still is needed to prevent hematoma formation with this procedure, thus avoiding recurrence . Schier initially performed laparoscopic hernia repair only in girls for fear of damaging the vas deferens and testicular vessels in boys . After refining his technique by using interrupted stitches to close the internal ring, thus avoiding this risk, he included boys for this operation . A space can lead to recurrence, whereas too narrow a space near the vas deferens or testicular vessels can cause obstruction . Prasad et al recently described the use of a 1.7-mm needle laparoscope, and a special curved stainless steel awl introduced percutaneously anteralateral to the internal ring for childhood hernia repair . This method still has the difficulty of separating the vas deferens and testicular vessels from the peritoneum, and the risk of damaging these important structures persists . Yep et al published a new laparoscopic technique in which the hernia opening was repaired with a peritoneal flip - flap anchored with a single tension - free intracorpo - real suture . The valve mechanism of the flip - flap helped to avoid scrotal collection and prevent hernia recurrence . The operative time was shorter in the open group than in the laparoscopic group, and the learning curve did not change the time which is comparable to that of the open technique due to the meticulous dissection required to create the flaps; however, even this caution did not guard against tearing of the flaps in 3 cases . Intraoperative complications in the open repair were nil in comparison with complications in the laparoscopic group in which one case of vassal injury occurred during creation of the flaps, and in 3 cases the flaps were torn during stitch fixation, which required closure of the internal ring by multiple stitches . Postoperative hospital stay was nearly the same in both groups, and all the patients were discharged the same day . Meanwhile, the postoperative analgesic requirements also did not differ in both groups, and this may be attributed to our policy of routine preemptive analgesia with nonsteroidal anti - inflammatory medication . We did not encounter any postoperative recurrence in the open group in the 3-month follow - up, while 4 cases of recurrence occurred in the laparoscopic group . From our experience, intraoperative complications and postoperative recurrence in the laparoscopic flip - flap technique were a major problem discouraging wide use of this new technique in comparison with the satisfactory results obtained by conventional open repair from all aspects including good cosmetic appearance and lower rate of recurrence . Although the incidence of contralateral hernia remains controversial, the advantage of using laparoscopy for inguinal hernia repair to diagnose the contralateral side should be associated with comparable results to that of the conventional open repair . Moreover, until we have satisfactory outcomes for laparoscopic hernia repair in children, the open repair remains the gold standard for pediatric surgeons, and other options for managing the contralateral side are still available through either observation and repair of a contralateral hernia only if it later becomes apparent, routine contralateral groin exploration, and laparoscopy to evaluate the contralateral groin for a potential hernia that could be used through the inguinal incision or by using a mini - telescope through the umbilicus . Our preliminary experience showed unsatisfactory outcomes with the laparoscopic flip - flap hernia repair in children . In spite of the advancement in the application of laparoscopy in pediatric surgery, future studies with more numbers and long - term follow - up should be conducted.
Procedures involving animals were approved by the animal care and use committee of the schepens eye research institute . Male sprague - dawley rats (5 weeks of age; taconic farms, hudson, ny) were randomly assigned to one of the following groups: control, diabetic, diabetic treated with sorbinil (65 mg kg day; gift of pfizer, groton, ct), and diabetic treated with aspirin (30 mg kg day). Induction of diabetes with streptozotocin, administration of sorbinil and aspirin with the food, and treatment of diabetic rats with maintenance insulin were as previously described (10,11). Diabetic rats and age - matched nondiabetic control rats were studied after 3, 6, and 8 months of diabetes . A1c, plasma salicylates levels, and retinal levels of sorbitol and fructose were measured as previously described (6,10,11). Rat retinal vessels were isolated by hypotonic lysis of the fresh retina, a method documented to yield the retinal vascular network free of glia and neural contamination (10). The two retinas of each rat were dissected and incubated in ice - cold sterile distilled water for 1 h at 4c . After incubation with dnase to dissolve dna released from the lysed neural and glial cells, the vascular network was transferred to ice - cold pbs and cleaned of debris and remaining glial and neural elements by gentle pipetting . Total rna free of contaminating genomic dna was isolated using the rneasy mini kit (qiagen, valencia, ca) following the on - column dnase digestion protocol according to the manufacturer's instructions . The retinal vascular network was also prepared by trypsin digestion (3) of retinas fixed in formalin immediately after killing . Gene expression profiling of rat retinal vessels was performed using the genechip rat genome 230 2.0 array (affymetrix, santa clara, ca). Each sample subjected to microarray hybridization consisted of the rna extracted from the vessels of both retinas of one individual rat . Because the amount of rna was insufficient for the standard hybridization protocol, each rna sample was subjected to a round of amplification (ovation biotin system; nugen technologies, san carlos, ca). All procedures for microarray analysis were carried out at the harvard medical school / partners healthcare center for genetics and genomics core laboratory (http://www.hpcgg.org). Gene expression data were preprocessed with mas 5.0, and probes that were consistently labeled as absent in all samples were excluded from subsequent analysis . Analysis of the microarray data was performed using badge (bayesian analysis of differential gene expression; http://genomethods.org/badge), a bayesian approach to identify differentially expressed genes across two experimental conditions designed to yield high reproducibility at low sample size (14). The differential expression of each gene in two conditions is estimated by the fold change, and evidence of differential expression is measured by the probability that the fold change exceeds a fixed threshold, conditional on the data . To reduce the number of false positives due to multiple comparisons, the selection of significant changes of expression was based on a threshold on the posterior probability specified to achieve a preset false discovery rate, as determined by significance analysis of microarrays (15). To identify the genes whose expression is affected by diabetes, only the 19,054 probe sets present in all samples (eight diabetic and nine controls) probe sets with a probability> 0.985 (equivalent to a false discovery rate <0.01) were selected as differentially expressed in diabetes . To determine which of the diabetes - induced gene expression changes were prevented by the two drugs, we selected the probes identified as differentially expressed in diabetes and compared their expression in the sorbinil - treated diabetic group (n = 5) versus the control group, and in the aspirin - treated diabetic group (n = 9) versus the control group . Genes for which the posterior probability of differential expression versus control was> 0.95 were considered different from control, and the diabetes - induced changes were thus classified as not corrected by the drug . Conversely, when the posterior probability of differential expression versus control was <0.95, the expression was classified as similar to control and thus normalized by the drug . The threshold was higher than that used to identify differential expression caused by diabetes to increase the specificity of the conclusions about the drug effects . The probe sets differentially expressed in diabetes were annotated using netaffx (www.affymetrix.com/analysis), and the annotations were individually verified based on the latest unigene cluster of the corresponding representative sequence . The differentially expressed genes identified through the annotation process were assigned to functional categories based on the information provided by entrez gene (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) and additional information from the published literature . Cdna was synthesized (8) from 1 g of total rna isolated from retinal microvessels and total retina of rats different from those studied in the microarray experiments . Primers and probe sets for rat transforming growth factor (tgf)-1 and adamts1 (a disintegrin and metalloproteinase with thrombospondin motif type 1) were from applied biosystems (foster city, ca). Relative expression was determined by the comparative ct method using -actin as the endogenous control (16). Fresh retinal microvessels and whole retinas were homogenized in ripa buffer containing protease and phosphatase inhibitors (10). The blots were sequentially probed for tgf- receptor 1 (tgfr1), cyclooxygenase (cox)-1, and nuclear receptor coactivator 3 (ncoa3). Ras gtpase activating protein (17) or -actin were used to verify protein loading . For evaluation of smad2 phosphorylation, blots were probed first for phosphorylated smad2, stripped, and reprobed for total smad2 . The primary antibodies used are listed in supplemental table 1, which is available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db08-1008/dc1 . Retinal trypsin digests were rehydrated in pbs and incubated in citrate buffer (ph 6.1; dako, carpinteria, ca) at 95c for heat - mediated antigen retrieval . After permeabilization in 1% triton x-100, quenching of endogenous peroxidase in 3% h2o2, and blocking in 10% normal goat serum, endogenous biotin (unmasked by the antigen retrieval procedure) was blocked by incubation in avidin and biotin blocking solutions (vector laboratories, burlingame, ca). The trypsin digests were then incubated overnight at 4c with the tgfr1 primary antibody (v-22; santa cruz biotechnology) diluted to 1 g / ml in 2% goat serum/0.1% triton x-100 in pbs . The antigen - antibody complexes were detected by the avidin - biotin - peroxidase method (abc elite; vector laboratories) and visualized with diaminobenzidine . Each experiment included trypsin digests from each of the four groups of rats as well as a negative control obtained by substituting the primary antibody with an equivalent concentration of normal rabbit igg . The trypsin digests were photographed at 5 magnification (axiovision imaging system; carl zeiss, gottingen, germany) to reconstruct the entire vascular network from each retina (11). The networks were used to identify for each rat four areas in the midretina with equivalent location and free of artifacts . The four areas were photographed at 20 magnification using the same exposure and light setting to allow for comparison among samples . For evaluation of tgfri immunostaining, the images were grouped in panels of 12 images each, with each panel containing images from control, diabetic, and diabetic treated rats . The staining intensity of the capillaries within each image was scored on a continuous scale from 0 (no staining) to 4 (highest staining) by four masked observers . The scores attributed to each retina by the four observers were averaged to obtain one individual final score for each rat . The scoring was consistent among the four observers, as indicated by an overall coefficient of variation of 17% . Complement activation in rat retinal vessels was evaluated by membrane attack complex immunostaining on rat retinal sections as previously described (10). The results of the effect of sorbinil and aspirin on diabetes - induced gene expression changes are summarized as the number of genes whose expression was normalized by the drug within each functional category . The significance of the deviation of this number from the null hypothesis of no gene being normalized was determined by means of a fisher's exact test (2 2 contingency tables). Results of western blotting and real - time rt - pcr are summarized as the means sd; the results in the diabetic and control group were compared by means of a two - tailed unpaired student's t test . Results of tgfri immunostaining are summarized with the means sd; the results in the four groups of rats were compared with anova followed by fisher's protected least - significant differences test . Statview 5.0 software (sas institute, cary, nc) was used for all analyses . Rat retinal vessels were isolated by hypotonic lysis of the fresh retina, a method documented to yield the retinal vascular network free of glia and neural contamination (10). The two retinas of each rat were dissected and incubated in ice - cold sterile distilled water for 1 h at 4c . After incubation with dnase to dissolve dna released from the lysed neural and glial cells, the vascular network was transferred to ice - cold pbs and cleaned of debris and remaining glial and neural elements by gentle pipetting . Total rna free of contaminating genomic dna was isolated using the rneasy mini kit (qiagen, valencia, ca) following the on - column dnase digestion protocol according to the manufacturer's instructions . The retinal vascular network was also prepared by trypsin digestion (3) of retinas fixed in formalin immediately after killing . Gene expression profiling of rat retinal vessels was performed using the genechip rat genome 230 2.0 array (affymetrix, santa clara, ca). Each sample subjected to microarray hybridization consisted of the rna extracted from the vessels of both retinas of one individual rat . Because the amount of rna was insufficient for the standard hybridization protocol, each rna sample was subjected to a round of amplification (ovation biotin system; nugen technologies, san carlos, ca). All procedures for microarray analysis were carried out at the harvard medical school / partners healthcare center for genetics and genomics core laboratory (http://www.hpcgg.org). Gene expression data were preprocessed with mas 5.0, and probes that were consistently labeled as absent in all samples were excluded from subsequent analysis . Analysis of the microarray data was performed using badge (bayesian analysis of differential gene expression; http://genomethods.org/badge), a bayesian approach to identify differentially expressed genes across two experimental conditions designed to yield high reproducibility at low sample size (14). The differential expression of each gene in two conditions is estimated by the fold change, and evidence of differential expression is measured by the probability that the fold change exceeds a fixed threshold, conditional on the data . To reduce the number of false positives due to multiple comparisons, the selection of significant changes of expression was based on a threshold on the posterior probability specified to achieve a preset false discovery rate, as determined by significance analysis of microarrays (15). To identify the genes whose expression is affected by diabetes, only the 19,054 probe sets present in all samples (eight diabetic and nine controls) probe sets with a probability> 0.985 (equivalent to a false discovery rate <0.01) were selected as differentially expressed in diabetes . To determine which of the diabetes - induced gene expression changes were prevented by the two drugs, we selected the probes identified as differentially expressed in diabetes and compared their expression in the sorbinil - treated diabetic group (n = 5) versus the control group, and in the aspirin - treated diabetic group (n = 9) versus the control group . Genes for which the posterior probability of differential expression versus control was> 0.95 were considered different from control, and the diabetes - induced changes were thus classified as not corrected by the drug . Conversely, when the posterior probability of differential expression versus control was <0.95, the expression was classified as similar to control and thus normalized by the drug . The threshold was higher than that used to identify differential expression caused by diabetes to increase the specificity of the conclusions about the drug effects . The probe sets differentially expressed in diabetes were annotated using netaffx (www.affymetrix.com/analysis), and the annotations were individually verified based on the latest unigene cluster of the corresponding representative sequence . The differentially expressed genes identified through the annotation process were assigned to functional categories based on the information provided by entrez gene (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) and additional information from the published literature . Cdna was synthesized (8) from 1 g of total rna isolated from retinal microvessels and total retina of rats different from those studied in the microarray experiments . Primers and probe sets for rat transforming growth factor (tgf)-1 and adamts1 (a disintegrin and metalloproteinase with thrombospondin motif type 1) were from applied biosystems (foster city, ca). Relative expression was determined by the comparative ct method using -actin as the endogenous control (16). Fresh retinal microvessels and whole retinas were homogenized in ripa buffer containing protease and phosphatase inhibitors (10). The blots were sequentially probed for tgf- receptor 1 (tgfr1), cyclooxygenase (cox)-1, and nuclear receptor coactivator 3 (ncoa3). Ras gtpase activating protein (17) or -actin were used to verify protein loading . For evaluation of smad2 phosphorylation, blots were probed first for phosphorylated smad2, stripped, and reprobed for total smad2 . The primary antibodies used are listed in supplemental table 1, which is available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db08-1008/dc1 . Retinal trypsin digests were rehydrated in pbs and incubated in citrate buffer (ph 6.1; dako, carpinteria, ca) at 95c for heat - mediated antigen retrieval . After permeabilization in 1% triton x-100, quenching of endogenous peroxidase in 3% h2o2, and blocking in 10% normal goat serum, endogenous biotin (unmasked by the antigen retrieval procedure) was blocked by incubation in avidin and biotin blocking solutions (vector laboratories, burlingame, ca). The trypsin digests were then incubated overnight at 4c with the tgfr1 primary antibody (v-22; santa cruz biotechnology) diluted to 1 g / ml in 2% goat serum/0.1% triton x-100 in pbs . The antigen - antibody complexes were detected by the avidin - biotin - peroxidase method (abc elite; vector laboratories) and visualized with diaminobenzidine . Each experiment included trypsin digests from each of the four groups of rats as well as a negative control obtained by substituting the primary antibody with an equivalent concentration of normal rabbit igg . The trypsin digests were photographed at 5 magnification (axiovision imaging system; carl zeiss, gottingen, germany) to reconstruct the entire vascular network from each retina (11). The networks were used to identify for each rat four areas in the midretina with equivalent location and free of artifacts . The four areas were photographed at 20 magnification using the same exposure and light setting to allow for comparison among samples . For evaluation of tgfri immunostaining, the images were grouped in panels of 12 images each, with each panel containing images from control, diabetic, and diabetic treated rats . The staining intensity of the capillaries within each image was scored on a continuous scale from 0 (no staining) to 4 (highest staining) by four masked observers . The scores attributed to each retina by the four observers were averaged to obtain one individual final score for each rat . The scoring was consistent among the four observers, as indicated by an overall coefficient of variation of 17% . Complement activation in rat retinal vessels was evaluated by membrane attack complex immunostaining on rat retinal sections as previously described (10). The results of the effect of sorbinil and aspirin on diabetes - induced gene expression changes are summarized as the number of genes whose expression was normalized by the drug within each functional category . The significance of the deviation of this number from the null hypothesis of no gene being normalized was determined by means of a fisher's exact test (2 2 contingency tables). Results of western blotting and real - time rt - pcr are summarized as the means sd; the results in the diabetic and control group were compared by means of a two - tailed unpaired student's t test . Results of tgfri immunostaining are summarized with the means sd; the results in the four groups of rats were compared with anova followed by fisher's protected least - significant differences test . Statview 5.0 software (sas institute, cary, nc) was used for all analyses . The characteristics of the rats used in the microarray and related studies are presented in table 1 . The microarray study was based on 31 expression profiles of retinal microvessels isolated from diabetic rats (n = 8), diabetic rats treated with sorbinil (n = 5), diabetic rats treated with aspirin (n = 9), and control rats (n = 9). The duration of diabetes was 6 months . When compared with the nondiabetic rats, the retinal vessels of the diabetic rats showed significantly different expression of 127 known genes (131 probe sets) and 229 expressed sequence tags (ests) or genes with similarity to known genes but not yet fully characterized . The majority of the 127 known genes (105 upregulated and 22 downregulated by diabetes) could be grouped into several functional categories (table 2). Characteristics, retinal polyol pathway activity, and serum salicylate levels of study rats data are means sd . Statistical analysis was performed with anova followed by fisher's protected least - significant difference test . * measurements were performed in four rats from each group; measurements were performed in three diabetic rats treated with aspirin and one untreated diabetic rat; functional clustering of the known genes differentially expressed in retinal vessels in diabetes * the 127 known genes differentially expressed in diabetes were assigned to functional categories based on review of information from ncbi (national center for biotechnology information) entrez gene integrated with data from published literature . Individual genes are listed in all pertinent categories based on their known functions . The complete list of the genes included in each functional category is presented in supplemental tables 28 . Includes genes related to functions that cannot be assigned to any of the major categories; as stated in bibliographical sources . There was a prominent effect of diabetes on the expression of genes involved in signaling by members of the tgf- family or affected by such signaling (fig . 1 and supplemental table 2). A total of 20 genes could be attributed to this category, which made the tgf- pathway the single functional pathway most affected by diabetes in retinal vessels . Diabetes increased the expression of the type i receptor activin receptor like kinase 5 (alk-5), a widely distributed tgf- type i receptor that in endothelial cells signals inhibition of migration and proliferation (18). Alk-1, a tgf- type i receptor that is expressed more selectively in endothelium and induces migration and proliferation (18), was not upregulated by diabetes . There was increased expression of syndecan-2, which is upregulated by tgf- and, in turn, upregulates the tgf- type i and type ii receptors (19); of cd44, also a target of tgf- that facilitates the activation of tgf- and signaling by the type i receptor (20); and of ubiquitin conjugating enzyme 9, the key enzyme of the sumoylation reaction, which is known to enhance tgf- signaling (21). The diabetic retinal microvessels showed enhanced expression of connective tissue growth factor (ctgf), the main tgf- effector in the induction of fibrosis (22); and of other genes prominently regulated by tgf-, such as tissue inhibitor of metalloproteinase 1, the serine (or cysteine) proteinase inhibitor heat shock protein 47, prostaglandin - endoperoxide synthase 1 (cox-1), the histone acetyltransferase ncoa3, and thioredoxin - interacting protein (txnip) (2225). For some of these molecules, increased expression was confirmed at the protein level (supplemental fig . 1). Diabetes alters the expression of multiple genes of the tgf- pathway (left side of the panel) and bmps pathway (right side) in rat retinal vessels . The expected effect of overexpression of genes on smad signaling is shown by arrowed lines (stimulation) or blunt lines (inhibition). The expected effect of downregulation of hoxc8 is release of inhibition, and thus activation of signaling (dotted blunt line). Bmpr1a, bmp receptor 1a; hsp47, heat shock protein 47; ifnr2, -interferon receptor 2; mxi1, max interactor 1; soat1, sterol o - acyltransferase 1; timp1, tissue inhibitor of metalloproteinase 1; ubc9, ubiquitin conjugating enzyme 9 . Diabetic retinal vessels showed changes in gene expression pointing to increased signaling also by bone morphogenetic proteins (bmps) (fig . 1 and supplemental table 2). There was increased expression of bmp receptor type 1a, also named alk-3 and utilized by bmp-2 (26), and of periostin and frizzled homolog 1, both responsive to vascular injury and known targets of bmp-2 (27,28). Facilitated bmp signaling was also suggested by the downregulation of the homeobox c8 (hoxc8) transcription factor because hoxc8 is a negative regulator of transcription of smad1 and other bmp - specific receptor - regulated smads (29). The array included probe sets for bmp-1 through -7; only the bmp-2 transcript was detected in all samples, and its levels were not modified by diabetes . Of the three tgf- isoforms present in mammals, the array showed the tgf-2 and -3 transcripts; the levels of neither were altered by diabetes . The tgf-1 mrna was undetectable in all samples tested . When measured by the more sensitive real - time rt - pcr, tgf-1 mrna levels were increased 1.5-fold in the retinal microvessels of diabetic rats (p = 0.04) (fig . 2a). Increased levels of the tgf- type i receptor alk-5 was confirmed at the protein level (fig . 2b). Because alk-5 signals through smad2 and smad3, we sought evidence of alk-5 activation by measuring smad2 phosphorylation in the retinal vessels of diabetic and control rats . We found increased smad2 phosphorylation after both 6- and 3-month duration of diabetes (fig . 2c). Taken together, the data indicate that the complete loop for tgf-1 signaling is enhanced in diabetic retinal vessels . Total rna and protein lysates were prepared from retinal vessels isolated by hypotonic lysis from the retina of diabetic (d) and age - matched control (c) rats . A: tgf-1 mrna levels assayed by quantitative real - time pcr . Relative expression of tgf-1 mrna was calculated by the comparative ct method using -actin as endogenous control . B: representative western blot of tgfr1 (alk-5) and bar plot of the quantitative analysis . Blots were probed first for tgfr1 followed by ras gtpase activating protein (rasgap) as control for loading . C: representative western blots of phosphorylated smad2 (p - smad2) and total smad2 (smad2) and bar plot of the p - smad2to smad2 ratio . The p - smad2to smad2 ratio was examined in the retinal vessels of rats with 6 months (left panel) and 3 months of diabetes (right panel). The 5860-kda bands detected in both retinal microvessels and the positive control (+; tgf-treated hepg2 cells) corresponds to smad2; the 50-kda band present in the positive control only corresponds to smad3 . The bar plot presents the pooled results obtained in rats with 6 and 3 months of diabetes . * p = 0.05 . To learn whether the diabetes - induced changes in the tgf- pathway extended to nonvascular cells of the retina, we measured the levels of the tgf-1 transcript, tgf- type i receptor, and smad2 phosphorylation in the whole retina . C). Tgf-1 signaling is not increased in the neural retina of diabetic rats . Total rna and protein lysates were prepared from the whole retina of diabetic (d) and age - matched control (c) rats . B: representative western blot of tgfr1 (alk-5) and bar plot of the quantitative analysis . C: representative western blots of phosphorylated smad2 (p - smad2) and total smad2 (smad2) and bar plot of the p - smad2to smad2 ratio . Diabetes increased the expression of molecules that generate oxygen - derived free radicals and decreased the expression of antioxidants (supplemental table 3). Similar to the aorta of diabetic rats (30), the retinal vessels showed prominent upregulation of txnip, a negative regulator of the antioxidant thioredoxin . Diabetes also increased the expression of genes involved in inflammation and response to injury (supplemental table 4). The gene expression program was thus consistent with, and expanded, previous findings pointing to the occurrence of oxidative stress and inflammation in diabetic retinal vessels (9,31,32). Genes involved in tissue remodeling through formation of extracellular matrix, regulation of cell adhesion, and organization of the actin cytoskeleton were upregulated in the diabetic vessels (supplemental table 5). All changes related to actin were in the direction of increased polymerization and filament stability, which may contribute antiproliferative effects (33). Additional gene expression changes induced by diabetes pointed to inhibitory effects on cell cycle progression and cell proliferation (supplemental table 6). Multiple mechanisms of apoptosis appeared activated in diabetic vessels, many not suspected previously (supplemental table 7). Changes in the expression of six genes of the p53 pathway converged in the proapoptotic direction . Tgf- can induce apoptosis by multiple mechanisms (34), which include upregulation of fas and activation of c - jun nh2-terminal kinase (34,35), the expression of which was also increased in the diabetic retinal vessels (supplemental table 7). A potential mechanism for apoptosis by neglect was the downregulation of the g - protein coupled receptor of lysophosphatidic acid edg2 (endothelial differentiation gene 2), a survival factor for multiple cell types (36). The sorbinil and aspirin doses were previously documented to prevent the development of acellular capillaries in diabetic rats (10,11). In this study, the dose of sorbinil overnormalized sorbitol and reduced fructose accumulation by 96% in the retina of diabetic rats (table 1), thus achieving almost complete inhibition of glucose flux through the polyol pathway . The aspirin dose, known to inhibit by 90% thromboxane b2 formation during blood clotting in rats and thus exert an antiplatelet effect (11), resulted in serum salicylate levels (table 1) 10- to 20-fold lower than those achieved by anti - inflammatory doses of aspirin (rev . In (11). Sorbinil normalized the diabetes - induced expression changes in 56% of the known genes and aspirin normalized those in 32%; the figures for the total number of probe sets (known genes and ests) were 48 and 47%, respectively . Both sorbinil and aspirin attenuated significantly and to a similar extent the effects of diabetes on the tgf- pathway (fig . 4), preventing the expression changes of 55 and 40% of the genes, respectively . Of note, both drugs prevented the increased expression of tgf- receptor 1 and ctgf . Likewise, sorbinil and aspirin reduced significantly and to a similar extent the effects of diabetes on proapoptotic pathways, with concordant correction of changes in the p53 pathway and concordant restoration of survival signals . In contrast, sorbinil reduced the changes in gene expression relevant to oxidative stress and inflammation strikingly more than aspirin (fig . Sorbinil led to normalization of expression of 71% of genes related to oxidative stress and 62% of those related to inflammation, whereas the figures for aspirin were 29 and 3%, respectively (p <0.0001 for effect of sorbinil vs. aspirin on inflammation - related genes). The effect of sorbinil and aspirin on the expression of individual genes is reported in supplemental tables 28 . Effects of sorbinil and aspirin treatments on the gene expression changes induced by diabetes in rat retinal vessels . Each functional category includes all genes pertinent to that category, as indicated in table 2 ., the number of genes differentially expressed in the retinal vessels of diabetic rats (untreated or treated) compared with control rats; the number of genes whose expression is normalized by the drug (i.e., the expression in the retinal vessels of treated diabetic rats was not different from that in control rats). D, diabetic rats; d+sor, diabetic rats treated with sorbinil; d+asa, diabetic rats treated with aspirin; ns, nonsignificant . The effects of sorbinil and aspirin on diabetes - induced activation of the tgf- pathway were also tested on trypsin digests prepared from the retinas of rats with 8 months of diabetes . These preparations afforded the opportunity of studying the retinal vascular network fixed immediately after death and not subjected to hypotonic lysis while fresh . Figure 5 shows that the diffuse pattern of tgfr1 immunoreactivity was increased in the retinal capillaries of diabetic rats compared with control rats (p = 0.05) and that both aspirin and sorbinil treatment prevented the increase (p = 0.03 and p <0.0001 vs. diabetes, respectively), in agreement with the microarray results (supplemental table 2). Of note, whereas aspirin returned tgfr1 immunoreactivity to control values (p = 0.62 vs. control), sorbinil suppressed staining intensity below control values (p = 0.015 vs. control). Phosphorylated smad2 could not be detected in the fixed vascular preparations . Increased tgfri immunoreactivity in retinal vessels of diabetic rats tgfri was detected by immunohistochemistry in retinal trypsin digests from diabetic rats (8 months of diabetes duration), diabetic rats treated with sorbinil or aspirin, and age - matched control rats . The effect of diabetes and of the two drugs on tgfri immunostaining was quantitated by four masked observers . C: representative photographs of midretina fields showing tgfri immunostaining of retinal capillaries . A: control, score 2.0 0.7 (mean sd of scores by the different masked observers). Values are the means sd of the final scores computed for each individual rat . C, control rats, n = 11; d, diabetic rats, n = 10; d+sor, diabetic rats treated with sorbinil, n = 6; d+asa, diabetic rats treated with aspirin, n = 6 . * p = 0.05 vs. control rats; p <0.02 versus control rats, diabetic rats, and diabetic rats treated with aspirin, p <0.04 versus diabetic rats . The discordant effect of sorbinil and aspirin on inflammation, as shown by the mrna data, was consistent with the discordant effect on the increased levels of intercellular adhesion molecule 1 in the diabetic rat retina (11) and was confirmed by data on complement activation in retinal vessels . 2 shows that aspirin, at variance with sorbinil (10), failed to prevent deposition of membrane attack complex in retinal vessels of diabetic rats . We compared the effects of diabetes on gene expression in retinal vessels to those in mller cells, the principal glia of the retina that shows signs of activation / reactivity in both human (37) and experimental (6) diabetes . We had previously examined the gene expression profile of mller cells in the same model used in this study (rats with 6-month duration of streptozotocin diabetes) and on a comparable platform (genechip rat genome rg - u34a; affymetrix) (16). Supplemental table 9 shows that the functional gene categories affected by diabetes in retinal vessels differed from those affected in mller cells . Of the genes related to tgf- that we found affected by diabetes in retinal vessels, 80% were represented in the rg - u34a array, but only 6% of those were affected by diabetes in mller cells . Of the genes related to apoptosis affected by diabetes in retinal vessels, 68% were represented in the rg - u34a array, and 0% of those were affected by diabetes in mller cells . Only the inflammation - related genes showed overexpression in both the vascular and glial cell types (76% of the genes affected by diabetes in the vessels were represented in the rg - u34a, and 55% were upregulated by diabetes in mller cells). There was a prominent effect of diabetes on the expression of genes involved in signaling by members of the tgf- family or affected by such signaling (fig . 1 and supplemental table 2). A total of 20 genes could be attributed to this category, which made the tgf- pathway the single functional pathway most affected by diabetes in retinal vessels . Diabetes increased the expression of the type i receptor activin receptor like kinase 5 (alk-5), a widely distributed tgf- type i receptor that in endothelial cells signals inhibition of migration and proliferation (18). Alk-1, a tgf- type i receptor that is expressed more selectively in endothelium and induces migration and proliferation (18), was not upregulated by diabetes . There was increased expression of syndecan-2, which is upregulated by tgf- and, in turn, upregulates the tgf- type i and type ii receptors (19); of cd44, also a target of tgf- that facilitates the activation of tgf- and signaling by the type i receptor (20); and of ubiquitin conjugating enzyme 9, the key enzyme of the sumoylation reaction, which is known to enhance tgf- signaling (21). The diabetic retinal microvessels showed enhanced expression of connective tissue growth factor (ctgf), the main tgf- effector in the induction of fibrosis (22); and of other genes prominently regulated by tgf-, such as tissue inhibitor of metalloproteinase 1, the serine (or cysteine) proteinase inhibitor heat shock protein 47, prostaglandin - endoperoxide synthase 1 (cox-1), the histone acetyltransferase ncoa3, and thioredoxin - interacting protein (txnip) (2225). For some of these molecules, increased expression was confirmed at the protein level (supplemental fig . 1). Diabetes alters the expression of multiple genes of the tgf- pathway (left side of the panel) and bmps pathway (right side) in rat retinal vessels . The expected effect of overexpression of genes on smad signaling is shown by arrowed lines (stimulation) or blunt lines (inhibition). The expected effect of downregulation of hoxc8 is release of inhibition, and thus activation of signaling (dotted blunt line). Bmpr1a, bmp receptor 1a; hsp47, heat shock protein 47; ifnr2, -interferon receptor 2; mxi1, max interactor 1; soat1, sterol o - acyltransferase 1; timp1, tissue inhibitor of metalloproteinase 1; ubc9, ubiquitin conjugating enzyme 9 . Diabetic retinal vessels showed changes in gene expression pointing to increased signaling also by bone morphogenetic proteins (bmps) (fig . 1 and supplemental table 2). There was increased expression of bmp receptor type 1a, also named alk-3 and utilized by bmp-2 (26), and of periostin and frizzled homolog 1, both responsive to vascular injury and known targets of bmp-2 (27,28). Facilitated bmp signaling was also suggested by the downregulation of the homeobox c8 (hoxc8) transcription factor because hoxc8 is a negative regulator of transcription of smad1 and other bmp - specific receptor - regulated smads (29). The array included probe sets for bmp-1 through -7; only the bmp-2 transcript was detected in all samples, and its levels were not modified by diabetes . Of the three tgf- isoforms present in mammals, the array showed the tgf-2 and -3 transcripts; the levels of neither were altered by diabetes . The tgf-1 mrna was undetectable in all samples tested . When measured by the more sensitive real - time rt - pcr, tgf-1 mrna levels were increased 1.5-fold in the retinal microvessels of diabetic rats (p = 0.04) (fig . 2a). Increased levels of the tgf- type i receptor alk-5 was confirmed at the protein level (fig . Because alk-5 signals through smad2 and smad3, we sought evidence of alk-5 activation by measuring smad2 phosphorylation in the retinal vessels of diabetic and control rats . We found increased smad2 phosphorylation after both 6- and 3-month duration of diabetes (fig . 2c). Taken together, the data indicate that the complete loop for tgf-1 signaling is enhanced in diabetic retinal vessels . Total rna and protein lysates were prepared from retinal vessels isolated by hypotonic lysis from the retina of diabetic (d) and age - matched control (c) rats . A: tgf-1 mrna levels assayed by quantitative real - time pcr . Relative expression of tgf-1 mrna was calculated by the comparative ct method using -actin as endogenous control . B: representative western blot of tgfr1 (alk-5) and bar plot of the quantitative analysis . Blots were probed first for tgfr1 followed by ras gtpase activating protein (rasgap) as control for loading . C: representative western blots of phosphorylated smad2 (p - smad2) and total smad2 (smad2) and bar plot of the p - smad2to smad2 ratio . The p - smad2to smad2 ratio was examined in the retinal vessels of rats with 6 months (left panel) and 3 months of diabetes (right panel). The 5860-kda bands detected in both retinal microvessels and the positive control (+; tgf-treated hepg2 cells) corresponds to smad2; the 50-kda band present in the positive control only corresponds to smad3 . The bar plot presents the pooled results obtained in rats with 6 and 3 months of diabetes . * p = 0.05 . To learn whether the diabetes - induced changes in the tgf- pathway extended to nonvascular cells of the retina, we measured the levels of the tgf-1 transcript, tgf- type i receptor, and smad2 phosphorylation in the whole retina . Total rna and protein lysates were prepared from the whole retina of diabetic (d) and age - matched control (c) rats . B: representative western blot of tgfr1 (alk-5) and bar plot of the quantitative analysis . C: representative western blots of phosphorylated smad2 (p - smad2) and total smad2 (smad2) and bar plot of the p - smad2to smad2 ratio . Diabetes increased the expression of molecules that generate oxygen - derived free radicals and decreased the expression of antioxidants (supplemental table 3). Similar to the aorta of diabetic rats (30), the retinal vessels showed prominent upregulation of txnip, a negative regulator of the antioxidant thioredoxin . Diabetes also increased the expression of genes involved in inflammation and response to injury (supplemental table 4). The gene expression program was thus consistent with, and expanded, previous findings pointing to the occurrence of oxidative stress and inflammation in diabetic retinal vessels (9,31,32). Genes involved in tissue remodeling through formation of extracellular matrix, regulation of cell adhesion, and organization of the actin cytoskeleton were upregulated in the diabetic vessels (supplemental table 5). All changes related to actin were in the direction of increased polymerization and filament stability, which may contribute antiproliferative effects (33). Additional gene expression changes induced by diabetes pointed to inhibitory effects on cell cycle progression and cell proliferation (supplemental table 6). Multiple mechanisms of apoptosis appeared activated in diabetic vessels, many not suspected previously (supplemental table 7). Changes in the expression of six genes of the p53 pathway converged in the proapoptotic direction . Tgf- can induce apoptosis by multiple mechanisms (34), which include upregulation of fas and activation of c - jun nh2-terminal kinase (34,35), the expression of which was also increased in the diabetic retinal vessels (supplemental table 7). A potential mechanism for apoptosis by neglect was the downregulation of the g - protein coupled receptor of lysophosphatidic acid edg2 (endothelial differentiation gene 2), a survival factor for multiple cell types (36). The sorbinil and aspirin doses were previously documented to prevent the development of acellular capillaries in diabetic rats (10,11). In this study, the dose of sorbinil overnormalized sorbitol and reduced fructose accumulation by 96% in the retina of diabetic rats (table 1), thus achieving almost complete inhibition of glucose flux through the polyol pathway . The aspirin dose, known to inhibit by 90% thromboxane b2 formation during blood clotting in rats and thus exert an antiplatelet effect (11), resulted in serum salicylate levels (table 1) 10- to 20-fold lower than those achieved by anti - inflammatory doses of aspirin (rev . In (11). Sorbinil normalized the diabetes - induced expression changes in 56% of the known genes and aspirin normalized those in 32%; the figures for the total number of probe sets (known genes and ests) were 48 and 47%, respectively . Both sorbinil and aspirin attenuated significantly and to a similar extent the effects of diabetes on the tgf- pathway (fig . 4), preventing the expression changes of 55 and 40% of the genes, respectively . Of note, both drugs prevented the increased expression of tgf- receptor 1 and ctgf . Likewise, sorbinil and aspirin reduced significantly and to a similar extent the effects of diabetes on proapoptotic pathways, with concordant correction of changes in the p53 pathway and concordant restoration of survival signals . In contrast, sorbinil reduced the changes in gene expression relevant to oxidative stress and inflammation strikingly more than aspirin (fig . Sorbinil led to normalization of expression of 71% of genes related to oxidative stress and 62% of those related to inflammation, whereas the figures for aspirin were 29 and 3%, respectively (p <0.0001 for effect of sorbinil vs. aspirin on inflammation - related genes). The effect of sorbinil and aspirin on the expression of individual genes is reported in supplemental tables 28 . Effects of sorbinil and aspirin treatments on the gene expression changes induced by diabetes in rat retinal vessels . Each functional category includes all genes pertinent to that category, as indicated in table 2 ., the number of genes differentially expressed in the retinal vessels of diabetic rats (untreated or treated) compared with control rats; the number of genes whose expression is normalized by the drug (i.e., the expression in the retinal vessels of treated diabetic rats was not different from that in control rats). D, diabetic rats; d+sor, diabetic rats treated with sorbinil; d+asa, diabetic rats treated with aspirin; ns, nonsignificant . The effects of sorbinil and aspirin on diabetes - induced activation of the tgf- pathway were also tested on trypsin digests prepared from the retinas of rats with 8 months of diabetes . These preparations afforded the opportunity of studying the retinal vascular network fixed immediately after death and not subjected to hypotonic lysis while fresh . Figure 5 shows that the diffuse pattern of tgfr1 immunoreactivity was increased in the retinal capillaries of diabetic rats compared with control rats (p = 0.05) and that both aspirin and sorbinil treatment prevented the increase (p = 0.03 and p <0.0001 vs. diabetes, respectively), in agreement with the microarray results (supplemental table 2). Of note, whereas aspirin returned tgfr1 immunoreactivity to control values (p = 0.62 vs. control), sorbinil suppressed staining intensity below control values (p = 0.015 vs. control). Increased tgfri immunoreactivity in retinal vessels of diabetic rats is prevented by sorbinil and aspirin treatments . Tgfri was detected by immunohistochemistry in retinal trypsin digests from diabetic rats (8 months of diabetes duration), diabetic rats treated with sorbinil or aspirin, and age - matched control rats . The effect of diabetes and of the two drugs on tgfri immunostaining was quantitated by four masked observers . C: representative photographs of midretina fields showing tgfri immunostaining of retinal capillaries . A: control, score 2.0 0.7 (mean sd of scores by the different masked observers). Values are the means sd of the final scores computed for each individual rat . C, control rats, n = 11; d, diabetic rats, n = 10; d+sor, diabetic rats treated with sorbinil, n = 6; d+asa, diabetic rats treated with aspirin, n = 6 . * p = 0.05 vs. control rats; p <0.02 versus control rats, diabetic rats, and diabetic rats treated with aspirin, p <0.04 versus diabetic rats . The discordant effect of sorbinil and aspirin on inflammation, as shown by the mrna data, was consistent with the discordant effect on the increased levels of intercellular adhesion molecule 1 in the diabetic rat retina (11) and was confirmed by data on complement activation in retinal vessels . 2 shows that aspirin, at variance with sorbinil (10), failed to prevent deposition of membrane attack complex in retinal vessels of diabetic rats . We compared the effects of diabetes on gene expression in retinal vessels to those in mller cells, the principal glia of the retina that shows signs of activation / reactivity in both human (37) and experimental (6) diabetes . We had previously examined the gene expression profile of mller cells in the same model used in this study (rats with 6-month duration of streptozotocin diabetes) and on a comparable platform (genechip rat genome rg - u34a; affymetrix) (16). Supplemental table 9 shows that the functional gene categories affected by diabetes in retinal vessels differed from those affected in mller cells . Of the genes related to tgf- that we found affected by diabetes in retinal vessels, 80% were represented in the rg - u34a array, but only 6% of those were affected by diabetes in mller cells . Of the genes related to apoptosis affected by diabetes in retinal vessels, 68% were represented in the rg - u34a array, and 0% of those were affected by diabetes in mller cells . Only the inflammation - related genes showed overexpression in both the vascular and glial cell types (76% of the genes affected by diabetes in the vessels were represented in the rg - u34a, and 55% were upregulated by diabetes in mller cells). The results of this work indicate that retinal microangiopathy in diabetic rats is the product of several molecular pathways that are interconnected but not of equal pathogenic importance . Attenuation of increased activity of the tgf- pathway and apoptosis, but not of oxidative stress and inflammation, appear to be required to prevent the microangiopathy . In view of its role in wound healing, extracellular matrix deposition, and fibrosis (22), tgf- has over the years been an obvious candidate mechanism for the basement membrane thickening and matrix accumulation that are the hallmark of diabetes on blood vessels and vascular structures . However, the studies pointing to a role of excess tgf- have been performed solely in relation to diabetic nephropathy (38). Diabetic retinopathy has not been previously linked to excess tgf- signaling, likely because the link has been more difficult to capture . We found increased tgf- and tgf- signaling selectively in microvessels but not in cells more abundantly represented in the retina, such as mller glial cells (16) or neurons, that would make an increase manifest in whole retinal extracts (this work). The few suggestions to date of a contribution of tgf- to diabetic retinal microangiopathy have come from the observations that the retinas of diabetic rats show the presence of oncofetal fibronectin (39), an isoform typically stimulated by tgf- during tissue healing and remodeling, and the retinas of diabetic patients show expression of ctgf in pericytes, a shift to the vascular compartment from the microglial location of ctgf in the retinas of nondiabetic individuals (40). Our finding of increased smad2 phosphorylation indicates that increased tgf- signaling does in fact occur in retinal vessels of diabetic rats, and the concordant attenuation of such signaling by drugs that protect the vessels from the effects of diabetes through different mechanisms suggests that the increased signaling contributes to the vascular pathology . It may be argued that the diabetic retinal vessels showed overexpression of inflammation - related genes, whereas tgf- is viewed, in general, as anti - inflammatory . In this respect, the increased tgf-1 expression and activity in diabetes could even represent a compensatory response and, as such, be protective for the retinal vessels . However, the finding that both sorbinil and aspirin attenuated the tgf- pathway, but only sorbinil reduced inflammation, indicates that the tgf- pathway was upregulated independently of the genes related to inflammation . This agrees with findings in mller cells, which show in diabetic rats changes in the expression of genes related to inflammation but not to tgf- (16). Fibrosis is not necessarily driven by inflammation, which might explain the general lack of efficacy of anti - inflammatory mediators in the treatment of fibrotic disease (41). In diabetes in particular, tgf- upregulation could be induced and sustained by high glucose levels, which stimulate tgf- promoter activity in vascular cells (42). Comparison of the effects of sorbinil and aspirin led to the identification of the signature of the polyol pathway on retinal vessels . The ari prevented or attenuated changes in all functional categories of genes, indicating that the polyol pathway mediates most molecular abnormalities induced by hyperglycemia in rat retinal vessels, including tgf- overexpression . The role of the polyol pathway in mediating glucose - induced increases in tgf- has been documented in cultured cells (43). Only the ari, and not aspirin, prevented the pro - oxidant and proinflammatory changes in gene expression, consistent with the discordant effects of the two drugs on other indicators of diabetes - induced oxidative stress and inflammation (this work and (10,11). This finding, combined with the known pro - oxidant effects of the polyol pathway (44) and the fact that reactive oxygen species induced by hyperglycemia or other stimuli beget inflammation (45), points to the combination of oxidative stress and inflammation as the distinct signature of the polyol pathway on retinal vessels . The effects of aspirin indicated that attenuation of pro - oxidant and proinflammatory gene expression is not necessary to prevent retinal vascular cell apoptosis and vessel histopathology (11) in diabetes . We cannot exclude that aspirin may have attenuated aspects of inflammation not reflected in the gene expression data, but we note that the concentration of aspirin achieved in our rats (two orders of magnitude lower than those exerting typical anti - inflammatory effects) (rev . In (11) also failed to attenuate complement deposition in diabetic retinal vessels . However, it remains conceivable that attenuation of oxidative stress and/or inflammation may be sufficient to prevent retinopathy if these processes in diabetes are upstream of, and contribute to, apoptosis . What the effect of aspirin highlights is that in diseases resulting from the contribution of multiple pathogenic events, the attenuation of some of the events is sufficient to prevent the ultimate phenotype, despite persistence of the other known contributors . An example of such interplay was recently documented in a model of alzheimer's disease (46). The mechanism(s) whereby aspirin attenuated the effects of diabetes on the tgf- and pro - apoptotic pathways in retinal vessels remain speculative . The dose effective in our diabetic rats was 20-fold lower than the dose reported to decrease the excess tgf- and ctgf expression induced by diabetes in the rat kidney and by high glucose in cultured mesangial cells (47). Comparison with the selective antiplatelet agent clopidogrel had suggested that our dose of aspirin protects retinal vessels in diabetes by mechanisms other than antiplatelet activity (11). Aspirin could have worked by inhibiting the activity of cox-1 in vascular cells, which expressed higher levels of the enzyme in diabetes . However, it is not known whether increased cox-1 is harmful to vascular cells, and the fact that aspirin prevented the overexpression of cox-1 (not a previously known effect of aspirin) suggests that the effects on cox-1 were downstream of some other beneficial effect . At concentrations consistent with those achieved in our experiments knowledge that the combination of oxidative stress and inflammation is the distinct signature of the polyol pathway on retinal vessels makes it possible to seek such a signature in human diabetes to help determine whether there is a rationale for adjunct therapy with aris . The action of aspirin, which prevents the vascular histopathology resulting from a complex process by attenuating only selected pathways, establishes a valuable paradigm for development of diversified drug approaches to the complications of diabetes . The observation that increased tgf- signaling begins early in diabetic retinal vessels proposes that capillary remodeling also begins early . Early onset of pathological remodeling can be one of the reasons for the memory that retinal vessels carry of exposure to the diabetic milieu (5,49), which translates into poor reversibility of even the initial lesions of diabetic retinopathy and justifies emphasis on prevention (5). Targeting the tgf- pathway may become a rational strategy in the prevention of the two main microvascular complications of diabetes, retinopathy and nephropathy . In view of the homeostatic importance of tgf-, the targeting modalities would need not to interfere with physiological activity . Phenotypes attributable to increased tgf- signaling can be rescued by inactivation of a single tgf-1 allele (50), and in our study prevention of retinal diabetic microangiopathy did not require complete normalization of gene expression changes in the tgf- pathway . Tgf- drugs insofar as they were recently reported to reduce the progression of vascular pathologies caused by excessive tgf- signaling (51). This effect of angiotensin ii blockers, coupled with our findings of an overactive tgf- pathway in diabetic retinopathy, may need to be taken into account in interpreting the encouraging results obtained with candesartan in the primary prevention of retinopathy in normotensive type 1 diabetic patients (52). Orally active inhibitors of tgf- signaling are in development (53) and would add options for highly targeted interventions . The next steps in these studies are to define the effects of selective normalization of tgf- signaling in retinal vessels and ascertain whether the tgf- pathway is overactive in human diabetic retinopathy.
The arrival of the precision medicine era brings new opportunities and challenges for patients undergoing precision diagnosis and treatment . The morbidity and mortality rates associated with malignant tumors have increased year by year, and the burden of malignant neoplasms is increasing . Minimally invasive surgical treatment, minimally invasive treatment guided by imaging navigation, and specific therapy of targeted drugs are important aspects of precision oncology . With further development of medical imaging technology, information from different imaging modalities can be integrated and comprehensively analyzed by the imaging fusion system, which provides more image information of tumors from different angles and dimensions to accurately make qualitative and quantitative diagnoses and achieve the aim of precision tumor treatment . Multimodality image fusion technology has become the main trend in the development of future imaging technology . This article reviews the application of several imaging fusion techniques in the diagnosis and treatment of tumors . In 2015, the precision medicine initiative proposed by the us had a profound impact all over the world . Precision medicine is based on individual diagnosis and treatment; in essence, it analyzes and verifies the occurrence and development of diseases from a genetic and molecular level, thereby accurately identifying the cause of diseases and the target of drug action, to achieve maximum therapeutic effect and minimum adverse reactions . Nowadays, malignant tumors are a global health concern; also, the morbidity and mortality rates associated with malignant disease have increased considerably in recent years . Owing to the complexity of tumors and their development mechanisms, completely different genes may determine similar signs and symptoms while the same gene may cause different symptoms and signs . It has been observed that the destructive and one size fits all treatment methods for malignant tumors often result in a serious waste of medical resources, cause considerable economic burden to patients, and seriously affect patients quality of life . Precision oncology is an important branch of precision medicine, that uses individual treatment, has the advantage of causing less trauma, causing fewer complications, and offering good prognosis; therefore, precision oncology has become the cutting - edge field that needs prioritization in precision medicine . Precision oncology must always consider three aspects of malignant tumors: prevention, diagnosis, and treatment, none of which is dispensable . In the background of the current social medicine, any clinical method that can achieve individual diagnosis and treatment of malignant tumors, minimally invasive treatment guided by medical imaging navigation is one precise treatment method for malignant tumors . Medical imaging plays an important role in the qualitative and quantitative assessment of tumors, the precise and individual design of a surgical plan, and preoperative surgical simulation . While different imaging modalities provide diagnostic information at different levels, each imaging method has its own advantages, disadvantages, and specific indications . Many types of imaging technologies complement each other to assist in precise qualitative and quantitative diagnosis of tumors and to achieve a more reasonable and comprehensive treatment plan . The multimodality imaging fusion system takes contrastive analysis of the same lesion in different imaging modalities and provides more imaging information from different dimensions and angles; further, it uses a complementation assay and cross - validation to achieve accurate qualitative and quantitative diagnosis of tumors and offers a strong technical support for the implementation of precision oncology . Computed tomography (ct) is based on anatomical imaging while position emission tomography (pet) imaging is based on functional imaging that imparts information on tissue metabolism and physiological function . A pet / ct imaging fusion is, thus, an integration of functional and anatomical imaging at a cellular and molecular level, and it reflects the physiological and biochemical characteristics and anatomical structure of the diseased tissue . Pet / ct has an important guiding significance in tumor localization and qualitative diagnosis, tumor staging, tumor - biopsy - site selection, and development of radiotherapy planning . Pet / ct can accurately locate tumors through high spatial resolution of ct; in addition, it can reflect the metabolic information of tumor cells to make an accurate judgment of the tumor's pathologic type . With only single pet imaging, it is difficult to precisely locate the tumor site; pet / ct fusion imaging improves the accuracy of tumor localization, especially in the head and neck region and for abdominal tumors . Pet / ct reduces the misdiagnosis and missed diagnosis rate of early - stage tumors, which can guide the choice of surgical methods and the resection scope of tumors, and also improve the quality of life for patients . A study showed that pet / ct was highly accurate in characterizing indeterminate pulmonary nodules detected in lung cancer screening with low - dose ct, and it was helpful for the early diagnosis and treatment of lung cancer . Because there are various tumor types in the head and neck region, pet imaging alone is not sufficient to accurately locate these tumors . However, a pet / ct scan can provide precise positioning and qualitative diagnosis of such tumors . It is necessary to strictly control the radiation dose when planning radiotherapy for tumors in the head and neck region where are many important organs; moreover, precise delineation of gross tumor volume is the basis for reducing the radiation injury of normal tissue . Hideghty et al . Reported that pet / ct - based gross tumor volume delineation helped make modifications to radiotherapy planning in head and neck squamous cell carcinoma . The results showed that on the basis of the initial and post - ict pet / ct comparison in 15/20 patients more than 50% volume reduction and in 6/20 cases complete response were achieved . Besides that, pet / ct is significantly valuable in monitoring tumor recurrence and distant metastasis . Suenaga et al . Reported that pet / ct had higher value in the surveillance of head and neck squamous cell carcinoma recurrence and distant metastasis than contrast - enhanced ct . The aim of most treatment methods is to maximally improve the local control rate of tumors and reduce the damage to normal tissue, while simultaneously increasing the cure rate of the tumor; precise staging plays a decisive role in formulating tumor - therapy strategies . A whole - body pet / ct scan can detect the existence of early lesions before morphological changes . Pet / ct has the advantage of a large scanning range, and it can reveal tumor occurrence and metastasis in all tissues and organs and precisely locate the primary tumor and distant metastases . In addition, pet / ct can detect lymph node metastases measuring <1.0 cm in diameter anywhere in the body and help accurate tnm staging of tumors . The purpose of radiotherapy is to provide the target area with an adequate curative radiation dose while exerting the least possible irradiation dose to the surrounding normal tissue . Pet / ct can clearly indicate active regions of tumor metabolism and liquefaction of a necrotic area, in addition to accurately delineating tumor radiotherapy target volume that can help guide the radiation field and radiation dose distribution design, in order to prevent normal tissues from receiving unnecessary irradiation and improve the efficacy of tumor radiotherapy . Reported that the pet / ct plan was better than the conventional point a plan in terms of target coverage without subjecting the normal tissue to an increased dose, which made optimized three - dimensional (3d) brachytherapy treatment planning possible . Whole - body pet / ct scans can also reduce missed diagnosis of metastatic lesions, help formulate individual treatment programs, and reduce the failure of locoregional therapy, thereby improving patient prognosis . Pet / ct functional imaging can show the most active part of tumor metabolism and locate it accurately during the process of sampling, such that components of liquefaction necrosis and secondary infection sites of tumors can be avoided . Further, it assists in obtaining biopsy samples from the most active part of the tumor to reduce false negative results . In addition, pet / ct can make use of tumor cell radiation concentration characteristics when biopsying the required sample before morphological changes, to aid in accurate early qualitative diagnosis of lesions and increase the cure rate of tumors . Applied pet / ct - guided percutaneous biopsy with 51 patients of suspected lung cancer and bone metastases . The results showed that the first - time diagnostic success rate of the biopsy was 96.1% (49/51), and the overall diagnostic success rate and sensitivity were both 100.0%; furthermore, no serious complications were encountered . Pet / ct imaging fusion technology has obvious advantages over magnetic resonance imaging (mri), ct, radiography, and other single modality imaging techniques in tumor diagnosis and treatment, which can significantly reduce misdiagnosis and missed diagnosis rates, but pet / ct can also cause false negative and false positive results in tumor diagnosis . The imaging quality of pet / ct is affected by respiratory motion and it is easy to generate motion artifacts in the course of examination of thoracic tumors . To improve the quality of pet / ct fusion images, the selection of ct system still needs further clinical standardization; further, the methods to obtain a higher accuracy of pet / ct images should be addressed in further research studies . Pet / ct can accurately locate tumors through high spatial resolution of ct; in addition, it can reflect the metabolic information of tumor cells to make an accurate judgment of the tumor's pathologic type . With only single pet imaging, it is difficult to precisely locate the tumor site; pet / ct fusion imaging improves the accuracy of tumor localization, especially in the head and neck region and for abdominal tumors . Pet / ct reduces the misdiagnosis and missed diagnosis rate of early - stage tumors, which can guide the choice of surgical methods and the resection scope of tumors, and also improve the quality of life for patients . A study showed that pet / ct was highly accurate in characterizing indeterminate pulmonary nodules detected in lung cancer screening with low - dose ct, and it was helpful for the early diagnosis and treatment of lung cancer . Because there are various tumor types in the head and neck region, pet imaging alone is not sufficient to accurately locate these tumors . However, a pet / ct scan can provide precise positioning and qualitative diagnosis of such tumors . It is necessary to strictly control the radiation dose when planning radiotherapy for tumors in the head and neck region where are many important organs; moreover, precise delineation of gross tumor volume is the basis for reducing the radiation injury of normal tissue . Reported that pet / ct - based gross tumor volume delineation helped make modifications to radiotherapy planning in head and neck squamous cell carcinoma . The results showed that on the basis of the initial and post - ict pet / ct comparison in 15/20 patients more than 50% volume reduction and in 6/20 cases complete response were achieved . Besides that, pet / ct is significantly valuable in monitoring tumor recurrence and distant metastasis . Suenaga et al . Reported that pet / ct had higher value in the surveillance of head and neck squamous cell carcinoma recurrence and distant metastasis than contrast - enhanced ct . The aim of most treatment methods is to maximally improve the local control rate of tumors and reduce the damage to normal tissue, while simultaneously increasing the cure rate of the tumor; precise staging plays a decisive role in formulating tumor - therapy strategies . A whole - body pet / ct scan can detect the existence of early lesions before morphological changes . Pet / ct has the advantage of a large scanning range, and it can reveal tumor occurrence and metastasis in all tissues and organs and precisely locate the primary tumor and distant metastases . In addition, pet / ct can detect lymph node metastases measuring <1.0 cm in diameter anywhere in the body and help accurate tnm staging of tumors . The purpose of radiotherapy is to provide the target area with an adequate curative radiation dose while exerting the least possible irradiation dose to the surrounding normal tissue . Pet / ct can clearly indicate active regions of tumor metabolism and liquefaction of a necrotic area, in addition to accurately delineating tumor radiotherapy target volume that can help guide the radiation field and radiation dose distribution design, in order to prevent normal tissues from receiving unnecessary irradiation and improve the efficacy of tumor radiotherapy . Reported that the pet / ct plan was better than the conventional point a plan in terms of target coverage without subjecting the normal tissue to an increased dose, which made optimized three - dimensional (3d) brachytherapy treatment planning possible . Whole - body pet / ct scans can also reduce missed diagnosis of metastatic lesions, help formulate individual treatment programs, and reduce the failure of locoregional therapy, thereby improving patient prognosis . Pet / ct functional imaging can show the most active part of tumor metabolism and locate it accurately during the process of sampling, such that components of liquefaction necrosis and secondary infection sites of tumors can be avoided . Further, it assists in obtaining biopsy samples from the most active part of the tumor to reduce false negative results . In addition, pet / ct can make use of tumor cell radiation concentration characteristics when biopsying the required sample before morphological changes, to aid in accurate early qualitative diagnosis of lesions and increase the cure rate of tumors . Applied pet / ct - guided percutaneous biopsy with 51 patients of suspected lung cancer and bone metastases . The results showed that the first - time diagnostic success rate of the biopsy was 96.1% (49/51), and the overall diagnostic success rate and sensitivity were both 100.0%; furthermore, no serious complications were encountered . Pet / ct imaging fusion technology has obvious advantages over magnetic resonance imaging (mri), ct, radiography, and other single modality imaging techniques in tumor diagnosis and treatment, which can significantly reduce misdiagnosis and missed diagnosis rates, but pet / ct can also cause false negative and false positive results in tumor diagnosis . The imaging quality of pet / ct is affected by respiratory motion and it is easy to generate motion artifacts in the course of examination of thoracic tumors . To improve the quality of pet / ct fusion images, the selection of ct system still needs further clinical standardization; further, the methods to obtain a higher accuracy of pet / ct images should be addressed in further research studies . Pet / mri fusion imaging can detect not only the anatomical, morphological, and functional information from mri but also the cellular and physiological metabolism and molecular information from pet . Mri permits multi - sequencing and multi - parametric imaging . Compared with pet / ct, pet / mri can achieve quasi - physiological synchronization and multi - functional information imaging . Pet / mri can provide more detailed tumor information and is likely more suitable for early screening and follow - up observation after treatment . Pet / mri has greater potential for qualitative diagnosis of early tumors than pet / ct, and it plays an important role in displaying local and/or distant lymph node metastasis and determining the tnm stage of tumors . Integrative pet / mri has both high soft - tissue resolution of mri and high specificity of pet imaging, thus confirming that it has obvious advantages in the early diagnosis of head and neck tumors, breast cancers, and abdominal and pelvic tumors . Heusch et al . Compared pet / mri and pet / ct examination results and tnm staging in 22 patients with nonsmall cell lung cancer . Both imaging modalities showed consistent t - staging in 16 patients, and all 16 patients were correctly staged with respect to histopathology and n - staging . Overall, the accuracy of pet / mri and pet / ct were 91% (20/22) and 82% (18/22), respectively, which indicated that pet / mri had some advantages over pet / ct . Reported that the accuracy of whole - body pet / mri with signal intensity (si) assessment was superior to that of pet / mri without si assessment and pet / ct, for identification of tnm classification, clinical stage, and operability evaluation of patients with tumors . Growing research suggests that pet / mri could be a new positive noninvasive alternative approach as compared to pet / ct for tumor staging . In recent years, pet / mri examination has also been used for the delineation of gross target volume in tumor radiotherapy, which provides a reliable means for the accurate estimation of tumor volume, and has achieved remarkable results . At present, the research of pet / mri is still in its infancy, and there is a certain gap between clinical research and clinical promotion . In addition, pet / mri is expensive and the imaging speed is slow, and both imaging modalities sometimes interfere with each other to influence the clarity and accuracy of the image . Besides, the operation of pet / mri scanning requires an experienced radiologist while skilled and specialized physicians of medical imaging and nuclear medicine are typically scarce . However, despite this, pet / mri examination has high application value in the diagnosis and treatment of tumors, especially for soft - tissue tumors . When whole - body pet / mri scan is used as a means of imaging fusion technology, it integrates functional imaging and cellular biological metabolism imaging and has great research potential for tumor biological characteristics and targeted therapy, tumor angiogenesis, and tumor tissue metabolism . With the development of an integrative system and improved attenuation correction technique, pet / mri, as a kind of multimodality molecular imaging technology, has the potential to initiate a future revolution in tumor diagnosis and treatment . Precise and minimally invasive treatment of tumors is the development trend of clinical medicine, preoperative evaluation, and intraoperative monitoring . Tissue biopsy guided by imaging technology improves the diagnostic accuracy, and imaging - guided tumor therapy is the embodiment of the individual and precise treatment of tumors . Ultrasound is the preferred imaging modality for puncture biopsy guidance and dynamic monitoring during operation of tumors, because of its convenience, real - time, high repeatability, and nonradiation . Ultrasound has its own shortcomings such as lack of spatial resolution, small imaging field, and many interference factors, whereas ct / mri possesses high spatial resolution, larger imaging field, and less disturbances . Ultrasound - ct / mri fusion technology achieves the integration of real - time ultrasound, convenience, and high spatial resolution and perfectly combines the advantages of different imaging modalities . At present, ultrasound - ct / mri fusion technology has become one of the research focuses in the field of interventional oncology . The spatial resolution of conventional ultrasound is low, and it is affected by gas, breathing movement, and body size . In addition, the ultrasonic wave produces attenuation in the process of transmission, hence making it difficult to access tumors in remote locations, leading to false negative results . With the development of multimodality ultrasound imaging technology however, ultrasound examination can still only poorly reveal tumors in isolated locations; therefore, tumor real - time puncture guidance, real - time monitoring of tumor ablation therapy, and other applications are limited . Ultrasound - ct / mri fusion imaging brings a new entry point for puncture guidance and ablation monitoring of tumors, which are difficult to be visualize in single ultrasound examination . First, locating tumors using ct / mri scans and then making an alignment and fusion with real - time ultrasound examination that accomplishes real - time needle biopsy guidance for precise location puncture of isolated tumors, real - time ultrasound imaging can also be used to monitor tumor ablation under fusion imaging mode, which avoids damage to surrounding tissues . Liu et al . Showed that all 18 target hepatocellular carcinoma (hcc) nodules could be detected with fusion images in all patients, but could not be detected on conventional ultrasound . The efficiency rate of microwave ablation assisted by real - time fusion imaging navigation system for hccs undetectable by conventional ultrasound was 94.4% (17/18). It often needs multi - point repeated placement of electrode needles for larger tumors to achieve complete ablation . The atomization gas produced in the process of tumor ablation can interfere with the display of lesions, which is known to influence the accuracy of the subsequent needle arrangement, thereby leading to residual tumor and recurrence . At present, however, during the process of ablation, rfa requires an experienced operator to obtain the 3d anatomical imaging of tumors to make the needle arrangements and design the puncture route in his mind, which is poor of objectiveness . Ultrasound - ct / mri fusion technology can be used to make a reasonable needle arrangement and design a suitable puncture route for the ablation of tumors by ct / mri 3d reconstruction technology . To reach minimum needle arrangement times and maximum ablation effect under the navigation of real - time ultrasound - ct / mri fusion imaging, the needle arrangement, needle insertion, and the ablation process should not be affected by the atomization gas . The operator subjective dependence is low, and the ablation process can be monitored in real time, which helps finally achieve the purpose of precise tumor ablation . Ultrasound - ct//mri fusion imaging has an important significance in the evaluation of tumor ablation effect, detection of residual and recurrent tumor lesions, and guidance of re - treatment . Although contrast - enhanced ultrasound (ceus) can display real - time tumor micro blood flow perfusion, it is difficult to detect tumors with lack of blood flow using this imaging technique, owing to requirements of a highly skilled and experienced operator . Moreover, scanning range limits can easily lead to missed diagnosis, and hence, this method cannot accurately assess the tumor ablation effect . The tumor ablation process needs to inactivate not only the whole tumor tissue but also the surrounding 5 mm normal tissue, namely the ablation margin (am). Reaching the am or not is an independent risk factor for tumor recurrence . At present, no single modality imaging can accurately assess the am . Fusion imaging technology can be used to fuse or compare the images obtained before and after tumor ablation, thus making the assessment of am possible . Applied real - time virtual navigation system with ultrasound - ct / mri fusion imaging and found 295 liver tumors that were completely undetectable with nonenhanced ultrasound from 2002 to 2012 . A total of 282 of 295 (95.6%) tumors were correctly targeted, and successful ablation was achieved in 266 of 295 (90.2%) tumors . This showed that ultrasound - ct / mri fusion imaging is precise for targeting and achieving successful ablation of target tumors undetectable with ultrasound alone . Song et al . Were able to detect 120 hccs not visible on conventional ultrasound in 96 patients when fusion imaging was applied, and 38 (31.7%) of the 120 hccs could be seen and rfa was feasible . Among the remaining 82 hccs still not visible after image fusion, 26 (31.7%) were ablated under the guidance of fusion imaging, the technique based on peritumoral anatomic landmarks . Overall, 64 (53.3%) of 120 hccs not visible on conventional ultrasound could be ablated under the guidance of the fusion imaging technique . Ultrasound - ct / mri fusion imaging cannot only be used to evaluate whether the tumor ablation can reach the am but also be used to assess the ablation effect in real time through ceus . Once residual lesions are detected, ablation was carried out in a timely manner until the tumor completely disappeared; this reduces the recurrence rate of tumors and improves the prognosis of patients . The accuracy of ultrasound - ct / mri fusion imaging registration is affected by many factors such as patient posture and respiratory phase . To ensure the accuracy of registration, it is essential to maintain the same body position and breathing stage as far as possible during the ct / mri scans . During ultrasound examination, the force applied by the operator should be gentle; otherwise, it will cause deformation of local tissue and affect the accuracy of image registration . The application of respiratory gating and motion tracking compensation technology in ultrasound examination has greatly improved the accuracy of image registration . Ongoing research indicates that ultrasound-3d ultrasound / ceus based on ultrasound images for fusion will overcome complex operation, image - alignment difficulty, and other shortcomings as compared to ultrasound - ct / mri imaging fusion . Imaging fusion with real - time ultrasound and new ultrasonic techniques will provide a more convenient, inexpensive, and precisely mediated platform for minimally invasive treatment of tumors . Precision medicine plan brings new opportunities and challenges to the diagnosis and treatment of malignant tumors with advances both in the field of human genomics, metabolomics, and proteomics and considers data from other large - scale biological databases . With our current understanding and background of medicine, methods of precision tumor treatment such as minimally invasive surgical treatment, minimally invasive interventional therapy, and targeted drug therapy are not possible without support from medical imaging . Multimodality medical imaging fusion technology has been widely applied in accurate tumor location, qualitative tumor diagnosis, tumor staging, treatment plan design, and real - time intraoperative monitoring . Multimodality medical imaging fusion technology will provide powerful technical support for the precise diagnosis and treatment of tumors in future clinical application . Currently, accurate registration and fusion method is still the most challenging aspect of imaging fusion technology . Cultivating compound imaging talents is one of the challenges in fusion imaging technology . Finally, the selection of objective and optimal fusion imaging methods for different kinds of tumors is also a significant issue that needs to be addressed . With the continuous development of imaging and computer technology, multimodality imaging fusion technology will likely increasingly improve and mature with respect to fusion speed, stability, and accuracy . In addition, the development and application of multimodality contrast agents also opens new research avenues in multimodality imaging fusion technology . Multimodality fusion imaging brings more personalized and diversified diagnosis and treatment options to precision medicine, and these new diagnosis and treatment methods can accelerate the development of precision medicine . Therefore, the additional use of fusion imaging should be considered when single modality imaging is not satisfactory for tumor diagnosis and treatment.
Adult - onset hearing loss is a highly prevalent yet relatively underrecognised health problem in the older adult australian population [1, 2]. Because hearing loss is often progressive and gradual in its onset in most individuals, it is typically diagnosed and managed several years after its onset, often only after having led to multiple negative consequences including effects on employment, poor quality of life, social isolation, depressive symptoms, increased mortality risk, and reduced independence [39]. It is one of the leading causes of burden of disease prior to older age, for ages 4564 years, in men and women . Further, as hearing loss interferes with so many of life's activities, it may prove to be a major impediment to society's need to have people remain longer in the workforce as the proportion of working age the annual cost of lost earnings due to workplace separation and early retirement from hearing loss was estimated at $6.7 billion, which is over half of the calculated economic impact of hearing loss ($11.75 billion, representing 1.4% of gdp). Therefore there is a need to better understand the barriers that may exist to help seek an effective remediation for hearing loss in this population . Hearing loss is a chronic problem and, contrary to current community perception and funding models of hearing services, hearing aids are typically a part of a rehabilitation program rather than provide a single and simple restorative solution to hearing loss . As such, hearing loss needs to be effectively managed under a biopsychosocial model of care, following the framework for intervention and treatment of the international classification of functioning disability, and health model . This framework not only considers the impairment per se, but also the impact that it has on the individual in terms of activity limitations (such as inability to perceive speech in noisy environments) and participation restrictions (such as the ability to fully participate in communication and conversational activities). Nonetheless, hearing aid use is a measurable quantity and, therefore, the majority of studies that have evaluated functional and quality - of - life outcomes of rehabilitation programs for individuals with hearing loss have used this as a marker . Multiple studies have identified that rehabilitation interventions can effectively address many of the difficulties associated with impaired hearing [1620]. Importantly, evidence shows that the later hearing rehabilitation occurs in the course of hearing loss, the less likely older adults are to continue to use and derive benefit from hearing aids . Despite this, several studies [22, 23] indicate high levels of unmet need for hearing health services and poor use of prescribed hearing aids . Denial or nonacceptance of hearing loss and the stigma associated with hearing loss other reasons include an underestimation of the negative impacts of hearing impairment on overall health by general practitioners (gps) and older adults, leading to poor referral to appropriate medical and allied health practitioners, such as ear, nose, and throat specialists and audiologists . To date, the australian public health system does not have an effective and sustainable hearing loss screening strategy for late - onset hearing loss in adults to manage this problem . This paper aims to review the current pathway of detection, referral, and management of late - onset adult hearing loss in australia and to identify an alternative, more effective pathway for the future . Australian population - based data describing prevalence, incidence, and risk factors for hearing loss have been identified in the blue mountains hearing study (bmhs) in 19972000 among 2956 participants of the blue mountains eye study (bmes) cohort (an overall response rate of 75.5% for the cross - section) [25, 26]. Of these, hearing thresholds were measured in audiometric soundproof rooms by qualified audiologists and bilateral hearing loss was described by the pure - tone average of air - conduction thresholds at octave frequencies between 500 and 4000 hz (pta0.54 khz) in the better ear . Risk factors measured (either via self - report or practitioner measurement) included self - reported health, noise exposure, and family history of hearing loss . In this study, we identified that a 33.0% prevalence of bilateral hearing loss existed in persons aged 50 + years (51% showed hearing loss in the worse ear) consistent with that measured in the us - based epidemiology of hearing loss study (ehls). More specifically, mild hearing loss was present in 22.4% of participants, moderate in 8.9% and severe in 1.7% participants . For each decade beyond age 50, further, a history of having worked in a noisy environment predicted a 70% increased likelihood of any hearing loss, whereas family history predicted a 68% increased risk of hearing loss, which increased with greater magnitudes of loss . The overall 5-year progression of hearing loss, defined as a difference in pta> 10 db, was moderately high at 15.7%, with the highest rate being evident in adults aged 80 years or older . Additionally, for each decade of age over 60 years, the risk of incident hearing loss increased threefold . As well as health - related influences, our epidemiological study also assessed quality - of - life and mental health factors, such as cognitive function and depression . Bmhs - i data showed that bilateral hearing loss was associated with poorer sf-36 scores in both physical and mental domains (decrease in physical component score (pcs) of 1.4 points, p = 0.025; decrease in mental component score (mcs) of 1.0 point, p = 0.13); with poorer scores associated with more severe levels of impairment (pcs ptrend = 0.04, mcs ptrend = 0.003). Persons with moderate - to - severe hearing loss had slightly lower mean cognitive function scores than those without hearing loss (p <0.001). Therefore, while milder levels of hearing losses were significantly more common in working - aged older adults, a lack of responsiveness to manage this early can lead to significant negative effects on quality of life, personal relationships, and ability to continue to work effectively . As the risk of hearing loss increases with advancing age, it seems that early detection and management would be critical to minimising any longer - term effects . Stephens et al . Suggest that the average consumer presenting at a hearing aid or rehabilitation clinic for the first time is aged ~70 years and has had hearing problems for about 10 years . As hearing loss significantly impacts on communication ability and communication is necessary for developing and maintaining effective relationships, it is likely that within this prolonged timeframe the individual and his / her family have experienced considerable frustration from the disability . The us national council on aging survey of 2069 hearing - impaired individuals and 1710 of family and friends demonstrated that hearing aid use is associated with lesser degrees of anger and frustration reported by family members . Further, stark and hickson demonstrated benefits in hearing - related quality - of - life scales for both the individual with hearing loss and their significant other after hearing aid fitting, despite only 1/3 of the individuals with hearing loss showing initial motivation to attend the hearing appointment . Certainly, we found that bmhs participants who used their hearing aid at least 1 hour / day or more were only one - third as likely to report depressive symptoms as infrequent users, multivariate adjusted or 0.32 (95% ci 0.140.76). Despite this, bmhs findings showed that of 33.0% persons with measured bilateral hearing loss, only 33% owned hearing aids and, of these, only 25% used them habitually, similar to the rates of use reported in the ehls study . When stratified into magnitudes of hearing loss, bmhs data showed that hearing aids were owned by only 16.4% of individuals with a mild loss, compared with 55.8% with a moderate loss and 91.3% with a severe - profound loss (figure 1), suggesting that either there is a critical unmet need for hearing services in individuals with mild - moderate levels of hearing loss or that hearing aids are not needed for all individuals with lower magnitudes of loss or that the technology is too difficult to manage in this population . Nonetheless, bmhs data showed that 33.4% of older adults with average hearing levels greater than 40 dbhl in the better ear did not own a hearing aid . While milder forms of hearing loss may be less correlated with hearing disability, dillon showed that more significant losses do show higher levels of benefit . Further, bmhs data demonstrate that 53.5% of older adults with severe losses wear their hearing aids for over 8 hours per day compared to 24% of those with moderate losses and 13.5% with mild losses, suggesting an increased need for amplification for greater magnitudes of loss (figure 2). Low rates for use of hearing services and hearing aids highlight barriers including cost and/or reluctance by many to accept their hearing loss (or those without self - perceived hearing disability). However, similar low rates of hearing service uptake and device use have been observed in the australian federal government office of hearing services program, where hearing services are largely provided free of charge to eligible older adults . Therefore, we assume that under use of fitted aids by older adults in australia may suggest either poor targeting of individuals with hearing loss or fitting at too late a stage for derived benefit . Substantial delays in accessing hearing services may impact effective hearing aid use because advancing age is associated with poorer auditory and cognitive processing, physical dexterity, and learning abilities making it more challenging to perceive sounds in competing noise environments, position a hearing aid in the ear, and to learn how to use new technology [3841]. Additionally, there is an increased likelihood of other health problems coexisting so that the management of hearing loss may be considered less of a priority and prove to be burdensome . As the consequences of the hearing loss are more significant, this may lead to poorer motivation to manage the impairment and/or its impacts . There remains a large proportion of hearing - impaired adults who would benefit from hearing aids but who decide not to seek help . Further, while bmhs showed that approximately one - third of people aged 50 years with measured bilateral hearing loss reported seeking help from their general practitioner (gp), a random cross - sectional survey of gp activity in australia between 2003 and 2008 identified that only approximately 3/1000 consultations for older adults included hearing loss management . Similar studies of gps undertaken in uk identified that the chance of referral to hearing services for older adults who reported hearing loss was only about 50% . Screening and intervention programmes have been recommended to improve this situation [21, 43]. Screening programs are not systematically implemented throughout the australian population, their success at meeting the needs of the target population is not assured, and they have no automatic link to action if the need for action is detected . Audiograms conducted by trained audiologists in soundproof booths, are currently used to diagnose hearing impairment and largely determine whether or not an individual is offered hearing rehabilitation . Audiometry is expensive and may not necessarily be accessible to those needing it . Particularly for late - onset hearing loss, it provides little information about effects of hearing loss on everyday functioning [45, 46]. It is important to note that while hearing impairment is extremely common in older adults, not all are significantly disturbed by this . The bmhs findings collectively show that severe hearing disability is strongly associated with measured hearing loss, poorer qol, and probable depression . This suggests that identifying hearing - related activity limitations and participation restrictions could potentially be effective in identifying persons more likely to have suffered an important impact from their hearing impairment and, thus, would be most likely to benefit most from using hearing aids . Self - perception of a hearing disability (e.g., increasing social isolation) can often be an important reason to seek aural rehabilitation . In fact, dillon showed that the benefits reported by individuals with hearing aids appear to be only weakly correlated with hearing loss, particularly for mild - moderate losses . This may in part explain why at least 20% of individuals fitted with hearing aids do not wear them . On the other hand, benefits are actually more highly correlated with initial motivation and perceived listening difficulty [10, 47, 48]. Thus, greater engagement by gps in hearing health could potentially be a cost - saving strategy, as gps are ideally placed to better motivate and identify older people with hearing loss disability, that is, those likely to benefit the most from a hearing aid, thereby improving the targeting of hearing - impaired patients for rehabilitation . There exists a need for a readily accessible screening test assessing hearing disability which could more accurately identify rehabilitation need, rather than just measurement of hearing loss . Validated, self - administered questionnaires about hearing disability have been shown to detect functional hearing impairment accurately and, so, have been recommended as potential screening tools [4952]. In particular, it has been suggested that primary care services could cost - effectively be used to identify hearing disability using targeted questions, possibly alongside other screening interventions [21, 43]. Previous work through uk gp - based case finding, which targeted people in the 5065-year age group, showed that effective hearing aid use can be at least tripled [30, 54]. One study assessed the patient's take - up of hearing disability screening and the subsequent take - up of hearing aids as an intervention for hearing disability . Substantial benefits were reported in hearing aid benefit outcome inventories and moderate benefits in health utilities index and quality - of - life scores from amplification for this target group . Another uk study of 604 gp patients, aged 5065 years, showed that the first posting of hearing disability questionnaires detected 78% of those prepared to accept hearing aids for the first time . The possession of hearing aids rose from 7% (at baseline) to 24% (after intervention), and 6 months later the hearing aids were being used regularly . The authors concluded that simple questionnaires are effective in detecting hearing disability in older adults and that this intervention was acceptable by many of those reporting significant hearing difficulties . Given the pivotal role of the gp in the early identification and management of chronic health problems, at least in australia, the implementation of a gp - based hearing screening program for adults> 50 years of age would be beneficial in addressing this problem . Further, with the inadequacies of the medical model in the treatment of chronic health conditions and the move towards a model of patient - centred care, gps are effectively placed to assist with the minimisation of the stigma associated with hearing loss and enhancing patient self - motivation to manage this . Current research identifies a critical role for gps in both detection and appropriate referral of many other disorders / diseases such as obesity [5658]. However, several such studies identified that the knowledge and attitudes of gps can be a major barrier to effective intervention within this process . Hence, underlying reasons for low rates of gp involvement in hearing health could include lack of awareness / understanding of (a) simple tools to identify hearing loss and associated disability; (b) risk factors for age - related hearing loss and ways to use this information to identify at - risk patients; (c) adverse impacts caused by hearing loss on the mental and physical well - being of older adults (i.e., disability); and (d) the benefits of aural rehabilitation . Given the increasing prevalence and disease burden of undetected hearing loss in older adults and the availability of effective interventions (e.g., hearing aids and/or assisted listening devices), there are 3 potential critical roles for the gp in hearing health: (1) early identification of patients with age - related hearing loss, as well as recognition of whether any negative consequences / disability has resulted; (2) assistance in reducing the stigma of hearing loss and motivating patients to seek further help; and (3) appropriate referral of these patients to hearing health providers . This could be achieved by sensitising gps to recognise at - risk individuals and providing targeted questions to identify hearing loss disability . We have identified an important role of gps in the process of targeting individuals with late - onset hearing loss and referral; however, the challenge that remains is how to effectively increase gps knowledge and practice behaviour in this area . Possibly the most obvious method is through development of a continuing medical education (cme) program that targets the impacts of hearing loss and remediation and provides a reliable method of hearing screening in adults . The evidence for good outcomes of cmes measured by factors including increased knowledge and skills as well as altered attitudes and practice behaviours is varied and possibly depends partly on the learners and learning context . A review of the literature has identified that while the quality of evidence is not high, generally cme provides a strategy that increases knowledge and may elicit a change in practice behaviour . However, in a meta - analysis of the cme literature, forsetlund and colleagues report that education meetings are likely to only have a moderate effect on professional practice and a smaller improvement on patient outcomes . Despite this, cook et al . Demonstrated that while internet - based programs have a significant effect on knowledge and behaviour compared with no - intervention, there is limited evidence to suggest that it is superior to other methods of delivery of learning materials . Therefore it is possible that both educational meetings and internet - based programs will have only a moderate impact in enhancing referrals to hearing healthcare providers . An alternative method of screening of hearing loss and disability which does not require gp involvement is telephone and/or internet screening using digits in noise [6264], which provides a quick, effective, and relatively inexpensive technique to detect hearing loss in adults [62, 63]. In addition, this presumably has a broader reach than gp screening because of the program's accessibility to individuals in rural and remote areas where worldwide shortages of healthcare professionals and services exist . Further, it provides information about the individual's hearing to the significant proportion of individuals who were not intending to see a gp or hearing healthcare provider (as shown in). Smits and colleagues [62, 64] developed the first telephone screening test which was introduced into the netherlands in 2003 as the national hearing test . The screening test used 23 monosyllabic digit triplets presented by a female speaker, adaptively varying in level by 4 db (to determine audibility) and then 2 db (to seek threshold) and embedded in a 73 dba speech noise, shaped to match the long - term average speech spectrum . They estimated the average signal - to - noise ratio (snr) for speech reception threshold (srt; 50% correctly identified) and characterised normal hearing using a criterion of 4.1 db snr, insufficient hearing between 4.1 and 1.4 db snr, and poor hearing> 1.4 db snr . In 38 participants with varying levels of hearing, this screening test showed excellent test - retest reliability (<1 db error), sensitivity (0.91), and specificity (0.93) when compared to an equivalent speech - in - noise test conducted under headphones and took approximately 3 minutes to complete . A similar telephone screening test telscreen using digit triplets embedded in spectrally shaped noise was developed and implemented in australia in 2007 . The noise was amplitude modulated by a 20 hz sinusoid and had gaps in the frequency spectrum to increase the sensitivity of this test to identify sensorineural hearing losses (described in). Significant correlations were found between telscreen and the individual's four - frequency pure - tone average (r = 0.77, p <0.001) and telscreen and the presence of subjectively rated disability (r = 0.65, p <0.001). Smits and colleagues demonstrated that over 50% of those referred to medical or other professional hearing healthcare in the netherlands were compliant in following this advice . On the other hand, meyer and colleagues showed that only 36% of the 193 individuals who failed the telscreen in australia went on to receive medical or other professional support . It is not clear whether such differences in health - seeking behaviour are explained by cultural, social, or economic factors . Another hearing screening program is the use of an automated face - to - face monosyllabic speech - in - noise test which aims to evaluate hearing disability in adults . The speech understanding in noise (sun) test was developed by paglialonga and colleagues [66, 67] and has been evaluated in multiple nonclinical sites with varying levels of ambient noise showing good sensitivity up to 65 dba . The sun test presents monosyllabic vowel - consonant - vowel sounds in a 3-alternative forced - choice paradigm . The response is provided through a touch screen, thereby avoiding tester scoring errors, and takes approximately 2 minutes to evaluate both ears . Good associations were found between pure - tone audiometry and referral on the sun test which indicates the benefit of this test as a screening test for adult hearing loss . Given the ageing demographic and increasing average life span in western countries, chronic hearing loss is projected to increase . A renewed focus on targeting the provision of hearing rehabilitation to people with self - perceived hearing disability, rather than those with only measured hearing loss, may lead to better long - term retention and use of aids . Therefore, over time the costs saved by provision of an effective and better - targeted health intervention enabling improved daily functioning among older adults will no doubt demonstrate this strategy and will provide
Respiratory distress syndrome (rds) is an acute lung disorder caused by developmental insufficiency of surfactant production and structural immaturity of the lungs in preterm neonates . Reduced surfactant level leads to insufficient surface tension in the alveolus during expiration, resulting in atelectasis, decreased gas exchange, severe hypoxia, and acidosis, clinically manifested in neonates as serious difficulty in breathing . Rds primarily occurs in infants with a gestational age of less than 32 weeks and weight of less than 1200 g . In this context, the mortality rates in rds are closely linked to disease severity, gestational age, and racial differences, although the average mortality rate is broadly placed at 2050% . Nevertheless, rds affects approximately 7% of newborn infants and is the leading cause of preterm infant death worldwide . Ards is a common cause of respiratory failure in children and euroneostat data shows that the incidence of rds in infants is 52% at 3031 weeks, 74% at 2829 weeks, 88% at 2627 weeks, and 91% at 2325-week gestation . Apart from gestational age, inherited and environmental risk factors impact rds development, including sex, birth weight, asphyxia neonatorum, age of the pregnant woman, and gestational diabetes mellitus [1012]. A previous document also reported that amantadine could induce respiratory failure, contributing to the occurrence of rds . Importantly, rds is greatly influenced by genetic mutations that result in abnormal production or synthesis of surfactant - associated proteins [1416]. Pulmonary surfactant (ps) is present at the alveolar surface and stabilizes the alveoli involved in gas exchange, preventing alveolar collapse at the end of expiration . Ps prevents airway collapse by decreasing the surface tension and also acts as a natural barrier for inhaled pathogens or other harmful aerosols and particles . Ps is mainly composed of 90% lipids and 10% protein, and contains 4 surfactant proteins: sp - a, sp - b, sp - c, and sp - d . These surfactant proteins interact with surfactant phospholipids and are essential for the ultrastructure, metabolism, and surface tension in lungs . Sp - a and sp - d bind to pathogens and regulate microbial phagocytosis through activation or deactivation of inflammatory responses in alveolar macrophages . Human sp - a is encoded by sftpa gene located on chromosome 10q22.3 and is a member of a subfamily of c - type lectins known as collectins . Sp - a binds carbohydrate moieties found on lipids and on the surface of microorganisms, playing a vital role in surfactant homeostasis and in defense against respiratory pathogens . Human sp - b protein is encoded by sftpb gene located on chromosome 2p12-p11.2 and contains 14 exons . Sp - b increases the stability of surfactant monolayer and sp - b mutations resulting in sp - b protein deficiency are associated with respiratory distress in neonates . Human sp - d protein is encoded by sftpd gene located on chromosome 10q22.223.1 and contains 8 exons . Lung epithelial cells secrete sps into the pulmonary airspaces and sps are important in host defense against microorganisms inhaled into the lungs . Mutations in sp - a, sp - b, and sp - d gene are also associated with idiopathic pulmonary fibrosis and other human pulmonary diseases [2931]. However, the link between sp - a, sp - b, and sp - d polymorphisms and the risk of rds in preterm neonates is not thoroughly established . In this study, we examined the association between sp - a, sp - b, and sp - d polymorphisms and the risk of rds in premature infants to understand the contribution of sp - a, sp - b, and sp - d polymorphisms to rds risk . Between july 2010 and november 2014, a total of 100 neonates with rds from the han population were recruited to this study as the case group at the general hospital of the pla rocket force, the 306 hospital of pla, and haidian maternal and the child healthcare hospital . The case group contained 58 male infants and 42 female infants, with an average age of 30.803.40 weeks . During the same period, a group of 120 age- and sex - matched preterm neonates without rds were selected as the control group, with 72 male infants and 48 female infants and average age of 31.704.10 weeks . The reasons for preterm delivery included twin pregnancy, placental abruption, gestational hypertension, and congenital heart disease . Patients enrolled in our study had no sibship, and no significant difference was found in comparisons of gestational age, sex, birth weight, mode of delivery, and glucocorticoid hormones used before delivery between the 2 groups (all p>0.05). The study procedures were approved by the ethics committee of the general hospital of the pla rocket force . The diagnosis of rds in preterm newborns was based on the standard clinical diagnostic criteria described in practical neonatology (third edition). The eligible rds patients met the following inclusion criteria: (1) gestational age less than 37 weeks; (2) acute and sudden onset of respiratory distress appearing within 12 h after birth, manifested as shortness of breath, cyanosis, nasal flaring, and 3 depression signs; (3) a typical chest x- ray finding: reduction of universal radiolucency of lungs, mesh and granular shadows, air bronchogram, and consolidation observed as white lung . Subjects in the case and control groups were excluded if they: (1) were infants of diabetic mothers; (2) had a history of severe fetal distress or were born with severe asphyxia; (3) had used prophylactic surfactants in prenatal or postnatal period; (4) suffered from congenital malformations or severe intrauterine infection . Peripheral venous blood samples (4 ml) a portion of the collected blood (2 ml) was placed in tubes containing ethylenediamine tetraacetic acid (edta) as an anticoagulant and stored at 20c until further use . This portion of blood was used for genomic dna isolation using a whole - blood genomic dna extraction kit (tiangen biotech co., ltd). The remaining blood samples (2 ml) without anticoagulant were placed at room temperature for 1 h and centrifuged at 3000 rpm for 10 min to collect the serum . Serum levels of il-6 and tnf- were measured to assess the inflammation status using enzyme - linked immunosorbent assay (elisa). The ventilator parameters in neonates were also collected, including fraction of inspired oxygen (fio2), peak inspiratory pressure (pip), positive end - expiratory pressures (peep), and mean arterial pressure (map). Calculations were performed to obtain the values of compliance of respiratory system (c value), oxygenation index (oi), respiratory index (ri), and alveolar - arterial oxygen partial pressure difference [(a - a) do2]. Polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) was used to analyze sp - a, sp - b, and sp - d genetic polymorphisms . Pcr primers were designed using primer premier version 5.0 software (premier biosoft, palo alto, ca) and synthesized by shanghai sangon biological engineering technology co., ltd . Pcr reaction was carried out in 20-l volume, containing 10 lpcr plus mix (dbi), 1.2 l dna templates, 0.5 l upstream primer, and 0.5 l downstream primer . Pcr conditions were: 95c initial denaturation for 5 min, 95c denaturation for 30 s, 60c annealing for 45 s, and 72c extension for 50 s. after 35 cycles, the final extension was at 72c for 10 min . Pcr amplification products (6 ml) were digested with 4 u fsp i (toyobo) in a 15-l volume and incubated overnight at 37c . A volume of 5 l of the digested product was mixed with 3 l 6buffer solution and analyzed on 3% agarose gel, followed by uv photography to record the results . Locus - specific restriction enzymes hincii, xspi, and ase i (takara) were also used to validate pcr products from different loci, and agarose gel electrophoresis was employed to analyze and record the results . Serum levels of sp - a, sp - b, and sp - d were detected by elisa . All related reagents were purchased from immudiagnostik ag bensheim, and the experimental procedures were carried out in strict accordance with kit instructions . The concentrations were calculated by applying the linear regression equation of the standard calibration curve . All specimens were measured twice and averaged, and the procedures conformed to all clinical laboratory quality control standards . Statistical analyses were conducted with spss 18.0 statistical software (spss, chicago, il). Deviations from hardy - weinberg equilibrium of the genetic polymorphisms were evaluated by the chi - square test . The comparison of genotypic distribution and allele frequencies between groups was assessed by the rc contingency table chi - square test . The odds ratio (or) of different genotypes and 95% confidence interval (ci) were calculated to represent the relative risk . Between july 2010 and november 2014, a total of 100 neonates with rds from the han population were recruited to this study as the case group at the general hospital of the pla rocket force, the 306 hospital of pla, and haidian maternal and the child healthcare hospital . The case group contained 58 male infants and 42 female infants, with an average age of 30.803.40 weeks . During the same period, a group of 120 age- and sex - matched preterm neonates without rds were selected as the control group, with 72 male infants and 48 female infants and average age of 31.704.10 weeks . The reasons for preterm delivery included twin pregnancy, placental abruption, gestational hypertension, and congenital heart disease . Patients enrolled in our study had no sibship, and no significant difference was found in comparisons of gestational age, sex, birth weight, mode of delivery, and glucocorticoid hormones used before delivery between the 2 groups (all p>0.05). The study procedures were approved by the ethics committee of the general hospital of the pla rocket force . The diagnosis of rds in preterm newborns was based on the standard clinical diagnostic criteria described in practical neonatology (third edition). The eligible rds patients met the following inclusion criteria: (1) gestational age less than 37 weeks; (2) acute and sudden onset of respiratory distress appearing within 12 h after birth, manifested as shortness of breath, cyanosis, nasal flaring, and 3 depression signs; (3) a typical chest x- ray finding: reduction of universal radiolucency of lungs, mesh and granular shadows, air bronchogram, and consolidation observed as white lung . Subjects in the case and control groups were excluded if they: (1) were infants of diabetic mothers; (2) had a history of severe fetal distress or were born with severe asphyxia; (3) had used prophylactic surfactants in prenatal or postnatal period; (4) suffered from congenital malformations or severe intrauterine infection . Peripheral venous blood samples (4 ml) were collected from all subjects within 3 days after birth . A portion of the collected blood (2 ml) was placed in tubes containing ethylenediamine tetraacetic acid (edta) as an anticoagulant and stored at 20c until further use . This portion of blood was used for genomic dna isolation using a whole - blood genomic dna extraction kit (tiangen biotech co., ltd). The remaining blood samples (2 ml) without anticoagulant were placed at room temperature for 1 h and centrifuged at 3000 rpm for 10 min to collect the serum . Serum levels of il-6 and tnf- were measured to assess the inflammation status using enzyme - linked immunosorbent assay (elisa). The ventilator parameters in neonates were also collected, including fraction of inspired oxygen (fio2), peak inspiratory pressure (pip), positive end - expiratory pressures (peep), and mean arterial pressure (map). Calculations were performed to obtain the values of compliance of respiratory system (c value), oxygenation index (oi), respiratory index (ri), and alveolar - arterial oxygen partial pressure difference [(a - a) do2]. Polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) was used to analyze sp - a, sp - b, and sp - d genetic polymorphisms . Pcr primers were designed using primer premier version 5.0 software (premier biosoft, palo alto, ca) and synthesized by shanghai sangon biological engineering technology co., ltd . Pcr reaction was carried out in 20-l volume, containing 10 lpcr plus mix (dbi), 1.2 l dna templates, 0.5 l upstream primer, and 0.5 l downstream primer . Pcr conditions were: 95c initial denaturation for 5 min, 95c denaturation for 30 s, 60c annealing for 45 s, and 72c extension for 50 s. after 35 cycles, the final extension was at 72c for 10 min . Pcr amplification products (6 ml) were digested with 4 u fsp i (toyobo) in a 15-l volume and incubated overnight at 37c . A volume of 5 l of the digested product was mixed with 3 l 6buffer solution and analyzed on 3% agarose gel, followed by uv photography to record the results . Locus - specific restriction enzymes hincii, xspi, and ase i (takara) were also used to validate pcr products from different loci, and agarose gel electrophoresis was employed to analyze and record the results . Serum levels of sp - a, sp - b, and sp - d were detected by elisa . All related reagents were purchased from immudiagnostik ag bensheim, and the experimental procedures were carried out in strict accordance with kit instructions . The concentrations were calculated by applying the linear regression equation of the standard calibration curve . All specimens were measured twice and averaged, and the procedures conformed to all clinical laboratory quality control standards . Statistical analyses were conducted with spss 18.0 statistical software (spss, chicago, il). Deviations from hardy - weinberg equilibrium of the genetic polymorphisms were evaluated by the chi - square test . The comparison of genotypic distribution and allele frequencies between groups was assessed by the rc contingency table chi - square test . The odds ratio (or) of different genotypes and 95% confidence interval (ci) were calculated to represent the relative risk . Baseline characteristics of preterm neonates with rds and preterm neonates without rds are shown in table 2 . We did not observe any statistically significant differences in sex, gestational age, or birth weight between the case group and control group (all p>0.05). Importantly, the c value in the case group was significantly lower than in the control group (p<0.05). Further, the average values of oi, ri, and (a - a) do2, and the mean serum levels of il-6 were significantly elevated in the case group, in comparison to the control group (all p<0.001). However, tnf- serum levels were similar between the 2 groups (p=0.068). The distribution of genotypes and allele frequencies of sp - a, sp - b, and sp - d snps in the case and control groups are shown in table 3 . The test for hardy - weinberg equilibrium showed that the frequency of all 3 genes reached equilibrium, indicating that the selected sample was representative of the population . Notably, no significant differences between the case group and control group were observed in the genotype frequencies of sp - b 18a / c snp and the 2 sp - d loci, met11thrt / c and ala160thrg / a (p>0.05). In addition, allele frequencies of sp - b 18a / c, sp - d met11thrt / c and sp - d ala160thrg / a did not exhibit statistically significant differences between the 2 groups (all p>0.05). Further study revealed statistically significant differences between the case group and the control group in the genotype and allele frequencies of 2 snps of sp - a, + 186a / g and + 655c / t, and sp - b 1580c / t (all p<0.05). The frequency of aa genotype of sp - a + 186a / g snp, the cc genotype of sp - a + 655c / t loci, and cc genotype of the sp - b 1580c / t loci were significantly higher in preterm neonates with rds (case group) compared to the control group (all p<0.05). The risk of rds in carriers of a allele of + 186a / g loci, and c allele of + 655c / t and 1580c / t loci was 3.117 times higher, 2.762 times higher, and 2.212 times higher, respectively, compared to the non - carriers (sp - a+186a / g: or=3.117, 95%ci: 1.9385.014, p<0.001; sp - a+655c / t: or=2.762, 95%ci: 1.4205.371, p<0.001; sp - b 1580c / t: or=2.212, 95%ci: 1.4793.309; p<0.001). Elisa results revealed that serum levels of sp - a and sp - b in rds neonates carrying the 2 snps of sp - a, + 186a / g and + 655c / t, and sp - b 1580c / t were significantly lower than in the control group (all p<0.05). However, serum levels of sp - b and sp - d in rds neonates carrying sp - b (18a / c) snp and 2 snps of sp - d (met11thrt / c and ala160thrg / a) were not significantly different from the control group (all p>0.05), as shown in table 4 . The haplotypes of sp - a, sp - b, and sp - d are shown in table 5 . Shesis software was used to analyze the different haplotypes of sp - a, sp - b, and sp - d in preterm neonates of the 2 groups, and haplotypes with frequencies of less than 3% were excluded . The results revealed that, among a total of 10 haplotypes, the frequencies of 3 haplotypes exhibited statistically significant differences between the case group and the control group . The frequency of acactg in the case group was significantly lower than in the control group (or=0.232, 95%ci: 0.0760.712, p=0.006), suggesting the acactg haplotype may be a protective factor against rds . On the other hand, the frequency of acacca and acaccg haplotypes in the case group was significantly higher than in the control group (or=3.606, 95%ci: 1.22010.660, p=0.014; or=11.567, 95%ci: 3.34140.045, p<0.001), indicating that acacca and acaccg haplotype increases risk of rds in preterm neonates . The incidence of rds was used as a dependent variable and aa genotype on sp - a + 186a / g, cc genotype on sp - a + 655c / t, cc genotype on sp - b 1580c / t, sp - a level, and sp - b level were tested as the independent variables . The results showed that the aa genotype of sp - a + 186a / g, the cc genotype of sp - b 1580c / t, and sp - a level were independently associated with the risk for rds (all p <0.05) (table 6). Baseline characteristics of preterm neonates with rds and preterm neonates without rds are shown in table 2 . We did not observe any statistically significant differences in sex, gestational age, or birth weight between the case group and control group (all p>0.05). Importantly, the c value in the case group was significantly lower than in the control group (p<0.05). Further, the average values of oi, ri, and (a - a) do2, and the mean serum levels of il-6 were significantly elevated in the case group, in comparison to the control group (all p<0.001). However, tnf- serum levels were similar between the 2 groups (p=0.068). The distribution of genotypes and allele frequencies of sp - a, sp - b, and sp - d snps in the case and control groups are shown in table 3 . The test for hardy - weinberg equilibrium showed that the frequency of all 3 genes reached equilibrium, indicating that the selected sample was representative of the population . Notably, no significant differences between the case group and control group were observed in the genotype frequencies of sp - b 18a / c snp and the 2 sp - d loci, met11thrt / c and ala160thrg / a (p>0.05). In addition, allele frequencies of sp - b 18a / c, sp - d met11thrt / c and sp - d ala160thrg / a did not exhibit statistically significant differences between the 2 groups (all p>0.05). Further study revealed statistically significant differences between the case group and the control group in the genotype and allele frequencies of 2 snps of sp - a, + 186a / g and + 655c / t, and sp - b 1580c / t (all p<0.05). The frequency of aa genotype of sp - a + 186a / g snp, the cc genotype of sp - a + 655c / t loci, and cc genotype of the sp - b 1580c / t loci were significantly higher in preterm neonates with rds (case group) compared to the control group (all p<0.05). The risk of rds in carriers of a allele of + 186a / g loci, and c allele of + 655c / t and 1580c / t loci was 3.117 times higher, 2.762 times higher, and 2.212 times higher, respectively, compared to the non - carriers (sp - a+186a / g: or=3.117, 95%ci: 1.9385.014, p<0.001; sp - a+655c / t: or=2.762, 95%ci: 1.4205.371, p<0.001; sp - b 1580c / t: or=2.212, 95%ci: 1.4793.309; p<0.001). Elisa results revealed that serum levels of sp - a and sp - b in rds neonates carrying the 2 snps of sp - a, + 186a / g and + 655c / t, and sp - b 1580c / t were significantly lower than in the control group (all p<0.05). However, serum levels of sp - b and sp - d in rds neonates carrying sp - b (18a / c) snp and 2 snps of sp - d (met11thrt / c and ala160thrg / a) were not significantly different from the control group (all p>0.05), as shown in table 4 . The haplotypes of sp - a, sp - b, and sp - d are shown in table 5 . Shesis software was used to analyze the different haplotypes of sp - a, sp - b, and sp - d in preterm neonates of the 2 groups, and haplotypes with frequencies of less than 3% were excluded . The results revealed that, among a total of 10 haplotypes, the frequencies of 3 haplotypes exhibited statistically significant differences between the case group and the control group . The frequency of acactg in the case group was significantly lower than in the control group (or=0.232, 95%ci: 0.0760.712, p=0.006), suggesting the acactg haplotype may be a protective factor against rds . On the other hand, the frequency of acacca and acaccg haplotypes in the case group was significantly higher than in the control group (or=3.606, 95%ci: 1.22010.660, p=0.014; or=11.567, 95%ci: 3.34140.045, p<0.001), indicating that acacca and acaccg haplotype increases risk of rds in preterm neonates . The incidence of rds was used as a dependent variable and aa genotype on sp - a + 186a / g, cc genotype on sp - a + 655c / t, cc genotype on sp - b 1580c / t, sp - a level, and sp - b level were tested as the independent variables . The results showed that the aa genotype of sp - a + 186a / g, the cc genotype of sp - b 1580c / t, and sp - a level were independently associated with the risk for rds (all p <0.05) (table 6). In the present study we examined multiple snps of sp - a (+ 186a / g and + 655c / t), sp - b (18a / c and 1580c / t) and sp - d (met11thrt / c and ala160thrg / a) for their association with rds risk in preterm infants . Our findings suggested that sp - a + 186a / g and sp - b 1580c / t polymorphisms are strongly correlated with an increased risk of rds, while sp - b 18a / c, sp - d met11 thrt / c, and ala160 thrg / a polymorphisms were not associated with rds in preterm infants . The results suggest a possibility that sp - a + 186a / g and sp - b 1580c / t polymorphisms and sp - a level could serve as biomarkers for rds for population - based screening . Sp is important for lung health and normal lung function throughout life, because of its surface tension lowering property and its critical function in innate immunity . Sp - a, sp - b, and sp - d are major components of sp and are the essential players in sp homeostasis . Sp - a and sp - d participate in host defense and inflammation, which are important in clearing a variety of lung pathogens such as respiratory syncytial virus, mycobacterium tuberculosis, bacteria, viruses, and fungi, and are also involved in the aggregation, agglutination, and inhibition of pathogen growth, which is a major component of pulmonary host defense mechanisms . As such, sp - a is the most abundant surfactant protein and regulates phospholipid insertion into the monolayer as well as the uptake and secretion of phospholipids by type ii cells . Previous studies found that sp - a deficiency affects normal lung function and surfactant metabolism, and leads to immunopathological defects . Consistent with a previous study, logistic regression analysis showed that sp - a was independently correlated with risk of rds . Similarly, a previous study demonstrated that significant increases in plasma sp - a were observed during acute lung injury, suggesting the role of sp - a in lung function . On the other hand, sp - b has an essential role in the formation of tubular myelin, and, along with sp - c, promotes rapid phospholipid insertion into the air - liquid interface . A c / t polymorphism at nucleotide 1580 alters amino acid 131 by substituting threonine with isoleucine, eliminating a potential n - linked glycosylation site . Previous studies showed that neonates carrying sp - b gene deletions or genetic mutations exhibit lethal respiratory distress after birth . Showed that c / t polymorphism at nucleotide + 1580 is linked with neonatal respiratory distress syndrome in the han population . Consistent with previous studies, our study also found that sp - a and sp - b polymorphisms are involved in the development of rds in preterm neonates, and aa and cc genotypes of sp - a, and cc genotype of sp - b were independently associated with rds risk . Another important finding is that the serum levels of sp - a and sp - b in rds patients carrying sp - a (+ 186a / g) and sp - b (1580c / t) snps were significantly lower than in the control group . Sp - a and sp - b polymorphisms may result in decreased transcription / translation of sp - a and sp - b proteins and consequently decrease sp - a and sp - b serum levels, increasing the risk of rds in preterm infants . As opposed to sp - a and sp - d, which are hydrophilic proteins and are primarily involved in innate host defense, the hydrophobic protein sp - b is essential for adsorption of surfactant film at the alveolar air - liquid interface and therefore is relevant to surfactant maturation [4749]. Elevated expression of sp - a in amniotic fluid in late gestation is a biomarker for determining lung maturity of the fetus and lack of sp - a expression may predict rds in infants . Prez - gil et al . Showed that lack of or reduced pulmonary surfactant proteins, including sp - a and sp - b, is a major factor in rds development . Similarly, decreased serum level of sp - b protein, caused by a hereditary deficiency in the sp - b gene, frequently results in unexplained respiratory distress in infants due to defective surfactant maturation . The inability to produce multiple sps due to autosomal recessive genetic alterations causes fatal respiratory insufficiency, indicating the fundamental role of sps in normal pulmonary function . Demonstrated that a major sp - a haplotype, interacting with sp - b ile131thr polymorphism and other environmental risk factors, increased the risk of rds . Clark et al . Reviewed the association between multiple genetic factors and rds, and showed that variations in the levels of sp proteins such as sp - a, sp - b, and sp - c, caused by corresponding genetic polymorphisms, elevated the risk of neonatal chronic lung diseases, including rds . Further, yin et al . Suggested that immature sp - b function is linked with the pathogenesis of neonatal rds in the chinese han population and homozygous c / c genetic polymorphism in sp - b gene was the underlying defect . Consistent with previous studies, our study showed that sp - a and sp - b polymorphisms increased the risk of rds in preterm neonates, and decreased serum levels of sp - a and sp - b are associated with elevated rds risk and may serve as biomarkers for rds . Importantly, we examined the association between genetic polymorphisms in 3 sps and the risk of rds in preterm infants . In addition, we studied the impact of the genetic polymorphism on the serum levels of sps, and their overall correlation with the development of rds in preterm neonatal . Kelli et al . Found that sp - d rs1923537 polymorphism may be associated with a significantly increased risk of rds in preterm infants with lower gestational ages, but such an association was not found in our study . On the other hand, lyra et al . Demonstrated that sp - b polymorphisms (18a / c and 1580c / t) were not associated with rds risk in premature infants, and the ag genotype in ag9306 polymorphism of sp - b gene is a protective factor against the development of rds . The conflicting results from previous studies suggest that allele and genotype frequencies of sp genes may differ between distinct human ethnic groups . Therefore, some differences between our study results and the results from previous studies may be attributed to ethnic differences or sample size . It is well known that ethnic background is an important interfering factor in studies involving analysis of allele and genotype frequencies . In this context our study also showed that rds patients had elevated serum levels of il-6 in comparisons to the case group, which suggests the involvement of il-6 in the development of rds . However, further logistic regression analysis failed to confirm that serum il-6 level was a risk factor for rds . Il-6 is a pro - inflammatory cytokine and il-6 measurement might reflect different biological actions . In contrast to our results, a previous study found no association between markers of placental or fetal infection and inflammation and cld risk . Additionally, a discrepancy was also found between our results and a previous study that suggested il-6 accelerates fetal lung maturity, thereby decreasing the incidence of rds in the preterm neonates . One possible explanation for the difference between our observations and these reports is that we used peripheral venous blood rather than funisitis and cord serum il-6 . To some extent, this study was consistent with our result confirming the implication of il-6 in rds . Nevertheless, the exact role and mechanism of il-6 in rds still need further exploration . Cortisol, produced in humans by the zona fasciculate, is a steroid hormone and functions to increase blood sugar through gluconeogenesis, to suppress the immune system, and to aid in the metabolism of fat, protein, and carbohydrates . Evidence documented that diurnal cortisol rhythm predicts early lung cancer death, suggesting the role of cortisol in lung function . Specifically, tsuda et al . Suggested that neonates who develop rds / ttn have significantly lower cortisol levels in the umbilical cord at birth than neonates without rds / ttn in twin pregnancies . The investigation of cortisol is a promising direction to pursue in order to better understand the possible mechanism of rds, but limitations on time and budget do not allow us to address that topic in this article and we plan to investigate this interesting aspect in more detail in the future . Our results provide strong evidence that polymorphisms of sp - a + 186a / g, and sp - b 1580c / t increase the risk of rds in preterm neonates . Notably, decreased sp - a and sp - b serum levels are associated with elevated rds risk and may serve as important biomarkers for rds . By contrast, sp - b 18a / c, sp - d met11 thrt / c, and ala160 thrg / a gene polymorphisms were not associated with the risk of rds in preterm infants . Further studies using larger sample sizes are required to achieve more accurate outcomes and independently validate the associations between sp - a, sp - b, and sp - d gene polymorphisms and rds risk.
In some clinical situations, such as root canal overinstrumentation, apical resorption and teeth with open apices, there may be extrusion of the filling material, either gutta - percha, root canal sealer or both, due to the difficulty or impossibility to lock the master gutta - percha cone . In these situations, fabrication of an apical plug with calcium hydroxide and, more recently, with mineral trioxide aggregate (mta) has been suggested2,4,13,21,25,26 . Mta was introduced in the early 1990's as an experimental material developed by dr . Mahmoud torabinejad at loma linda university, usa . This material was originally indicated as a retrograde filling material for use in endodontic surgery and cases of intraradicular and furcal perforations18 . Since then, it has been used in different clinical situations, such as communicating internal and external resorptions, as capping material in mechanically exposed pulps, as intracoronal barrier during internal bleaching of endodontically treated teeth, and as apical plug in case of difficulty to lock the master gutta - percha cone4 . These indications of mta are related to the possibility of use in moist environments, as in most aforementioned indications, and mainly to its biocompatibility4,26 . The sealing ability of mta apical plug and its thickness have been investigated by several authors2,13,16,17,20,21,26 . Four - millimeter - thick plugs have been shown to be the most efficient with respect to root canal sealing ability and resistance to displacement13,20,21,26 . All of these studies have sought an alternative to mta apical plug as well as its most appropriate thickness . Wucherpfennig and green27 (1999) have called the attention to the fact that mta and portland cement were similar materials . Other studies were conducted and confirmed this similarity by means of microbiological, chemical, physical and biological behavior tests1,5,8,10,11,15,23 . The purpose of this study was to evaluate the sealing ability of apical plugs made of white and gray mta - angelus (angelus solues em odontologia, londrina, pr, brazil) and white portland cement (votorantim cimentos, votorantim, so paulo, sp, brazil) placed via the root canal and having different thicknesses (2, 5 and 7 mm). This study was approved by the institutional review board of the dental school of bauru, university of so paulo, brazil . Ninety extracted human single - rooted teeth with intact roots and completely formed apices were used . The teeth were obtained from the files of the department of endodontics of the dental school of bauru and were kept in 10% aqueous formalin solution . The dental crowns were sectioned at the cementoenamel junction with a low - speed diamond saw (kg sorensen, so paulo, sp, brazil) under continuous water spray to obtain access to the root canal . The root canal length was determined by inserting a size 15 k - file (dentsply - maillefer instruments sa, ballaigues, switzerland) into the canal until its tip reached the apical foramen . The working length was established by subtracting 1 mm from this measurement . The stepback technique was employed and the root canal was flared using a size 60 k - file to the working length . Instrumentation was aided by irrigation with 1 ml of 1% sodium hypochlorite solution (biodinmica qumica e farmacutica ltda, ibipor, pr, brazil) alternated with the sequence of instruments, followed by a final flushing with 1 ml of sterile water . The teeth were assigned to 3 groups (n=30), according to the material used for fabrication of the apical plugs: group a = placement of gray mta - angelus plugs; group b = placement of white mta - angelus plugs; group c = placement of white portland cement plugs . The groups were further subdivided into groups of 10 teeth each, according to the thickness of the apical plugs, namely 2, 5 and 7 mm (table 1). Before standardization of the foraminal opening, the roots were made impermeable by application of a layer of epoxy adhesive (araldite - ciba - geigy s.a ., taboo da serra, sp, brazil), followed by two coats of nail polish (cosbra cosmeticos ltda ., so paulo, sp, brazil). The foramen diameter was standardized by inserting the 40 k - file 1 mm beyond the apical foramen, so that only the apical opening would not be impermeable . For fabrication of the apical plugs, the tested materials were applied with a size 4 lentulo spiral (dentsply - maillefer instruments sa, ballaigues, switzerland) at the apical end of the root, trying to fill it completely . The material was condensed with the tip of a size 40 k - file involved in cotton for achievement of the plug19 . Next, using a size 40 k - file with a rubber stop positioned 2, 5 and 7 mm shorter than the root canal length, the excess material was removed for fabrication of 2-, 5- and 7-mm - thick apical plugs, respectively . Finally, the root canal walls were cleaned with the tip of an instrument wrapped in moist cotton . Then, the canal entrances were sealed with epoxy adhesive and nail polish, and the roots were immersed in 0.2% rhodamine b solution (labsynth produtos para laboratrios ltda, diadema, sp, brazil) at ph 7.0 for 72 hours at 37c . After this period, the roots were removed from the dye, washed in running water for 24 hours, had the impermeable coating scraped away and were washed for additional 12 hours . The roots were then sectioned longitudinally in a buccolingual direction for exposure of the apical plugs, photographed with a digital camera (canon eos rebel 300 d) and analyzed by image tool software (university of texas health science center, san antonio, tx, usa). Table 2 shows the percent marginal leakage for the different materials for each plug thickness . Tables 3, 4 and 5 show the results of the kruskal - wallis statistical test for comparison among groups a (mta - angelus gray), b (mta - angelus white) and c (white portland cement) with respect to the plug thicknesses (2, 5 and 7 mm). A statistically significant difference (p<0.05) was observed between groups a and b as to the leakage in 2- mm - thick plugs, with better results for group a (table 3). Materials did not show statistically significant difference (p>0.05) when groups with 5-mm - thick plugs were compared to each other (table 4). Regarding the 7-mm - thick plugs, there was statistically significant difference (p<0.05) between groups a and b and between groups b and c, with worst results for group b in both comparisons (table 5). In figure 1 shows the graphic presentation of percent marginal leakage (0.2% rhodamine b) in the root canals, regarding the tested materials and plug thicknesses . It is possible to verify that 2-mm - thick plugs yielded the least satisfactory results in all groups, whereas the 7-mm - thick plugs yielded the best results for groups a and c, yet not for group b, in which the 5-mm - plugs had the best results as to dye leakage . This study investigated the sealing ability of gray mta, white mta and white portland cement used for fabrication of apical plugs, given that this procedure is often required in the clinical practice12,14,19, mainly in cases where appropriate adaptation of the master gutta - percha cone is difficult . Torabinejad and chivian25 (1999) pointed out that the goal of an apical plug is to induce hard tissue formation, in order to prevent filling material extrusion in teeth with open apices . The use of methylene blue in marginal sealing studies has been questioned, due to its incompatibility with alkaline substances, which may induce discoloration of the dye . When calcium oxide is mixed with water, it results in the formation of calcium hydroxide, with a subsequent increase in ph, as previously demonstrated by duarte, et al.9 (2003). Therefore, rhodamine b dye solution is more appropriate for evaluating the sealing ability of mta22,24 . In the present study, 5- and 7-mm - thick plugs were more efficient for apical sealing than 2-mm - thick plugs, regardless of the material utilized (table 1 and figure 1), which is in agreement with the findings of previous2,13,21,26 . In group b (white mta), the 5-mm - thick plugs had better performance than the 2- and 7-mm - thick plugs, yet without statistically significant difference . With regard to tested materials, no statistical difference was expected among them because the chemical components of mta and portland cement are the same, except for bismuth oxide, which provides radiopacity to mta8,10,11 . Camilleri, et al.7 (2005) evaluated the chemical constitution and biocompatibility of white and gray portland cement, white and gray mta, portland cement clinker without calcium sulfate and portland cement clinker without calcium sulfate with addition of bismuth oxide . They concluded that the chemical composition of the tested materials is similar, primarily containing tricalcium silicate and dicalcium silicate . The white cements differ from the gray cements by the small quantity of iron oxide (feo), while mta differs from portland cement due to the presence of bismuth oxide . There was no difference between white and gray mta, and the addition of bismuth oxide did not interfere with the biocompatibility of cements . In the present study, white mta showed poorer results than gray mta and white portland cement for all tested plug thicknesses (table 1). Asgary, et al.3 (2005) observed significant differences between gray and white mta, especially in the contents of aluminum trioxide (al2o3), magnesium oxide (mgo) and iron oxide (feo). However, these differences are not enough to explain the results of the present study, in which gray mta showed better results than white mta as to dye leakage . Matt, et al.21 (2004) observed similar results in their study, where apical plugs made with gray mta were more efficient than those made with white mta . As to the fabrication of apical plugs, torabinejad and chivian25 (1999) recommended carrying the mta with a large amalgam carrier to the root canal and then condensing the material to the apical end of the root with pluggers or paper points . Sometimes, this procedure is difficult due to the root canal diameter and anatomy . In the present study, mta and portland cement were carried to the root canal with a size 4 lentulo spiral, according to the technique proposed by bramante, et al.6 (2004). The lentulo spiral is used to carry the mta in paste consistence more easily to the root canal end in a fast and correct manner . Another important factor is material condensation at the end of the root canal because the apical plug should resist the filling material . This condensation is more effective when performed with a k - file compatible with the root canal diameter and with the tip wrapped in a cotton mesh . Cleaning of the root canal walls and removal of excess material must be performed with the same k - file wrapped in moist cotton in sterile saline, not to interfere with the proper root canal filling . The findings of the present study showed that gray mta and portland cement had better sealing ability than white mta when used as apical plugs . Dye leakage was smaller for 5- and 7-mm - thick plugs compared to 2-mm - thick plugs.
We designed a cohort study in catalonia (northeast spain) that included two groups of patients aged 3074 years: consecutive first acute myocardial infarction patients without diabetes who survived at least 28 days after index myocardial infarction symptom onset and a random sample of type 2 diabetic patients without coronary heart disease . The study was approved by a local ethics committee (institut municipal d'investigaci mdica) and complied with all the laws and international ethics guidelines (declaration of helsinki). Acute myocardial infarction patients were recruited consecutively between 1990 and 2003 in the context of the population - based regicor (registre giron del cor [girona heart registry]) study (17). All 10 public and private hospitals in the region participate in regicor, using the same standards to diagnose acute myocardial infarction: non q - wave and q - wave myocardial infarction, determined by a discharge electrocardiogram in patients who presented with chest pain lasting> 20 min on admission, followed by typical changes in serial electrocardiograms and an abnormal increase in the cardiac enzymes or troponin value curve . We excluded patients outside the selected age range and those meeting the national diabetes data group 1979 and american diabetes association 1997 criteria for diabetes (18,19), including patients with two consecutive fasting plasma glucose values 7.8 or 7 mmol / l during admission, before and after 1998, previous acute myocardial infarction, or other diseases that shortened life expectancy to <1 year . Type 2 diabetic patients were randomly recruited between 1993 and 1998 in the 53 primary health care centers in catalonia participating in the gedaps (grupo de estudio de la diabetes en atencin primaria de salud [primary health care diabetes study group]) network . Diagnosis of type 2 diabetes was based on national diabetes data group 1979 criteria, i.e., two fasting plasma glucose values 7.8 mmol / l or 2-h plasma glucose values 11.1 we excluded patients not within the age range of the study, with bmi <22 kg / m, diseases that shortened life expectancy to <1 year or history of any coronary heart event or ketoacidosis . For all participants, we recorded age, sex, follow - up (in days), and, from medical records, their history of dyslipidemia and hypertension and smoking habit . For diabetic patients we also collected a1c concentration (fructosamine levels were not considered), treatment (diet, oral drugs, or insulin), and duration of diabetes in years . In acute myocardial infarction patients,, we completed up to 10 years follow - up by telephone, medical examination, or clinical record review . The individual end points considered were all - cause death, coronary death, stroke death, cardiovascular death, nonfatal acute myocardial infarction, and unstable angina . Two composite end points were used in the analyses: cardiovascular mortality (coronary, stroke, and other cardiovascular deaths) and coronary heart disease incidence (unstable angina or fatal or nonfatal acute myocardial infarction). The first cardiovascular event, regardless of its severity, was considered in the analysis . Deaths were considered of coronary heart disease origin in cases of suggestive necropsy findings, clinical records of hospitalized patients, or the presence on death certificates of icd-9 codes 410412, 414, 429.9, 798.1, and 798.2 or icd-10 codes i210i214, i219i229, i236, i240i249, i250i259, i46.1, r960, and r961). Stroke was defined by suggestive necropsy findings, clinical records for hospitalized patients, or icd-9 codes 430434 and 436438 (excluding 437.4437.8) or icd-10 codes i619i639, i64, i670i679, i688, and i690i698 . Other cardiovascular deaths were similarly defined . The applicable icd-9 codes were 401405, 426428, and 429.1429.9 and icd-10 codes were i10i110, i50i52, i440i499, i500i509, i250, and i511i519 . Nonfatal acute myocardial infarction was diagnosed when patients presented with chest pain lasting> 20 min on admission, followed by typical changes in serial electrocardiograms and an abnormal increase in the cardiac enzymes or troponin value curve . Unstable angina during follow - up was diagnosed by the presence of angina symptoms without an abnormal increase in the cardiac enzymes or troponin and with electrocardiographic changes in serial electrocardiograms or when, with or without electrocardiographic changes, suggestive symptoms were recorded during the event and confirmed by a positive stress test, with or without isotopic stress gammagraphy, or a positive coronary angiogram (stenosis> 70%). Our study was sufficiently powered (> 90%) to identify a statistically significant hazard ratio (hr) 0.80 for type 2 diabetic patients compared with first acute myocardial infarction patients, assuming> 25% 10-year coronary heart disease incidence among the latter and a correlation <0.3 of the type 2 diabetes variable with potential confounders . The two groups were approximately equally represented in the study: 51% diabetic and 49% myocardial infarction patients . Differences between myocardial infarction and diabetic patients at 10 years were assessed by a test for categorical variables and by student's t test for continuous variables or the nonparametric equivalents, as appropriate . Cox proportional hazards models were fitted to estimate the adjusted hr of cardiovascular mortality and coronary heart disease incidence at 10 years . Demographic, comorbidity, clinical, and severity variables that showed at least marginally significant differences (p 0.10) between type 2 diabetes and acute myocardial infarction patients, as well as variables considered important based on clinical judgment, were included as potential confounders in the multivariate analyses . Because the two cohorts were conducted at different time points, the results were adjusted for recruitment year, and a sensitivity analyses was performed in 2,260 diabetic patients and 828 acute myocardial infarction patients recruited in the same time periods (19931998). We considered tertiles of diabetes duration, comparing the third tertile versus the first and second together and two groups of glycemic control (a1c <7% and 7%) and therapy (diet alone, only oral drugs, and insulin). Survival curves were estimated with the kaplan - meier method and compared by mantel - cox statistics . Calculations were made with r (2.6.2 package; the r foundation for statistical computing, free software foundation, boston, ma). For all participants, we recorded age, sex, follow - up (in days), and, from medical records, their history of dyslipidemia and hypertension and smoking habit . For diabetic patients we also collected a1c concentration (fructosamine levels were not considered), treatment (diet, oral drugs, or insulin), and duration of diabetes in years . In acute myocardial infarction patients, in 2008, we completed up to 10 years follow - up by telephone, medical examination, or clinical record review . The individual end points considered were all - cause death, coronary death, stroke death, cardiovascular death, nonfatal acute myocardial infarction, and unstable angina . Two composite end points were used in the analyses: cardiovascular mortality (coronary, stroke, and other cardiovascular deaths) and coronary heart disease incidence (unstable angina or fatal or nonfatal acute myocardial infarction). The first cardiovascular event, regardless of its severity, was considered in the analysis . Deaths were considered of coronary heart disease origin in cases of suggestive necropsy findings, clinical records of hospitalized patients, or the presence on death certificates of icd-9 codes 410412, 414, 429.9, 798.1, and 798.2 or icd-10 codes i210i214, i219i229, i236, i240i249, i250i259, i46.1, r960, and r961). Stroke was defined by suggestive necropsy findings, clinical records for hospitalized patients, or icd-9 codes 430434 and 436438 (excluding 437.4437.8) or icd-10 codes i619i639, i64, i670i679, i688, and i690i698 . Other cardiovascular deaths were similarly defined . The applicable icd-9 codes were 401405, 426428, and 429.1429.9 and icd-10 codes were i10i110, i50i52, i440i499, i500i509, i250, and i511i519 . Nonfatal acute myocardial infarction was diagnosed when patients presented with chest pain lasting> 20 min on admission, followed by typical changes in serial electrocardiograms and an abnormal increase in the cardiac enzymes or troponin value curve . Unstable angina during follow - up was diagnosed by the presence of angina symptoms without an abnormal increase in the cardiac enzymes or troponin and with electrocardiographic changes in serial electrocardiograms or when, with or without electrocardiographic changes, suggestive symptoms were recorded during the event and confirmed by a positive stress test, with or without isotopic stress gammagraphy, or a positive coronary angiogram (stenosis> 70%). Our study was sufficiently powered (> 90%) to identify a statistically significant hazard ratio (hr) 0.80 for type 2 diabetic patients compared with first acute myocardial infarction patients, assuming> 25% 10-year coronary heart disease incidence among the latter and a correlation <0.3 of the type 2 diabetes variable with potential confounders . The two groups were approximately equally represented in the study: 51% diabetic and 49% myocardial infarction patients . Differences between myocardial infarction and diabetic patients at 10 years were assessed by a test for categorical variables and by student's t test for continuous variables or the nonparametric equivalents, as appropriate . Cox proportional hazards models were fitted to estimate the adjusted hr of cardiovascular mortality and coronary heart disease incidence at 10 years . Demographic, comorbidity, clinical, and severity variables that showed at least marginally significant differences (p 0.10) between type 2 diabetes and acute myocardial infarction patients, as well as variables considered important based on clinical judgment, were included as potential confounders in the multivariate analyses . Because the two cohorts were conducted at different time points, the results were adjusted for recruitment year, and a sensitivity analyses was performed in 2,260 diabetic patients and 828 acute myocardial infarction patients recruited in the same time periods (19931998). We considered tertiles of diabetes duration, comparing the third tertile versus the first and second together and two groups of glycemic control (a1c <7% and 7%) and therapy (diet alone, only oral drugs, and insulin). Survival curves were estimated with the kaplan - meier method and compared by mantel - cox statistics . Calculations were made with r (2.6.2 package; the r foundation for statistical computing, free software foundation, boston, ma). The study included 2,260 type 2 diabetic patients and 2,154 first acute myocardial infarction patients who survived 28 days after symptom onset . Acute myocardial infarction patients were younger and less frequently than the diabetic participants were women, hypertensive, and dyslipidemic . Baseline characteristics in non coronary heart disease type 2 diabetic patients compared with nondiabetic first acute myocardial infarction survivors data are n (%) or means sd unless specified otherwise . Ami, acute myocardial infarction . * some missing values in these variables (<5%). Evaluated in a sample of 499 patients at 6 months . Included only patients with a1c: fructosamine alone was used in 497 (22%) patients . The incidence rate for all event types was significantly worse among acute myocardial infarction patients, except for stroke death and unstable angina (table 2). These differences held after adjustment for sex, age, and baseline dyslipidemia, hypertension, and recruitment year . These findings were similar in both sexes, except for unstable angina: diabetic women had a significantly lower risk . Incidence rate and adjusted hr of different cardiovascular end points at 10 years for initially non coronary heart disease diabetic patients compared with nondiabetic first acute myocardial infarction survivors in all participants and by sex data are n (%) or hr (95% ci). All models are adjusted for sex, age, recruitment year, and baseline dyslipidemia and hypertension . Figure 1 shows the kaplan - meier curves of cardiovascular mortality and coronary heart disease incidence, respectively, comparing myocardial infarction patients with diabetic patients stratified by duration of type 2 diabetes, a1c, and therapy . All type 2 diabetes strata had significantly lower risk of both end points than acute myocardial infarction patients . Free of cardiovascular mortality (panel 1) and free of coronary heart disease (panel 2) survival curves among initially non coronary heart disease diabetic patients compared with nondiabetic first acute myocardial infarction (ami) survivors . A: according to time of evolution of type 2 diabetes (cut point 8 years). C: according to diabetes treatment (diet alone, only oral drugs, or insulin). All subgroups of diabetic patients had significantly lower risk of both composite end points than their acute myocardial infarction counterparts (table 3). Patients with type 2 diabetes receiving insulin therapy, with> 8 years of disease duration and a1c 7% were at significantly higher risk of coronary heart disease incidence or cardiovascular mortality than were those receiving dietary therapy alone or only oral drugs, with 8 years of evolution and a1c <7%, respectively . Other cutoff points of a1c (<6.5 vs. 6.5% and <6.5, 6.57.5, and> 7.5%) showed similar hrs for cardiovascular mortality and coronary heart disease incidence . At the cutoff point <6.5 vs. 6.5%, the hr for cardiovascular mortality was 0.16 (95% ci 0.100.27) and 0.23 (0.170.32) and for coronary heart disease incidence was 0.34 (0.250.45) and 0.44 (0.360.53), respectively . For the cutoff points <6.5, 6.57.5, and> 7.5%, the hr for cardiovascular mortality was 0.16 (0.100.27), 0.22 (0.140.36), and 0.24 (0.160.35) and for coronary heart disease incidence was 0.34 (0.250.45), 0.37 (0.280.50), and 0.48 (0.380.60), respectively . Adjusted hr of 10-year end points for type 2 diabetes patients by baseline tertiles of duration, glycemic control, and therapy compared with nondiabetic first acute myocardial infarction survivors data are hr (95% ci). P <0.05 compared with reference category (myocardial infarction patients). P <0.05 compared with immediately previous category . All models are adjusted for sex, age, recruitment year, and baseline dyslipidemia and hypertension . For example, the adjusted hr for 10-year coronary heart disease incidence and cardiovascular mortality was 0.53 (95% ci 0.420.67) and 0.30 (0.200.44) and 0.24 (0.170.34) and 0.23 (0.120.44) in men and women, respectively . The results of our study indicate that type 2 diabetic patients without previous coronary heart disease not only have lower 10-year cardiovascular mortality but also have lower coronary heart disease incidence than first acute myocardial infarction patients without diabetes . Results of previous studies may have found similar cardiovascular event rates due to differences in prognosis . In some cases, the population - based diabetes samples included only patients receiving drug treatment, which would exclude up to 25% of the total diabetic population (35). In several cohorts of diabetic patients from finland, scotland, and u.s . Selected irrespective of treatment status, patients with myocardial infarction had more events than those with diabetes (610). In our study the results suggest that diabetic patients receiving insulin or oral drug treatment may have a worse prognosis at 10 years than those treated with diet alone . However, diabetic patients treated with any of these modalities had significantly lower risk than myocardial infarction patients . Duration of diabetes was a determinant of cardiovascular outcomes in our study, which concurs with other reports (7,11,16). Our cut point (third tertile) was 8 years; other authors found that twice this evolution time (16 years) was required to worsen prognosis at 25 years (11). Diabetic patients in our series had lower levels of mean a1c at baseline than those included in the intensive branch of the ukpds, 7.5 vs. 8.1%, respectively (14). Despite differences in study design, our results support the relationship reported by the ukpds between high levels of a1c and worse prognosis . The ukpds approach is probably more realistic and correct than the stricter targets (a1c <6.5%) proposed in some intervention studies (20) that did not find significant differences or an increased number of cardiovascular events in the more intensive intervention arm . Some studies with stricter targets have shown the important role of hypoglycemia episodes in the poor prognosis of patients randomly assigned to intensive treatment (21). We found that incidence of unstable angina was similar in men with myocardial infarction and in men with diabetes, but lower in diabetic women than in their myocardial infarction counterparts . Mortality due to stroke was similar in both groups of patients and in both sexes . This observation adds to the existing controversy, with some authors finding positive (3,11) and some negative (5,7) differences . The low number of events, due to the age range selection in our study and to the fact that only fatal events were considered, hampers a more conclusive result . Our findings, taken together with the opposite observation in some high cardiovascular mortality countries (35) and intermediate observations in central - western europe (6) and the u.s ., the south - to - north gradient is persistently observed in myocardial infarction incidence and mortality rates . The paradox of high cardiovascular risk factor prevalence that contrasts with relatively low acute myocardial infarction incidence rates has been described in spain (17). Our findings also support such a gradient: the risk of fatal and nonfatal myocardial infarction in diabetic patients was 0.33 (95% ci 0.270.41); in similar cohorts, it was 0.42 (0.330.54) in the u.k ., primary prevention measures may need to be adapted to the particularities of cardiovascular and diabetes diseases by country or region . We have shown that lipid profile and blood pressure diagnosis and control improved between 1995 and 2005 in the region we studied (22). Our sample of myocardial infarction and diabetic patients is population - based in a region of northeast spain, where risk factor prevalence and myocardial infarction incidence and mortality are well studied by the regicor group (23). Over the follow - up period, many improvements occurred simultaneously in the management of both acute myocardial infarction and type 2 diabetes, which may have influenced the outcomes . In spain, a decrease in 28-day and 1-year mortality between 1995 and 2000 has been associated with increased use of reperfusion strategies and medical therapies (23). These changes were paralleled by intensified management of patients' cardiovascular risk factors and glycemic targets, following the ukpds results (14) and international recommendations (american diabetes association and european association for the study of diabetes) in primary care centers within the gedaps network . The proportion of diabetic patients who smoked was very similar to the proportion of smokers at 6 months after the index event that was reported in a sample of the acute myocardial infarction patients . Finally, asymptomatic myocardial infarction is known to occur to a greater degree in patients with diabetes than in the general population . In our study type 2 diabetes is on the increase in developed countries, a trend related to the epidemics of obesity observed in the past two decades . Between 19941995 and 20032004, the annual incidence of diabetes increased by 23% and prevalence by 62% in individuals> 65 years (24). The economic and clinical practice consequences of considering diabetic patients, who represent> 10% of the adult population in developed countries, for secondary prevention therapies are very important: benefits and effectiveness must be assessed and balanced, particularly in regions with low coronary heart disease incidence and mortality . Type 2 diabetes is not a coronary heart disease equivalent for cardiovascular risk in the region studied . In fact, this equivalence also has not been found in countries with high cardiovascular risk (610). Although patients with diabetes are at higher risk than the general nondiabetic population (7), individual cardiovascular risk scores are required before implementation of the level of treatment (statins, antiplatelet therapy, and intensification of hyperglycemia treatment) that has been shown to be useful in high - risk patients (25). Our study confirms that type 2 diabetic patients initially free of coronary heart disease are at lower adjusted 10-year cardiovascular mortality and coronary heart disease incidence risk than patients with a first acute myocardial infarction without diabetes . Our findings also contribute to showing that length of diabetes, type of treatment, and glycemic control should be taken into account in future studies on prognosis of patients with type 2 diabetes initially free of coronary heart disease . We have shown that lipid profile and blood pressure diagnosis and control improved between 1995 and 2005 in the region we studied (22). Our sample of myocardial infarction and diabetic patients is population - based in a region of northeast spain, where risk factor prevalence and myocardial infarction incidence and mortality are well studied by the regicor group (23). Over the follow - up period, many improvements occurred simultaneously in the management of both acute myocardial infarction and type 2 diabetes, which may have influenced the outcomes . In spain, a decrease in 28-day and 1-year mortality between 1995 and 2000 has been associated with increased use of reperfusion strategies and medical therapies (23). These changes were paralleled by intensified management of patients' cardiovascular risk factors and glycemic targets, following the ukpds results (14) and international recommendations (american diabetes association and european association for the study of diabetes) in primary care centers within the gedaps network . The proportion of diabetic patients who smoked was very similar to the proportion of smokers at 6 months after the index event that was reported in a sample of the acute myocardial infarction patients . Finally, asymptomatic myocardial infarction is known to occur to a greater degree in patients with diabetes than in the general population . In our study type 2 diabetes is on the increase in developed countries, a trend related to the epidemics of obesity observed in the past two decades . For example, in the u.s . Between 19941995 and 20032004, the annual incidence of diabetes increased by 23% and prevalence by 62% in individuals> 65 years (24). The economic and clinical practice consequences of considering diabetic patients, who represent> 10% of the adult population in developed countries, for secondary prevention therapies are very important: benefits and effectiveness must be assessed and balanced, particularly in regions with low coronary heart disease incidence and mortality . Type 2 diabetes is not a coronary heart disease equivalent for cardiovascular risk in the region studied . In fact, this equivalence also has not been found in countries with high cardiovascular risk (610). Although patients with diabetes are at higher risk than the general nondiabetic population (7), individual cardiovascular risk scores are required before implementation of the level of treatment (statins, antiplatelet therapy, and intensification of hyperglycemia treatment) that has been shown to be useful in high - risk patients (25). Our study confirms that type 2 diabetic patients initially free of coronary heart disease are at lower adjusted 10-year cardiovascular mortality and coronary heart disease incidence risk than patients with a first acute myocardial infarction without diabetes . Our findings also contribute to showing that length of diabetes, type of treatment, and glycemic control should be taken into account in future studies on prognosis of patients with type 2 diabetes initially free of coronary heart disease.
Data of subjects participating in the third national health and nutritional examination survey 19881994 (nhanes iii), a nationally representative sample of civilian noninstitutionalized us population was used . Briefly, the nhanes surveys are cross - sectional, multistage, stratified, clustered probability samples of the noninstitutionalized us civilian population . The details of the study can be found at http://www.cdc.gov/nchs/nhanes.htm . For the current study, data of participants older than 18 years with complete data on blood pressure, serum phosphorus and mortality, and other relevant covariates (n=15 833) were examined . The national centres for health statistics ethics review board approved the study protocol, and each participant provided written informed consent . Blood pressure was measured by a trained research assistant 3 times during the in - home visits and 3 additional times by a trained clinician during the visit to the mobile examination clinic . In both settings, blood pressures were measured with the participant in the sitting position after 5 minutes of rest . For systolic and diastolic blood pressure, separately, the second and third measurements from each visit were averaged . Hypertension was defined as self - reported history of hypertension, measured systolic blood pressure 140 mm hg, measured diastolic blood pressure 90 mm hg, or self - reported use of blood pressure medications.10 serum phosphorus was measured using a hitachi model 737 multichannel analyzer (boehringer mannheim diagnostics) by reacting inorganic phosphorus with ammonium molybdate in an acidic solution to form ammonium phosphomolybdate, which was quantified in the uv range (340 nm) through the use of a sample - blanked end - point method.11 elevated serum phosphorus was defined as serum phosphorus concentration 1.36 probabilistic matching was used to link nhanes iii participants with the national death index to ascertain vital status . Matching was based on 12 identifiers for each participant (eg, social security number, gender, and date of birth), leading to correct matching in 96.1% of deceased participants and 99.4% of living participants . Cardiovascular mortality was defined by international classification of diseases, tenth edition, clinical modification codes 100 to 178 derived from death certificate data . Information on age, gender, race, cigarette smoking, history of cardiovascular disease, and family income was self - reported by the participants and was obtained via interview during in - home visits . Socioeconomic status was assessed by the poverty income ratio, which is the ratio of family income to the federal poverty threshold (specific to the year of the interview). Height and weight were measured using standardized methods, and body mass index was calculated as weight in kilograms divided by height in meters squared . Serum creatinine, cholesterol, and glucose were measured as previously described.12 kidney function was assessed from estimated glomerular filtration rate (egfr). Glomerular filtration rate was estimated using the chronic kidney disease epidemiology collaboration creatinine equation after calibrating serum creatinine values to cleveland clinic reference values.13 diabetes was defined in accordance with the criteria of the american diabetes association (ie, fasting plasma glucose 7 mmol / l [126 mg / dl] and/or current use of insulin or oral hypoglycemic agents).14 a cox proportional hazards model was used to estimate hazard ratios of cardiovascular and all - cause mortality with 95% ci . Because egfr is related to serum phosphorus and hypertension, relationship between hypertension, elevated serum phosphorus, and mortality was examined in a multivariate analysis adjusted for egfr along with age, gender, and race . Proportional hazards assumption was evaluated by schnfeld's residuals and by inspection of log - log plots . Given that the nature of a joint effect can be additive or multiplicative,15,16 the joint effect of hypertension and elevated serum phosphorus on risk of mortality was investigated by testing interaction between hypertension and elevated serum phosphorus on an additive and multiplicative scale . Additive interaction indicates that an effect is more than additive when the risk ratios found are greater than the sum of individual risk ratios . Interaction on the additive scale was assessed by calculating the relative excess risk caused by interaction (reri), using the algorithm of andersson et al.17 reri was calculated as (hr11hr10hr01)+1, where hr11 is the hazard ratio for both risk factors present, hr10 is the hazard ratio for hypertension but no elevated serum phosphorus, and hr01 is the hazard ratio for no hypertension but elevated serum phosphorus . Interaction on the multiplicative scale was assessed by comparing multiplicative models with and without an interaction term using the log - likelihood ratio test . First, to account for possible reverse causation, we performed analyses after excluding participants who died in the first 2 years of follow - up . Second, because use of diuretic antihypertensive medication can affect blood pressure, risk of mortality, as well as affect serum phosphorus levels, we performed analyses after excluding participants who were on diuretic medication . Third, we additionally adjusted for dietary phosphorus intake . Finally, interactions were tested after adjustment for additional covariates in association between hypertension, elevated serum phosphorus, and mortality . Models were additionally adjusted for socioeconomic status, body mass index, smoking, cardiovascular disease history, diabetes, serum cholesterol, and vitamin d. in accordance with nhanes analytic guidelines, nhanes iii specific sampling weights were incorporated to account for its complex survey design (http://www.cdc.gov/nchs/tutorials/nhanes/stata_tips.htm). All analyses were performed using stata statistical software (version 13.0; stata corp). Data of subjects participating in the third national health and nutritional examination survey 19881994 (nhanes iii), a nationally representative sample of civilian noninstitutionalized us population was used . Briefly, the nhanes surveys are cross - sectional, multistage, stratified, clustered probability samples of the noninstitutionalized us civilian population . The details of the study can be found at http://www.cdc.gov/nchs/nhanes.htm . For the current study, data of participants older than 18 years with complete data on blood pressure, serum phosphorus and mortality, and other relevant covariates (n=15 833) were examined . The national centres for health statistics ethics review board approved the study protocol, and each participant provided written informed consent . Blood pressure was measured by a trained research assistant 3 times during the in - home visits and 3 additional times by a trained clinician during the visit to the mobile examination clinic . In both settings, blood pressures were measured with the participant in the sitting position after 5 minutes of rest . For systolic and diastolic blood pressure, separately, hypertension was defined as self - reported history of hypertension, measured systolic blood pressure 140 mm hg, measured diastolic blood pressure 90 mm hg, or self - reported use of blood pressure medications.10 serum phosphorus was measured using a hitachi model 737 multichannel analyzer (boehringer mannheim diagnostics) by reacting inorganic phosphorus with ammonium molybdate in an acidic solution to form ammonium phosphomolybdate, which was quantified in the uv range (340 nm) through the use of a sample - blanked end - point method.11 elevated serum phosphorus was defined as serum phosphorus concentration 1.36 mmol / l (4.2 mg / dl). Probabilistic matching was used to link nhanes iii participants with the national death index to ascertain vital status . Matching was based on 12 identifiers for each participant (eg, social security number, gender, and date of birth), leading to correct matching in 96.1% of deceased participants and 99.4% of living participants . Cardiovascular mortality was defined by international classification of diseases, tenth edition, clinical modification codes 100 to 178 derived from death certificate data . Blood pressure was measured by a trained research assistant 3 times during the in - home visits and 3 additional times by a trained clinician during the visit to the mobile examination clinic . In both settings, blood pressures were measured with the participant in the sitting position after 5 minutes of rest . For systolic and diastolic blood pressure, separately, hypertension was defined as self - reported history of hypertension, measured systolic blood pressure 140 mm hg, measured diastolic blood pressure 90 mm hg, or self - reported use of blood pressure medications.10 serum phosphorus was measured using a hitachi model 737 multichannel analyzer (boehringer mannheim diagnostics) by reacting inorganic phosphorus with ammonium molybdate in an acidic solution to form ammonium phosphomolybdate, which was quantified in the uv range (340 nm) through the use of a sample - blanked end - point method.11 elevated serum phosphorus was defined as serum phosphorus concentration 1.36 mmol / l (4.2 mg / dl). Probabilistic matching was used to link nhanes iii participants with the national death index to ascertain vital status . Matching was based on 12 identifiers for each participant (eg, social security number, gender, and date of birth), leading to correct matching in 96.1% of deceased participants and 99.4% of living participants . Cardiovascular mortality was defined by international classification of diseases, tenth edition, clinical modification codes 100 to 178 derived from death certificate data . Information on age, gender, race, cigarette smoking, history of cardiovascular disease, and family income was self - reported by the participants and was obtained via interview during in - home visits . Socioeconomic status was assessed by the poverty income ratio, which is the ratio of family income to the federal poverty threshold (specific to the year of the interview). Height and weight were measured using standardized methods, and body mass index was calculated as weight in kilograms divided by height in meters squared . Serum creatinine, cholesterol, and glucose were measured as previously described.12 kidney function was assessed from estimated glomerular filtration rate (egfr). Glomerular filtration rate was estimated using the chronic kidney disease epidemiology collaboration creatinine equation after calibrating serum creatinine values to cleveland clinic reference values.13 diabetes was defined in accordance with the criteria of the american diabetes association (ie, fasting plasma glucose 7 mmol / l [126 mg / dl] and/or current use of insulin or oral hypoglycemic agents).14 a cox proportional hazards model was used to estimate hazard ratios of cardiovascular and all - cause mortality with 95% ci . Because egfr is related to serum phosphorus and hypertension, relationship between hypertension, elevated serum phosphorus, and mortality was examined in a multivariate analysis adjusted for egfr along with age, gender, and race . Proportional hazards assumption was evaluated by schnfeld's residuals and by inspection of log - log plots . Given that the nature of a joint effect can be additive or multiplicative,15,16 the joint effect of hypertension and elevated serum phosphorus on risk of mortality was investigated by testing interaction between hypertension and elevated serum phosphorus on an additive and multiplicative scale . Additive interaction indicates that an effect is more than additive when the risk ratios found are greater than the sum of individual risk ratios . Interaction on the additive scale was assessed by calculating the relative excess risk caused by interaction (reri), using the algorithm of andersson et al.17 reri was calculated as (hr11hr10hr01)+1, where hr11 is the hazard ratio for both risk factors present, hr10 is the hazard ratio for hypertension but no elevated serum phosphorus, and hr01 is the hazard ratio for no hypertension but elevated serum phosphorus . Interaction on the multiplicative scale was assessed by comparing multiplicative models with and without an interaction term using the log - likelihood ratio test . First, to account for possible reverse causation, we performed analyses after excluding participants who died in the first 2 years of follow - up . Second, because use of diuretic antihypertensive medication can affect blood pressure, risk of mortality, as well as affect serum phosphorus levels, we performed analyses after excluding participants who were on diuretic medication . Third, we additionally adjusted for dietary phosphorus intake . Finally, interactions were tested after adjustment for additional covariates in association between hypertension, elevated serum phosphorus, and mortality . Models were additionally adjusted for socioeconomic status, body mass index, smoking, cardiovascular disease history, diabetes, serum cholesterol, and vitamin d. in accordance with nhanes analytic guidelines, nhanes iii specific sampling weights were incorporated to account for its complex survey design (http://www.cdc.gov/nchs/tutorials/nhanes/stata_tips.htm). All analyses were performed using stata statistical software (version 13.0; stata corp). A total of 1691 cases of cardiovascular mortality and 3875 cases of all - cause mortality were identified over a median follow - up of 14.3 years . The hrs of hypertension and elevated serum phosphorus, adjusted for age, gender, race, and egfr, are shown in table 2 . Hypertension and elevated serum phosphorus separately were significantly associated with the risk of cardiovascular and all - cause mortality . The joint effect of hypertension and elevated serum phosphorus on the risk of mortality is shown in table 3, and is visualized in figure 1 for cardiovascular mortality . The joint effect of hypertension and elevated serum phosphorus on the risk of cardiovascular mortality and all - cause mortality was (hr=2.80, 95% ci: 2.09; 3.73) and (hr=1.76, 95% ci: 1.49; 2.07), respectively . For cardiovascular mortality, interaction was observed between hypertension and elevated serum phosphorus on the additive scale (reri=0.99, 95% ci: 0.06; 1.92, adjusted for age, gender, race, and egfr), suggesting that the joint effect of hypertension and elevated serum phosphorus is stronger than is the sum of the independent effects of individual risk factor . With respect to all - cause mortality, no clear evidence of interaction was observed between hypertension and elevated serum phosphorus on the additive scale (reri=0.16, 95% ci: 0.25; 0.57, adjusted for age, gender, race, and egfr). No substantial interaction was observed on the multiplicative scale between hypertension and elevated serum phosphorus either for cardiovascular mortality (p=0.130, log - likelihood ratio test) or all - cause mortality (p=0.741, log - likelihood ratio test). Baseline characteristics of hypertensive cases, elevated serum phosphorus cases, hypertensive cases with elevated serum phosphorus, and entire cohort continuous variables are presented as mean (standard error), categorical variables are presented as percentages . Cvd indicates cardiovascular disease; dbp, diastolic blood pressure; egfr, estimated glomerular filtration rate; sbp, systolic blood pressure . Crude incidence rate and adjusted * hazard ratios of cardiovascular and all - cause mortality for hypertension and elevated serum phosphorus adjusted for age, gender, race, and estimated glomerular filtration rate . Crude incidence rate and adjusted * hazard ratios of cardiovascular and all - cause mortality for joint effect of hypertension and elevated serum phosphorus adjusted for age, gender, race, and estimate glomerular filtration rate . Joint and individual effects of hypertension and elevated serum phosphorus on the risk of cardiovascular mortality . The joint effect of hypertension and elevated serum phosphorus on the risk of cardiovascular mortality and all - cause mortality after excluding participants who died in the first 2 years of follow - up is shown in table 4 . In this case, reri for cardiovascular mortality was significant (reri=1.21, 95% ci: 0.26; 2.17) and not for all - cause mortality (reri=0.17, 95% ci: 0.27; 0.62, adjusted for age, gender, race, and egfr). After excluding participants on diuretic medication, reri for cardiovascular mortality was statistically significant (reri=1.31, 95% ci: 0.30; 2.33) but not for all - cause mortality (reri=0.20, 95% ci: 0.22; 0.62). After adjusting for dietary phosphorus intake, reri for cardiovascular mortality was statistically significant (reri=1.10, 95% ci: 0.09; 2.10) and not for all - cause mortality (reri=0.18, 95% ci: 0.22; 0.58). Results after additional adjustment for socioeconomic status, body mass index, smoking, history of cardiovascular disease, diabetes, serum cholesterol, and vitamin d are shown in table 4 . In this case, reri for cardiovascular mortality, although not statistically significant, was positive and tended to be higher (reri=0.88, 95% ci: 0.07, 1.83) compared to all - cause mortality (reri=0.10, 95% ci: 0.35; 0.56). The interactions on the multiplicative scale were not significant in either of the sensitivity analyses . Adjusted hazard ratios of cardiovascular and all - cause mortality for joint effect of hypertension and elevated serum phosphorus adjusted for age, gender, race, estimated glomerular filtration rate, socioeconomic status, smoking, body mass index, history of cardiovascular disease, diabetes, serum cholesterol, and vitamin d. adjusted for age, gender, race, estimated glomerular filtration rate, and dietary phosphorus intake / day . In this prospective study, our findings suggest that regarding cardiovascular mortality the joint effect of hypertension and elevated serum phosphorus is larger than the sum of their individual effects but not regarding all - cause mortality . These findings suggest that controlling serum phosphorus levels in hypertensive individuals may lead to an extra risk reduction regarding cardiovascular mortality . Interaction in epidemiology tests whether the joint effect of 2 risk factors on a certain outcome differs from the sum or product of their independent effects . Therefore, interaction can be measured on an additive or a multiplicative scale . Interaction on the additive scale indicates the extent to which the observed risk for disease in those with both risk factors is greater (synergism) or lower (antagonism) than the sum of individual risk ratios of each risk factor separately . Interaction on the multiplicative scale concerns whether the risk for disease in those with both risk factors was greater or lower than was the multiplied risk ratios of each risk factor alone.17,18 it is possible that the interaction is present only on 1 scale or both scales . Although there is no consensus on whether the additive or multiplicative scale is the most appropriate, additive interaction may be of more relevance from the viewpoint of translating epidemiological findings into prevention of disease events, as it is readily translated into impact of intervention in terms of absolute number of preventable outcomes.19 the recent strobe statement advises to report interaction analyses on an additive and multiplicative scale 20 when evaluating joint effect of exposures . We, therefore, presented interaction results on both an additive and multiplicative scale . Moreover, it allows us to compare our results with existing literature where conventionally interaction is reported on a multiplicative scale . The implications of finding interaction on the additive scale between hypertension and elevated serum phosphorus and the risk of cardiovascular mortality may suggest that these risk factors share a common pathway in the pathogenesis of cardiovascular mortality, and that combining serum phosphorus and blood pressure lowering may have an extra risk reduction effect in preventing cardiovascular mortality . Some earlier studies had reported inverse association between high dietary serum phosphorus intake and risk of hypertension.21,22 however, in these studies either the main source of high serum phosphorus was dairy products / leafy vegetables or only high serum phosphorus from dairy products was associated with a reduced risk of hypertension . Dairy products and leafy vegetables are known to be rich in other nutrients such as magnesium, calcium, and potassium and are effective in reducing risk of hypertension,23 which might explain these findings . No previous study has formally examined the joint effect of hypertension and elevated serum phosphorus on the risk of mortality . Only 1 study by tonelli et al tested interaction between serum phosphorus and hypertension while examining the independent effect of serum phosphorus on mortality in the general population.3 tonelli et al found no joint effect of hypertension and elevated serum phosphorus on risk of mortality . Interaction, however, was tested on a multiplicative scale only . In our study, interaction of risk factors was tested on both an additive and a multiplicative scale, and it was found that interaction between hypertension and elevated serum phosphorus was present on the additive, but not the multiplicative, scale . We separately accounted for some of the factors that might affect serum phosphorus levels and risk of mortality (eg, dietary phosphorus intake, hypertensive medication use, and renal function). Unfortunately, nhanes - iii did not collect information on factors that tightly regulate phosphate metabolism (eg, fibroblast growth factor 23 or intact parathyroid hormone measurement) and, therefore, it was not possible to investigate the effect of phosphate metabolism on our findings . However, it should be noted that the aim of this study was not to investigate the causes of elevated serum phosphorus but to investigate the risk of mortality when individuals have elevated serum phosphorus as well as hypertension . After adjustment for additional covariates, the reri for cardiovascular mortality was not statistically significant, but it was positive and indicative of joint effect . The low power for reri after adjustment for additional covariates might be because not all individuals in the study had complete information on all of the covariates . Therefore, adjustment for additional covariates resulted in loss of a substantial number of individuals with elevated serum phosphorus and hypertension (13%). The major strength of this analysis is that it was conducted using data from a large, well - characterized cohort of general population . Potential confounders of the association between hypertension, elevated serum phosphorus, and mortality were carefully considered . Data were only available for onetime assessment of both hypertension and elevated serum phosphorus . Therefore, bias may have been introduced because of risk factor levels that may have changed after baseline measurements . Given the changes in health behaviors, disease awareness, and type of antihypertensive medication use in last 20 years, prevalence and incidence of hypertension and elevated serum phosphorus might have changed . However, we believe that the mechanism behind the joint effect of hypertension and serum phosphorus is unlikely to have changed . Like previous studies,24 we also found increased risk of cardiovascular - related mortality from elevated serum phosphorus in men but not women (data not shown). Because the main aim of this study was to investigate the joint effect of elevated serum phosphorus and hypertension, and there were a limited number of participants with both elevated serum phosphorus and hypertension, we could not investigate whether their joint effect differs by sex . It should be investigated in future studies with a larger sample size of participants with both elevated serum phosphorus and hypertension . Finally, as for all observational studies, the present findings do not conclusively demonstrate that the joint effect of hypertension and serum phosphorus for the risk of cardiovascular mortality is causal . A large body of evidence exists regarding excess risk of mortality risk in kidney disease patients who have high levels of serum phosphorus.2528 therefore, clinical management of serum phosphorus is a priority in kidney disease patients . Recently, suggestion has been made to control dietary intake of phosphorus - containing food.6 however, there are no established guidelines for serum phosphorus control in hypertensive individuals . Managing causes of elevated serum phosphorus and thereby controlling an existing elevated serum phosphorus level in hypertensive individuals may be a relevant therapeutic target in preventing excess risk of cardiovascular mortality . Future studies, however, should confirm causality of the relationship between simultaneous existence of serum phosphorus and hypertension and the risk of cardiovascular mortality . In conclusion, our study revealed that the joint effect of hypertension and elevated serum phosphorus is larger than the sum of their individual effects on cardiovascular mortality, but not in case of all - cause mortality . Future studies should investigate whether controlling elevated serum phosphorus in hypertensive individuals helps in prevention of extra risk of cardiovascular mortality.
Myofascial pain syndrome (mps) is described as the sensory, motor, and autonomic symptoms caused by myofascial trigger points (trps). Trps are defined as exquisitely tender spots in discrete taut bands of hardened muscle that produce local and referred pain, among other symptoms . An individual contraction knot appears as a segment of a muscle fiber with extremely contracted sarcomeres and an increased diameter . The integrated trp hypothesis postulates that in myofascial pain motor endplates release excessive acetylcholine, which is evidenced histopathologically by the presence of sarcomere shortening . It prevents full lengthening of the muscle, weakens the muscle, and mediates a local twitch response of muscle fibers when adequately stimulated . When compressed within the patient s pain tolerance, it produces referred motor and often autonomic phenomena, generally in its pain reference zone . A latent trp is clinically quiescent with respect to spontaneous pain; it is painful only when palpated . A latent trp may have all the other clinical characteristics of an active trp and always has a taut band that increases muscle tension and restricts range of motion . Trps are very common, although literature about their prevalence is sparse [37]. Knowing the potential causes of trps is important to prevent their development and recurrence, but also to inactivate and eliminate existing trps . There is general agreement that any kind of muscle overuse or direct trauma to the muscle can lead to the development of trps . Muscle overload is thought to be the result of sustained or repetitive low - level muscle contractions, eccentric muscle contractions, and maximal or submaximal concentric muscle contractions . Although muscle damage is not required for the development of trp, there may be a disruption of the cell membrane, damage to the sarcoplasmic reticulum with a subsequent release of high amounts of calcium - ions, and disruption of cytoskeletal proteins, such as desmin, titin, and dystrophin . Ragged red (rr) fibers and increased numbers of cytochrome - c - oxidase (cox) negative fibers are common in patients with myalgia, which are suggestive of an impaired oxidative metabolism . Mechanical muscle overuse can be defined as the result of muscle contractions that exceed muscle capacity . Since the capillary blood pressure ranges from approximately 35 mm hg at the beginning (arterial side) to 15 mm hg at the end of the capillary beds (venous side), the capillary blood flow is temporarily obstructed during muscle contractions . The blood flow recovers immediately with relaxation, which is consistent with its normal physiological mechanism . In dynamic rhythmic contractions, intramuscular blood flow is enhanced by this contraction - relaxation rhythm, also known as the muscular pump . During sustained muscle contractions, however, muscle metabolism is highly dependent upon oxygen and glucose, which are in short supply . Even contractions performed at only 10% and 25% of capacity or maximum voluntary contraction (mvc) may produce intramuscular pressures high enough to significantly impair the intramuscular blood circulation . The association of the percentage of mvc and intramuscular pressure (imp) is highly dependent upon the architecture of the muscle . It has been postulated that the percentage of mvc reported as the upper limit for localized muscle fatigue varies between muscles, because of the variation of imp during a contraction . For example, 10% of mvc of the supraspinatus muscle produces approximately 50 mm hg of imp, while 25% of mvc of the trapezius muscle is good for only 22 mm hg of pressure . The emg signs of localized muscle fatigue did not recover until the muscle contraction pressure was below 20 mm hg in the biceps muscle . According to otten, the increased pressure gradients during low - level exertions may contribute to the development of pain and eventually to the formation of trps (personal communication 2005). Since oxygen and glucose are required for the synthesis of adenosine triphosphate (atp), which provides the energy needed for muscle contractions, sustained contractions may cause a local energy crisis due to the lack of oxygen . To guarantee an adequate supply of atp, the muscle can switch within a few seconds to anaerobic glycolysis . During the initial phase of glycolysis (sugar splitting) 1 glucose molecule is broken down into 2 pyruvic molecules releasing enough energy to form 2 atp molecules . Under aerobic circumstances, oxygen reacts with pyruvic acid producing a high amount of atp (16 molecules per pyruvic acid molecule), carbon dioxide and water . Under anaerobic circumstances, however, most of the pyruvic acid produced during glycolysis is converted into lactic acid, thereby increasing the intramuscular acidity (ph). Most of the lactic acid diffuses out of the muscle into the bloodstream; post - exercise lactic acid is washed out within 30 minutes after exercise . Unfortunately, when the capillary circulation is restricted, as in sustained low - level contractions, this process comes to a standstill . Researchers at the us national institutes of health found that in the direct environment of active trps, the ph may be well below 5, which is more than sufficient to excite muscle nociceptors, including acid - sensing ion channels (eg, asic 1 and 3), and the transient receptor potential vanilloid receptor trpv1 [13, 14]. Small increases of the h concentration, as seen with inflammation, heavy muscle work, and ischemia, are sufficient to excite muscle group iv endings, contributing to mechanical hyperalgesia and central sensitization . Furthermore, a low ph downregulates acetylcholinesterase (ache), increases the efficacy of acetylcholine (ach), and maintains the sarcomere (super-) contraction . It also triggers the release of several nociceptive substances, such as calcitonin gene - related peptide (cgrp), which can enhance the release of ach from the motor endplate and simultaneously decrease the effectiveness of ache in the synaptic cleft . Cgrp also upregulates the ach - receptors (achr) at the muscle and thereby creates more docking stations for ach . Miniature endplate activity depends on the state of the achr and on the local concentration of ach, which is the result of ach - release, reuptake, and breakdown by ache . After atp is attached to the myosin molecule, the link between myosin and actin weakens, and the myosin head detaches from actin . In other words, the cross - bridge between myosin and actin breaks. Simultaneously, the ca ion detaches from the troponin molecule, which blocks tropomyosin . Under normal physiological circumstances, large amounts of free calcium - ions will reenter the sarcoplasmic reticulum by the ca pump (calcium atpase), which places a high demand on atp during relaxation . In case of severe energy depletion, the sarcomeres may stay contracted, until enough atp is available to resolve the intracellular ca accumulation . Ca accumulation due to sustained motor unit activity has been suggested to play a causative role in the development of muscle disorders and trps . In a preliminary study using doppler ultrasound, sikdar et al have shown that blood flow waveforms show significant differences between active trps, latent trps and normal sites . The flow waveforms near active sites showed increased systolic velocities and flow reversal with negative diastolic velocities [17]. They identified 2 contributing factors, namely an increase in the volume of the vascular compartment, and an increased outflow resistance . Increased outflow resistance could be due to muscle contractures at the trp that compress the capillary or venous bed . Sustained low - level contractions are common in the workplace where many occupations rely on prolonged postures, as seen in musicians, supermarket cashiers, computer operators, hairdressers, and dentists, among others . Henneman, working with anesthetized cats, showed that in response to increasing physiological excitation, motor neurons are recruited in order of increasing size . This fundamentally important finding was verified in humans by milner - brown et al and is now generally accepted . Smaller motor units have a smaller alpha - motor neuron cell body, smaller axons and fewer muscle fibers to activate compared with larger ones . According to the size principle, small motor units innervating type i red colored, slow oxidative fibers are recruited first, followed by red to pink colored, fast oxidative fibers, and finally by white colored, fast glycolytic fibers . Furthermore, small type i muscle fibers are activated during prolonged tasks, although a few studies have described some degree of motor unit substitution . Hgg suggested that the continuous activity of these motor units in sustained contractions causes overuse muscle fiber damage, especially to the type i fibers during low - level activities, which he summarized in his cinderella hypothesis . It is conceivable that in sustained low - level contractions and in dynamic repetitive contractions, ischemia, hypoxia and insufficient atp synthesis in type i motor unit fibers are responsible for increasing acidity, ca accumulation, and subsequently sarcomere contracture . Furthermore, starting with the sarcomere (super-) contraction, the intramuscular perfusion slows down and ischemia and hypoxia will occur . This may lead to the release of several sensitizing substances causing peripheral sensitization [15, 22]. Henneman, working with anesthetized cats, showed that in response to increasing physiological excitation, motor neurons are recruited in order of increasing size . This fundamentally important finding was verified in humans by milner - brown et al and is now generally accepted . Smaller motor units have a smaller alpha - motor neuron cell body, smaller axons and fewer muscle fibers to activate compared with larger ones . According to the size principle, small motor units innervating type i red colored, slow oxidative fibers are recruited first, followed by red to pink colored, fast oxidative fibers, and finally by white colored, fast glycolytic fibers . Furthermore, small type i muscle fibers are activated during prolonged tasks, although a few studies have described some degree of motor unit substitution . Hgg suggested that the continuous activity of these motor units in sustained contractions causes overuse muscle fiber damage, especially to the type i fibers during low - level activities, which he summarized in his cinderella hypothesis . It is conceivable that in sustained low - level contractions and in dynamic repetitive contractions, ischemia, hypoxia and insufficient atp synthesis in type i motor unit fibers are responsible for increasing acidity, ca accumulation, and subsequently sarcomere contracture . Furthermore, starting with the sarcomere (super-) contraction, the intramuscular perfusion slows down and ischemia and hypoxia will occur . This may lead to the release of several sensitizing substances causing peripheral sensitization [15, 22]. During (sub-) maximal concentric contractions high amounts of energy (atp) are required . After about 46 seconds, the muscle shifts to direct phosphorylation of adp by creatine phosphate (cp). Phosphorylation produces atp by coupling 1 phosphate group to an adp molecule catalyzed by the enzyme creatine kinase . Stored atp and cp provide enough energy for maximum muscle power for approximately 1416 seconds . Hereafter, a short period of rest is needed to replenish the exhausted reserves of intracellular atp and cp . When ongoing atp demands are within the capacity of the aerobic pathway, muscular activity can continue for hours in well - conditioned individuals . However, when the demands of exercise begin to exceed the ability of the muscle cells to carry out the necessary reactions quickly enough, anaerobe glycolysis will contribute more and more of the total generated atp . Finally, the muscle will run out of atp and sustained sarcomere contractions may occur, starting the development of trps . Classic examples of this are walking, whereby the knee extensors are active while being lengthened just after heel strike while the knee flexes; placing an object gently downwards, whereby the arm flexors are active while being lengthened to control the rate of movement of the object . In other words, eccentric contractions are commonly used to control the rate of movement . In another scenario, the load on the muscle may increase to the point where the external force on the muscle is greater than the force that the muscle can generate . In that case there is no solid evidence that eccentric loading would lead to the development of trps, but as gerwin et al summarized, there is much overlap between the mechanisms of eccentric contraction and the development of trps . In one study, itoh et al found that healthy volunteers presented with tender ropy taut bands, which were painful on compression, immediately after 3 sets of eccentric exercises of the middle finger extensor muscle . After 7 days the muscles were recovered . While this study suggests that eccentric loading does contribute to the development of trps, the study also employed isometric loading in addition to eccentric loading, which precludes definitive conclusions . There is evidence from biopsy studies that during eccentric contractions disruption of the cytoskeletal structures occurs, especially of the protein desmin that interconnects the adjacent myofibrils [26, 27] and of titin, the largest protein in the human body . Titin connects myosin filaments to the z - bands and is also linked to actin filaments [28, 29]. Microscopic research revealed increased sections of abnormal fibers in eccentrically exercised muscles [30, 31], which appeared to be approximately 4 times the normal size . This was only observed with eccentric exercise, and not with concentric exercise or passive stretching . Furthermore, all enlarged fibers were exclusively of the fast glycolytic type or fast contracting type ii fibers that are highly fatigable . It is hypothesized that early in the exercise period fast glycolytic fibers fatigue as they are unable to regenerate atp and as a result they enter into a rigor or high stiffness state . Subsequent stretching of the stiff fibers mechanically disrupts the fibers resulting in cytoskeletal and myofibrillar damage . There is also evidence that in eccentric exercised muscles the intracellular concentration of calcium is increased, probably because of disruption of the sarcoplasmic reticulum . As we have seen before high concentrations of furthermore, increased ca has the potential for activating several mechanisms that leads to further damage of the cell membrane and cytoskeletal disruption . In contrast, hocking has postulated that eccentric loading does not provide a good model for the trp pathogenesis . He suggested that sustained partial depolarization or plateau depolarization of an -motor neuron dendrites leads to lasting alterations in the function of the entire -motor neuron due to an upregulation of l- or n- type voltage dependent calcium channels and 1-adrenergic receptors and a downregulation of calcium - activated potassium channels, which would lead to an increase in the motor terminal cytosolic calcium ion concentration (personal communication, 2012). In other words, according to hocking, the increased ach release would be the cause and not the result of the energy crisis . Alpha-1-adrenergic receptors are linked to l - type voltage dependent calcium channels, which would suggest that sympathetic activity would increase the cytosolic calcium ion concentration and the excessive release of ach . Rather than looking at overuse mechanisms, hocking maintains that persistent nociceptive input causes the formation of trps through central sensitization of the c - fiber nociceptive withdrawal reflex and plateau depolarization of withdrawal agonist alpha - motor neurons and compensatory reticulospinal motor facilitation of antigravity muscles and plateau depolarization of withdrawal antagonist alpha - motor neurons . Eccentric contractions occur during all activities of daily life, but have been researched especially in sports . Overhead throwing, spiking in tennis and volleyball, javelin - throwing, downhill running, jumping and running (landing phase) involve eccentric activities . During the deceleration phase of throwing, the posterior shoulder muscles such as the infraspinatus, teres minor and major, posterior deltoid, and latissimus dorsi muscles, contract eccentrically not only to decelerate horizontal adduction and internal rotation of the arm, but also to resist shoulder distraction and anterior subluxation forces . A shoulder compressive force slightly greater than bodyweight is generated to resist shoulder distraction, while a posterior shear force of 40% 50% bodyweight is generated to resist shoulder anterior subluxation . Consequently, high activity is generated by the posterior shoulder musculature, in particular the rotator cuff muscles . The percentage of maximum voluntary isometric contraction (mvic) is calculated in studies using needle electromyography . For example, the teres minor exhibits its maximum activity (84% of mvic), the infraspinatus 37%, supraspinatus 51%, posterior deltoid 69%, latissimus 88%, subscapularis 115%, and triceps 89% during the deceleration phase . Scapular muscles also exhibit high activity to control scapular elevation, protraction, and rotation during this phase . The lower trapezius muscle, which exerts force on the scapula in the direction of depression, retraction and upward rotation, generated its highest activity during the eccentric phase [35]. High emg activity from glenohumeral and scapular musculature during eccentric contraction illustrates the vulnerability of the posterior musculature for developing trps in the overhead - throwing athlete . Posterior shoulder pain is a common complaint of throwing athletes and is mostly explained by posterior impingement [36, 37]. Interestingly, stretching exercises have been shown to assist in reducing the athlete s pain and to normalize shoulder motion, particularly shoulder internal rotation and horizontal adduction . It is common for the overhead thrower to exhibit loss of internal rotation of 20 or more, referred to as glenohumeral internal rotation deficit (gird). This internal rotation deficit has been suggested to be a cause of specific shoulder injuries . Wilk expressed that loss of internal rotation is most often due to osseous adaptations of the humerus and posterior muscle tightness, and not posterior capsular tightness . Stretching exercises, including the sleeper s stretch and supine horizontal adduction with internal rotation, are advised to improve the flexibility of the posterior musculature, which may become tight because of the muscle contraction during the deceleration phase of throwing . Trps can be found in many sportsmen, including elite volleyball players, swimmers, and runners [3941]. In spite of a lack of well - designed studies, the best available evidence supports that trps develop after muscle overuse . Several potential mechanisms may play a role, such as eccentric overload, submaximal sustained, and (sub)-maximal concentric contractions . A key factor is the local ischemia, which leads to a lowered ph and a subsequent release of several inflammatory mediators in muscle tissue . Whether overuse mechanisms are the crucial initiating factor or persistent nociceptive input remains a point of debate and further study.
Pilomatricoma also known as benign calcifying epithelioma of malherbe is an uncommon slow growing benign adnexal skin tumor, which differentiates towards the hair matrix ., we report a case of pilomatricoma with bullous appearance in a young female which is rare, and also review the literature on bullous pilomatricoma . A 17-year - old girl presented with a solitary reddish semi - transparent blister over her right upper arm since 3 months . Initially, the lesion started as a solid pin - head sized papule, which gradually progressed to form a semi - transparent blister with a hard nodule inside . No history of similar lesions in the past or in family members . On examination, single 2 cm sized erythematous semi - transparent bullae with a white nodule was seen on the medial aspect of the right upper arm [figure 1a and b]. On palpation, the bullae had a jelly like consistency and the nodule underneath was hard . On puncturing the lesion, it oozed a jelly like material . (a and b) 2 cm sized erythematous semi - transparent bullae with a white nodule inside photograph of a bouncy ball routine investigations were normal and excision biopsy was carried out [figure 3]. Histopathology revealed islands of basaloid cells with central abrupt keratinization and dilated lymphatics with dermal edema [figures 4 and 5a]. Ghost cells (shadow cells) [figure 5b], foreign body giant cell reaction [figure 5c], and small foci of calcification were also seen [figure 5d]. With these findings, gross specimen of excised material histopathological features of excision biopsy showing islands of basaloid cells with central abrupt keratinization, shadow (ghost) cells, and dilated lymphatics with dermal edema (h and e, 10) (a) basaloid cells undergoing abrupt keratinization; (b) ghost cells / shadow cells; (c) foreign body giant cell reaction; (d) small foci of calcification (h and e, 40) pilomatricoma is a benign tumor considered to be a hamartoma of the hair matrix composed of cells resembling those of the hair matrix, cortex, and inner root sheath . It makes up around 20% of all hair follicle - related tumors and is the commonest hair follicle tumor . Majority of patients are under 20 years of age, and females are affected more often than males . It is usually a solitary, deep dermal or subcutaneous tumor 3 - 30 mm in diameter situated on the head, neck or upper extremities . Clinical variants include, large (giant), extruding or perforating lesions, ulcerative lesions, multiple eruptive cases, familial cases, superficial pilomatricoma presenting as a cutaneous horn and bullous pilomatricoma . Association with myotonic dystrophy, gardner's syndrome, turner's syndrome, and rubinstein taybi syndrome have been reported with pilomatricoma . However, no such associations with bullous pilomatricoma has been reported . Reports on bullous variant of pilomatricoma is sparse, and only 17 cases have been reported worldwide until date out of which only two cases are reported from india . Bullous pilomatricoma has a predilection for occurring on the shoulder and upper arm regions, predominantly in females . It can also be found on the neck, trunk, eyelid, and scalp . The peak age of presentation is 10 - 20 years, and mostly of sizes 1 - 3 cm . Usually presents as a solitary, flaccid, thick - walled red bullae with an underlying palpable hard nodule . Our present case is consistent with these reports . Since our case resembled a bouncy ball, we would like to coin this as the bouncy ball sign in bullous pilomatricoma . Histopathological hallmarks of pilomatricoma are the tumor nests of basophilic cells and eosinophilic shadow cells . Bullous variant in addition to these findings, show dilated lymphatic vessels, giant cell reaction, lymphoedema, disruption of collagen fibers, dilated blood vessels, fibrous capsule, calcification, nests of transitional cells, and necrosis . Our case is consistent with these findings which showed islands of basaloid cells with central abrupt keratinization, shadow cells, dilated lymphatics with dermal edema, foreign body giant cell reaction and small foci of calcification . It could be due to mechanical irritation, or it could be a pseudoblister since the bullae - like spaces are filled with lymphatic fluid . Another theory, suggests that the tumor cells or infiltrating inflammatory cells produce elastinolytic enzymes which disrupt the collagen fibers and destruct and dilate the lymphatic vessels, which lead to accumulation of lymph fluid in the dermis causing a bullae . However, the widely accepted theory is that obstruction of lymphatic vessels and congestion of lymphatic fluid caused by the growth of tumor nodule causes dilatation of lymphatic vessels, leakage of lymphatic fluid, and edema in the dermis surrounding the tumor resulting in the bullous appearance . Malignant change (pilomatricarcinoma) is recorded in several cases, and appears to arise chiefly in large pilomatricomas that have been present for a long time ., dermatologists should be aware of this rare variant of pilomatricoma and excision biopsy is the norm for diagnosis and treatment of this condition.
Our patient, a 60-year - old woman, originally presented to her primary care physician for pelvic discomfort in september 2006 . At that time, she underwent a pelvic ultrasound as well as a computed tomography (ct) scan of the abdomen and pelvis, which showed a complex ovarian mass . A pap smear performed one month later was positive for atypical glandular cells suspicious for adenocarcinoma . In november 2006, she underwent a total abdominal hysterectomy with bilateral salpingoophorectomy, omentectomy, and periaortic lymphadenectomy . Upon intraoperative exam, her surgeons noted involvement of the omentum and appendix, as well as studding of the small bowel mesentery and right diaphragm . The pathologic specimen showed extension of the tumor throughout the fallopian tubes, appendix, omentum, and 5 out of 5 positive lymph nodes . The patient underwent placement of an intraperitoneal catheter and an intravenous port - a - cath for initiation of chemotherapy in december 2006 . The remainder of the patient's past medical history is noncontributory as she was previously in excellent health prior to the diagnosis of ovarian cancer . Unfortunately, she suffered a hypersensitivity reaction to the taxol, and was therefore switched to carboplatin and abraxane . She received a total of 8 cycles of that combination, with only 1 cycle postponed secondary to neutropenia . Just before the 8th cycle, in order to assess her response to treatment, she underwent a ct scan of the chest, abdomen, and pelvis . In addition, her ca-125 level, which had previously reached a plateau of 30 u / ml, had risen to 123 u / ml . Concerned about the possibility of drug - resistant disease, she was evaluated for enrollment in a trial of avastin and tarceva . She underwent a new ct scan of the chest, abdomen, and pelvis, in order to obtain baseline data for the trial (3 months after her previous ct scan). The new ct scan showed interval development of right axillary lymphadenopathy; the largest lymph node was 1.1 x 1.8 cm and suspicion of a new primary breast cancer was raised . We proceeded with breast magnetic resonance imaging (mri) with gadolinium which showed no suspicious lesions or masses . However, she developed a significant rash in association with these drugs and required dose reduction . In october 2007, almost a year after her initial ovarian cancer diagnosis, the patient reported the new - onset of right breast edema . Although she had been previously followed for the right axillary lymphadenopathy, she had recently noticed an increase in erythema, thickness, and warmth of the skin of her right breast [fig 1, 2]. She was treated with a 10-day course of antibiotics, with no change in symptoms . She then underwent an ultrasound of her breast that showed an ill - defined hypoechoic area in the right upper outer quadrant with multiple enlarged lymph nodes . A subsequent mammogram showed scattered fibroglandular densities and an area of architectural distortion with a few small punctate calcifications . Her gynecologic oncologist performed a fine - needle aspiration of the breast, which showed cells consistent with adenocarcinoma . She then underwent a second bilateral breast mri, which confirmed the presence of an area of heterogeneous enhancement measuring 8 x 4 cm, highly suggestive of cancer, with areas suspicious for tumoral extension to the chest wall [fig 3, 4]. Because of these findings, the patient was referred to a breast surgical oncologist, who performed a punch biopsy of her right breast . Pathologic analysis showed multiple foci of high - grade adenocarcinoma with dermal lymphatic invasion, with morphology similar to that of the previous ovarian cancer [fig 5]. Furthermore, the breast tissue specimen immunohistochemistry results were positive for ca-125, but negative for estrogen receptor (er), progestin receptor (pr), and the her-2/neu oncoprotein . Upon review by our institution's multidisciplinary tumor board, it was concluded that these results were consistent with an ovarian primary tumor . Breast cancer is one of the most common primary malignancies in women, yet metastatic tumors to the breast are infrequent, accounting for only 0.5% to 1.3% of breast cancer cases 1 . The most common source of metastasis to the breast is a contralateral primary breast tumor, frequently from transthoracic or lymphatic spread . A study by hadju and urban involving 4,051 breast cancer patients found an overall incidence of primary gynecologic cancers metastatic to the breast of 0.17%, with only 0.07% of metastatic disease originating from a primary ovarian tumor 3 . The first case report of ovarian cancer with metastasis to the breast was in 1907 by sitzenfrey 4 . To date, a total of only 39 such cases have been reported in the english - language literature 5 . Ovarian metastasis to the breast mimicking primary inflammatory breast carcinoma is even more infrequent, with only 6 previous cases reported [table 1]. Inflammatory metastasis to a single breast was first reported by ibach in 1964 6, followed by 5 other case reports 2, 4, 7, 8, 9 . Of note, the most recent patients, including ours, all had a diagnosis of stage iiic papillary serous adenocarcinoma . In contrast to primary breast tumors, metastasis to the breast generally consists of firm, well - circumscribed, multinodular masses . In addition, the masses are usually superficial and less fixed to surrounding tissues, with the overlying skin generally of normal consistency . The most common form of clinical presentation (in 85% of patients) was a solitary tumor; only 4% of patients had diffuse involvement 4 . Furthermore, the most common location was the upper outer quadrant in 62% of patients 3 . Metastatic tumors to the breast more frequently present as well - circumscribed, non - calcified dense masses . They generally lack spiculation and microcalcifications as well as architectural distortion and other skin changes . However, because of the presence of psammoma bodies associated with some ovarian cancers, microcalcifications can be seen with ovarian metastasis 3, 10 . Breast metastasis from a primary ovarian tumor, however, commonly lacks a characteristic pattern, may be morphologically indistinguishable from its primary, and is associated with widespread dissemination . In addition, breast metastasis from a primary ovarian tumor generally is diagnosed an average of 2 years after the initial diagnosis of ovarian cancer 2 . Our patient developed a markedly diffuse inflammatory process of her right breast with features consistent with inflammatory breast cancer within 1 year after her initial diagnosis . Histopathological analysis of breast and ovarian tumors can yield similar results . Therefore, accurate differentiation is necessary, because treatment and prognosis differ significantly . The most common histologic variant of ovarian cancer associated with metastatic disease to the breast is papillary serous adenocarcinoma 3 . Immunohistochemistry may provide additional insight into the origin of the tumor . By combining the tumor markers oc125 and ov632, yamaski et al . Found a sensitivity of 86% and a specificity of 89% for the diagnosis of metastatic ovarian cancer 10 . Secondary breast involvement from an ovarian tumor suggests widespread dissemination and is associated with a poor prognosis . According to several studies, after the detection of metastatic breast disease secondary to an ovarian primary tumor, survival times ranged from 13 days to 3.5 years 2, with most patients dying within 1 year 11 . Another study found a 1-year survival rate of 40% for patients with ovarian cancer who also had breast metastasis, as opposed to a 4-year survival rate of 75% for patients with primary breast cancer 12 . Inflammatory metastatic disease to the breast also confers a grave prognosis: patient survival ranges from 3 to 18 months (median, 6 months) after diagnosis of the metastasis to the breast [table 1]. Extramammary tumors should be distinguished from primary breast tumors to avoid any unnecessary surgical procedures . Correct diagnosis is vital: surgical interventions for patients with secondary breast cancers are potentially both diagnostic and palliative . Ovarian metastasis to the breast should be treated as a systemic disease, with appropriate chemotherapeutic agents . Mastectomy of the breast mass is likely best reserved for patients who are unresponsive to systemic therapy and require palliation 11 . Although ovarian metastasis to the breast presenting as inflammatory breast cancer is rare, it should be included in the differential diagnosis for any patient with a personal history of ovarian cancer . Accurate differentiation is necessary because treatment differs significantly for patients with ovarian metastasis to the breast, as compared with patients with primary inflammatory breast cancer . Ovarian metastasis to the breast confers a poor prognosis: patient survival ranged from 3 to 18 months, with a median survival of 6 months after the diagnosis of the breast metastasis.
National medicaid outpatient drug expenditure and utilization data are available from two sources: cms' medicaid statistical information system (msis), and the medicaid drug rebate program data files . Since 1999, all states are required to submit electronically certain fields of claims data from their medicaid claims processing systems to cms' msis . Data files containing utilization and expenditure data on prescribed drugs from the drug rebate program beginning in federal fiscal year (ffy) 1996 can be accessed at internet address: http://www.cms.hhs.gov/medicaid/msis/msis99sr.asp . Outpatient drug expenditure, but not utilization data, are also available through the quarterly medicaid statement of expenditures (cms-64). Figures 1 and 2 display trends expenditure data on medicaid prescribed drugs under ffs payment arrangements . Between ffys 1985 and 2001, medicaid ffs expenditures for prescribed drugs increased from $2.3 to $24.7 billion, approximately a 10-fold increase . During the same period, medicaid ffs expenditures for all service types increased from $37.5 to $216.2 billion, slightly less than a 6-fold increase . Between ffys 1997 and 2001, medicaid ffs prescribed drug expenditures nearly doubled from $12.4 to $24.7 billion . During the same period, total medicaid expenditures increased from $160.3 to $216.2 billion, a growth of 35 percent . In ffy 1985, prescribed drug expenditures in ffs comprised about 6 percent of total ffs medicaid expenditures . By ffy 2001, in that same year, the medicaid ffs program provided pharmaceuticals to over 20 million beneficiaries . Twenty - two percent of the increase in total medicaid expenditures between ffys 1997 and 2001 was accounted for by increases in expenditures for ffs prescribed drugs . Figure 3 displays ffs medicaid prescribed drug expenditures by age in ffy 2000, and figure 4 shows the percent change by age in ffs medicaid drug expenditures between ffys 1999 and 2000 . Beneficiaries age 65 or over accounted for the largest share of total medicaid ffs prescribed drug expenditures in ffy 2000 (32 percent of the total). Beneficiaries age 45 - 64 accounted for 30 percent of the total medicaid ffs prescribed drug expenditures followed by beneficiaries age 21 - 44 (26 percent of total), and beneficiaries age 0 - 20 (12 percent of the total). Between ffys 1999 and 2000, medicaid ffs expenditures for prescribed drugs increased by 20 percent for the age group 65 or over . Age group 45 - 64 beneficiaries experienced the greatest increase in medicaid ffs prescription drug expenditures between ffys 1999 and 2000 (26 percent) followed by the age group 0 - 20 (18 percent), and the age group 21 - 44 (18 percent). The following information relates to the medicaid ffs population for ffy 2001 (october 1, 2000-september 30, 2001), unless otherwise stated (figure 5). This section looks at the 10 states with the highest ffs prescribed drug expenditures in ffy 2001 . Prescription payment data in medicaid ffs includes services to individuals in primary care case management . The composition and characteristics of the beneficiaries vary by state, in large part because the proportion of beneficiaries in capitated m+c varies by state . Because most beneficiaries in capitated m+c are children and non - elderly adults, states with predominantly ffs populations will have a disproportionate number of elderly medicaid beneficiaries included in the data reported . There were $14.1 billion in ffs pharmaceutical expenditures for the top 10 states in ffy 2001 . The $14.1 billion figure represented 57 percent of the total ffs medicaid prescribed drug expenditures for that year . California had the greatest amount of prescribed drug expenditures at $3.1 billion followed by new york at $2.9 billion . Florida was third in expenditures at $1.5 billion, slightly more than one - half of that of california . Missouri was tenth in expenditures at $0.5 billion, less than one - fifth that of the top two states, california and new york . Figure 6 shows the relationship between percent of the medicaid population who were (age 65 or over) by state, and the average number of prescriptions per medicaid beneficiary . Figure 7 demonstrates the average number of claims per ffs beneficiary for the top 10 states . The number of pharmacy beneficiaries listed represents medicaid enrollees who received at least 1 prescription through a state's ffs program . New jersey had the highest average number of prescriptions per beneficiary in 2001 at 33, followed by ohio at 28, and massachusetts at 23 . There appears to be a relationship between percent aged in the medicaid population and ffs pharmacy utilization (figure 8); however this relationship is not necessarily a simple linear one . Medicaid ffs beneficiaries (age 65 or over) accounted for 14 percent of the population and 33 percent of the expenditures . New jersey had both the highest average number of prescriptions per beneficiary and the highest percent aged in the medicaid population among the top 10 states . Texas was lowest in average claims per beneficiary and third lowest in percent aged among the top 10 states . The medicaid drug rebate program operates according to the statutory requirements found in section 1927 of the social security act (social security administration, 2003). States invoice pharmaceutical manufacturers for both innovator or brand - name drugs, as well as non - innovator or generic drugs . While other states have established state supplemental rebate programs, california is the only state that showed an appreciable collection in ffy 2001 . During that year, the state of california collected additional state rebate amounts that totaled 7 percent of their entire pharmacy expenditure (figure 8). In ffy 2001, nationwide, the federal rebate program collected approximately $4.9 billion . The average payment per prescription nationwide was roughly $55 without the rebate . When the federal rebate program is taken into consideration, the average cost per ffs medicaid prescription nationwide was reduced by approximately 20 percent to $44.
In october 2004, a 3-month - old kitten (felis domesticus) was adopted from the society of the prevention of cruelty to animals (east london, eastern cape province, south africa) and lived with its owner on a farm 23 km outside the city . It had been neutered and had been vaccinated at 10 months of age with an adjuvanted inactivated vaccine against rabies (rabisin; merial, lyon, france), but no subsequent vaccinations were given . The cat spent most of the day indoors, but went out at night and returned in the morning . It appeared dull and physically unbalanced and its pupils were dilated but it was not aggressive . The cat was humanely killed, and its brain was sent to the onderstepoort veterinary institute for rabies testing . On june 17, 2005, a 6-month - old puppy (canis familiaris) was brought by its owner to a veterinarian in the rural town of nkomazi (mpumalanga province, south africa). After symptoms were treated, the dog was discharged, but it was brought back 11 days later because it was paralyzed, dehydrated, and had a fixed stare . The dog was humanely killed, and its brain was sent to the onderstepoort veterinary institute for rabies testing . Direct immunofluorescent antibody test with an anti - rabies conjugate cross - reactive with african lyssaviruses showed numerous and strongly stained inclusion bodies in every field of impression smears of both brain samples . Isolation of virus was attempted by suckling mouse brain passage and cell culture (neuroblastoma cells; diagnostic hybrids, athens, oh, usa); both methods were successful for the cat sample . However, neither method yielded an isolate from the dog sample, despite a lyssavirus - specific reaction in the original brain sample by direct immunofluorescent antibody test . Subsequently, antigenic characterization was performed with a panel of 16 monoclonal antibodies to the nucleocapsid protein of rabies virus (canadian food inspection agency, nepean, ontario, canada). Gt, genotype; +, positive reactivity; var, reactivity with some regional variants;, negative reactivity . Final confirmation of mokv in both case samples was obtained by reverse transcription pcr, nucleotide sequencing, and phylogenetic analysis as described (12). Phylogenetic analysis (figure) showed that the virus isolated from the cat sample (designated mokv173/06) belonged to the same lineage of mokv isolates that were recovered from cats in the same region of south africa (12). However, the virus detected in the dog sample (designated mokv404/05) appeared to represent a different south african mokv lineage that was phylogenetically positioned between known south african and zimbabwean lineages . This mokv had nucleotide similarities of 88.1%90.4% and 85.3%88.5% with viruses from zimbabwe and south africa, respectively . Phylogenetic tree based on 267 nt of partial nucleoprotein gene sequences of moloka virus (mokv) identified with the n1-n2 primer set as described (12). The tree shows phylogenetic positions of 2 recently identified cases of mokv infection from south africa (mokv173/06 from a cat and mokv404/05 from a dog) (in boldface) relative to previously characterized mokv isolates from south africa (sa) and zimbabwe (zim) and lagos bat virus (lbv) as the outgroup . Infections with mokv are rare; only 23 isolates are known . During the past 2 decades, because these viruses are not exclusive to south africa (212), lack of isolates from other regions of africa indicates a lack of active surveillance and limited diagnostic capabilities in many african laboratories . To our knowledge, the 2 cases of infection with mokv we report are the first in 8 years from south africa . Clinical signs in the dog and cat, including general neurologic manifestations with a lack of aggression, are often signs that warrant submitting samples for rabies testing . Whether these variations are caused by neutral genetic drift or reflect different epidemiologic features is not known . Phylogenetically, divergence of these viruses into different lineages indicates active cycles and evolutionary changes that occur independently, but in close proximity (a few hundred kilometers apart). Although the epidemiology of mokv is incomplete, the case for a reservoir host(s) among small terrestrial animals of limited range together with recent isolations of rabies - related lyssaviruses in a human (13) and wild animals (14,15), these reports emphasize the endemicity of these lyssaviruses in south africa . Public health implications of african rabies - related lyssaviruses should be recognized by laboratory workers, researchers, veterinarians, wildlife personnel, gamekeepers, and pet owners . A better understanding of the epidemiology of these viruses is vital and can only be achieved by improved surveillance and awareness.
Lumbar spinal stenosis (lss) is a common cause of low back pain and lower extremity pain in older adults and is responsible for 1.2 million physician office visits each year in the us.1 degenerative lss is characterized by narrowing of the spinal canal as a result of bony and intraspinal soft tissue degeneration, including intervertebral disc herniation, facet joint and ligamentum flavum hypertrophy, and/or osteophyte formation.2 lss may involve the central spinal canal, lateral recess, or foramina, with affected areas frequently coexisting in the elderly.3 the characteristic clinical manifestations of lss include periodic exacerbations of low back and leg pain and neurogenic claudication,4 with resulting compromise in health - related quality of life, mobility, and independence.5 the burden of lss is expected to rise concomitant with quadrupling of the number of persons 60 years and older by the year 2050 . Nonoperative treatment including limitation of activity, posture modification, and epidural steroid injections, are useful for lss symptoms of mild - to - moderate severity.6 decompressive surgery is indicated for severe neurogenic claudication, neural compromise, and/or intractable radicular symptoms . Surgical decompression of symptomatic lss is the most common indication for low back surgery in patients over 65 years of age.7 decompression with spinal fusion is utilized as the primary treatment in selected lss patients, even in the absence of gross instability, in part because of the limitations of traditional decompression tools . The effectiveness of decompression surgery for the treatment of lateral recess or foraminal stenosis is hindered by the difficulty associated with gaining access to these areas using standard rigid instrumentation, such as kerrison ronguers, without partial or total resection of the posterior elements . Resection of the facet joints and midline structures during decompression surgery increases the risk of spinal instability, which may eventually necessitate revision surgery with fusion.8,9 a minimally invasive approach that selectively targets the lateral recess and foramen with preservation of the posterior elements may be useful in reducing perioperative complications and minimizing the need for revision surgery after lumbar decompression . The io - flex system (baxano inc, san jose, ca) utilizes a flexible, over - the - wire microblade shaver instrument designed to allow for direct decompression of impinging tissue, particularly in the lateral recess and foramen, while avoiding disruption of the pars interarticularis, the facet joints, and the midline structures . The purpose of this feasibility study was to quantify changes in lumbar spinal anatomy using computed tomography (ct) following decompression with the io - flex system versus hemilaminotomy with foraminotomy (hl) in cadaveric lumbar specimens . This study was conducted using 36 lumbar motion segments in nine human cadaveric lumbar (l1 to sacrum) specimens . Six specimens (mean cadaveric age 78 years) exhibited age - appropriate degenerative changes and three specimens (mean cadaveric age 87 years) had ct - verified degenerative lss with moderate - to - severe multilevel foraminal stenosis . The cranial portion of l1 and the caudal sacrum were stripped of muscle and adipose tissue and secured in glass - reinforced filler (bondo; 3 m bondo, atlanta, ga). Each specimen was then fixed in an acrylic frame in the neutral position and scanned intact using thin - slice (0.75 mm) axial ct (somatom sensation 4; siemens, munich, germany). After completion of the ct scan, the specimen was removed and bilateral tissue exposure of the l2s1 lamina was performed by one of five experienced surgeons (three neurosurgeons and two orthopedic spinal surgeons). All procedures on a given specimen were performed using a consistent technique by the same surgeon in order to minimize bias . The decompression procedure, ie, io - flex system or standard hl, was randomly selected and performed for the first level and the alternate procedure was performed next on the contralateral side for each level from l2/3 to l5/s1 . Surgeons aimed to remove the central ligamentum flavum and any excess bone and soft tissue in the lateral recess and foramen that would typically be expected to contribute to lss symptoms, while sparing as much of the nonoffending structures as possible (eg, lamina, pars, and facet joints). A total of 36 levels were treated (six nondiseased and three stenotic specimens, with four treated levels each). The io - flex system is a set of over - the - wire instruments that allows ventral - to - dorsal decompression of impinged neural elements in the lumbar spine while sparing uninvolved bone and soft tissue with controlled bimanual reciprocations . Specifically, the microblade shaver instrument allows for inside - out decompression by removing the ligamentum flavum and shaving bony overgrowth on the superior articular process and under the pars interarticularis, while requiring minimal resection of the facet joints and midline structures . Up to four nerve roots (traversing and exiting roots on the ipsilateral and contralateral side) may be decompressed via a single interlaminar access point using the microblade shaver (figure 1). The procedural steps for the io - flex system consist of an io - flex probe to gain access, the neuro check device for localization of neural structures, and the microblade shaver instrument for targeted tissue removal and decompression (figure 2). In this study, surgeons typically performed a small ipsilateral laminotomy to create an interlaminar window just large enough to remove the central ligament, directly visualize the dura, and to fit the microblade shaver instrument so that it could be used to remove bone and soft tissue in the lateral recess and foramen . Additional structural anatomy was not removed because, in a clinical setting, the neuro check device is used to confirm positions dorsal to the lateral neural structures without direct visualization of the traversing root and because the flexible nature of the shaver allows for decompression around the facet joints without their removal . After the laminotomy, the probe, with its cannulated catheter in the retracted position, is passed out of the neural foramen, just rostral to the caudal pedicle . The position of the probe inside the foramen is confirmed using lateral fluoroscopy and the inner catheter is then deployed . A nitinol wire is passed through the probe and out through the dorsal skin where it is locked into a distal handle . The catheter is then retracted and the probe removed, leaving the wire in place . In a clinical setting, the neural localization step with the neuro check device would follow next, but was not performed in this cadaver study . The microblade shaver instrument is then attached to the wire via a proximal exchange tip and pulled into the lateral recess and foramen using a distal handle (figure 3). The dorsal side of the instrument has small cutting teeth designed to excise bone and ligament, while the ventral side is smooth to protect the neural structures . Decompression is achieved using gentle upward tension and a bimanual reciprocating motion with the handle of the microblade shaver instrument and the distal handle . Tissue removal and completeness of decompression is assessed using foraminal probes and lateral fluoroscopy (figure 4). Surgeons used a traditional hl technique to decompress the neural elements adequately.10,11 traditional surgical instruments, such as spinal curettes, high speed burrs, tissue, and kerrison rongeurs were used . After the laminae were exposed, bone from the inferior edge of the superior lamina as well as the superior edge of the inferior lamina was removed . Kerrison rongeurs were used to decompress further laterally by removing the medial aspect of the facet . Visual inspection and standard foraminal probes were used throughout the procedure to assess the amount of space in the neural foramen and to determine when the decompression was complete . Surgeons typically removed significant portions of the lamina and the facet joints in order to visualize the shoulder of the traversing nerve root for safety and due to the constraints of rigid instruments, such as kerrison rongeurs, in attempting to access impinging tissue in the lateral recess and foramen . However, each surgeon carefully balanced the tradeoff between the ability to reach this impinging tissue with the desire to spare the structural anatomy for stability . For nondiseased specimens, ct scans were evaluated by an independent core laboratory (medical metrics, houston, tx) using validated quantitative image analysis software.12 all measurements were taken from a digital composite of the pretreatment and post - treatment scans, an automated technique that reflects only the change attributable to treatment by eliminating the error arising from manual selection of anatomical landmarks on separate examinations . Reconstructed left and right parasagittal images were used to measure anteroposterior foramen width and area (figure 5). Reconstructed mid - disc axial images were used to measure the spinal canal area, soft tissue canal area, ligamentum flavum area, laminar width (medial to lateral distance), lateral recess diameter (anterior - posterior diameter of lateral recess), and facet width (medial to lateral articulation distance), as shown in figure 6 . Joint (articulation) cross - sectional area was measured from a plane bisecting the facet joint at each level . The magnitude of laminar removal was measured as a change in width from an axial slice or as a change in area from a coronal slice . Stenotic specimens were evaluated by an independent neuroradiologist using identical ct image analysis software . Following initial analysis of nondiseased lumbar specimens, only the most clinically relevant outcomes were preselected for analysis in the stenotic specimens . The wilcoxon signed - rank test was used to assess anatomical changes before and after each procedure, as well as to compare anatomical changes between treatments on paired sides of each treated level . This study was conducted using 36 lumbar motion segments in nine human cadaveric lumbar (l1 to sacrum) specimens . Six specimens (mean cadaveric age 78 years) exhibited age - appropriate degenerative changes and three specimens (mean cadaveric age 87 years) had ct - verified degenerative lss with moderate - to - severe multilevel foraminal stenosis . The cranial portion of l1 and the caudal sacrum were stripped of muscle and adipose tissue and secured in glass - reinforced filler (bondo; 3 m bondo, atlanta, ga). Each specimen was then fixed in an acrylic frame in the neutral position and scanned intact using thin - slice (0.75 mm) axial ct (somatom sensation 4; siemens, munich, germany). After completion of the ct scan, the specimen was removed and bilateral tissue exposure of the l2s1 lamina was performed by one of five experienced surgeons (three neurosurgeons and two orthopedic spinal surgeons). All procedures on a given specimen were performed using a consistent technique by the same surgeon in order to minimize bias . The decompression procedure, ie, io - flex system or standard hl, was randomly selected and performed for the first level and the alternate procedure was performed next on the contralateral side for each level from l2/3 to l5/s1 . Surgeons aimed to remove the central ligamentum flavum and any excess bone and soft tissue in the lateral recess and foramen that would typically be expected to contribute to lss symptoms, while sparing as much of the nonoffending structures as possible (eg, lamina, pars, and facet joints). A total of 36 levels were treated (six nondiseased and three stenotic specimens, with four treated levels each). The io - flex system is a set of over - the - wire instruments that allows ventral - to - dorsal decompression of impinged neural elements in the lumbar spine while sparing uninvolved bone and soft tissue with controlled bimanual reciprocations . Specifically, the microblade shaver instrument allows for inside - out decompression by removing the ligamentum flavum and shaving bony overgrowth on the superior articular process and under the pars interarticularis, while requiring minimal resection of the facet joints and midline structures . Up to four nerve roots (traversing and exiting roots on the ipsilateral and contralateral side) may be decompressed via a single interlaminar access point using the microblade shaver (figure 1). The procedural steps for the io - flex system consist of an io - flex probe to gain access, the neuro check device for localization of neural structures, and the microblade shaver instrument for targeted tissue removal and decompression (figure 2). In this study, surgeons typically performed a small ipsilateral laminotomy to create an interlaminar window just large enough to remove the central ligament, directly visualize the dura, and to fit the microblade shaver instrument so that it could be used to remove bone and soft tissue in the lateral recess and foramen . Additional structural anatomy was not removed because, in a clinical setting, the neuro check device is used to confirm positions dorsal to the lateral neural structures without direct visualization of the traversing root and because the flexible nature of the shaver allows for decompression around the facet joints without their removal . After the laminotomy, the probe, with its cannulated catheter in the retracted position, is passed out of the neural foramen, just rostral to the caudal pedicle . The position of the probe inside the foramen is confirmed using lateral fluoroscopy and the inner catheter is then deployed . A nitinol wire is passed through the probe and out through the dorsal skin where it is locked into a distal handle . The catheter is then retracted and the probe removed, leaving the wire in place . In a clinical setting, the neural localization step with the neuro check device would follow next, but was not performed in this cadaver study . The microblade shaver instrument is then attached to the wire via a proximal exchange tip and pulled into the lateral recess and foramen using a distal handle (figure 3). The dorsal side of the instrument has small cutting teeth designed to excise bone and ligament, while the ventral side is smooth to protect the neural structures . Decompression is achieved using gentle upward tension and a bimanual reciprocating motion with the handle of the microblade shaver instrument and the distal handle . Tissue removal and completeness of decompression is assessed using foraminal probes and lateral fluoroscopy (figure 4). When the surgeon deems that decompression is complete, surgeons used a traditional hl technique to decompress the neural elements adequately.10,11 traditional surgical instruments, such as spinal curettes, high speed burrs, tissue, and kerrison rongeurs were used . After the laminae were exposed, bone from the inferior edge of the superior lamina as well as the superior edge of the inferior lamina was removed . Kerrison rongeurs were used to decompress further laterally by removing the medial aspect of the facet . Visual inspection and standard foraminal probes were used throughout the procedure to assess the amount of space in the neural foramen and to determine when the decompression was complete . Surgeons typically removed significant portions of the lamina and the facet joints in order to visualize the shoulder of the traversing nerve root for safety and due to the constraints of rigid instruments, such as kerrison rongeurs, in attempting to access impinging tissue in the lateral recess and foramen . However, each surgeon carefully balanced the tradeoff between the ability to reach this impinging tissue with the desire to spare the structural anatomy for stability . For nondiseased specimens, ct scans were evaluated by an independent core laboratory (medical metrics, houston, tx) using validated quantitative image analysis software.12 all measurements were taken from a digital composite of the pretreatment and post - treatment scans, an automated technique that reflects only the change attributable to treatment by eliminating the error arising from manual selection of anatomical landmarks on separate examinations . Reconstructed left and right parasagittal images were used to measure anteroposterior foramen width and area (figure 5). Reconstructed mid - disc axial images were used to measure the spinal canal area, soft tissue canal area, ligamentum flavum area, laminar width (medial to lateral distance), lateral recess diameter (anterior - posterior diameter of lateral recess), and facet width (medial to lateral articulation distance), as shown in figure 6 . Joint (articulation) cross - sectional area was measured from a plane bisecting the facet joint at each level . The magnitude of laminar removal was measured as a change in width from an axial slice or as a change in area from a coronal slice . Following initial analysis of nondiseased lumbar specimens, only the most clinically relevant outcomes were preselected for analysis in the stenotic specimens . The wilcoxon signed - rank test was used to assess anatomical changes before and after each procedure, as well as to compare anatomical changes between treatments on paired sides of each treated level . In nondiseased cadaver specimens, open hl required excision of significantly more bone than did the io - flex system . Hl resulted in removal of 83% greater laminar area (p <0.01), including resection of the structural pars interarticularis and removal of 95% more bone in the central canal (p <0.001) versus the io - flex system . Similarly, in stenotic specimens, hl resulted in significantly more resection of structural posterior elements, with a 54% decrease in laminar width versus 28% when using the io - flex system (p = 0.03, tables 1 and 2). Decreases in facet width were approximately threefold greater in nondiseased (p <0.01) and stenotic (p = 0.03) specimens following hl versus the io - flex system . In nondiseased specimens, facet area was similarly decreased after each treatment (11% versus 14%, p = 0.94). However, decreases in joint cross - sectional area were significantly less following use of the io - flex system (3%) versus hl (7%, p <0.01, tables 1 and 2). In nondiseased cadaver specimens, similar increases in soft tissue canal area (47% and 56%, p = 0.16) and decreases in ligamentum flavum area (79% and 83%, p = 0.94) were observed for the io - flex system and hl, respectively . Lateral recess diameter increased by 33% (p <0.001) in the nondiseased specimens following use of the io - flex system versus a 20% increase (p <0.01) with hl (table 1). Lateral recess diameter changes were particularly pronounced in stenotic specimens following decompression using the io - flex system, with an increase of 43%, which was significantly greater (p = 0.02) compared with hl (7%, table 2). Foraminal width (p = 0.06) and area (p = 0.09) were marginally greater following use of the io - flex system in nondiseased specimens compared with hl (table 1). However, in the stenotic specimens, hl resulted in a nonsignificant 4% increase in foraminal width . In contrast, decompression using the io - flex system resulted in a significant 24% (p <0.001) improvement in foraminal width (p = 0.01 between groups, table 2). In nondiseased cadaver specimens, open hl required excision of significantly more bone than did the io - flex system . Hl resulted in removal of 83% greater laminar area (p <0.01), including resection of the structural pars interarticularis and removal of 95% more bone in the central canal (p <0.001) versus the io - flex system . Similarly, in stenotic specimens, hl resulted in significantly more resection of structural posterior elements, with a 54% decrease in laminar width versus 28% when using the io - flex system (p = 0.03, tables 1 and 2). Decreases in facet width were approximately threefold greater in nondiseased (p <0.01) and stenotic (p = 0.03) specimens following hl versus the io - flex system . In nondiseased specimens, facet area was similarly decreased after each treatment (11% versus 14%, p = 0.94). However, decreases in joint cross - sectional area were significantly less following use of the io - flex system (3%) versus hl (7%, p <0.01, tables 1 and 2). In nondiseased cadaver specimens, similar increases in soft tissue canal area (47% and 56%, p = 0.16) and decreases in ligamentum flavum area (79% and 83%, p = 0.94) were observed for the io - flex system and hl, respectively . Lateral recess diameter increased by 33% (p <0.001) in the nondiseased specimens following use of the io - flex system versus a 20% increase (p <0.01) with hl (table 1). Lateral recess diameter changes were particularly pronounced in stenotic specimens following decompression using the io - flex system, with an increase of 43%, which was significantly greater (p = 0.02) compared with hl (7%, table 2). Foraminal width (p = 0.06) and area (p = 0.09) were marginally greater following use of the io - flex system in nondiseased specimens compared with hl (table 1). However, in the stenotic specimens, hl resulted in a nonsignificant 4% increase in foraminal width . In contrast, decompression using the io - flex system resulted in a significant 24% (p <0.001) improvement in foraminal width (p = 0.01 between groups, table 2). The io - flex system is a minimally invasive, facet - sparing approach that allows for direct decompression of impinging tissue via a ventral - to - dorsal action of the microblade shaver . In contrast, traditional decompression procedures utilize an invasive medial - to - lateral approach that removes a significant portion of the posterior elements at the treated level . Another advantage of the io - flex system is that, unlike open laminectomy that utilizes fixed - angle tools with a limited ability to address lateral recess and foraminal stenosis, the io - flex system uses low - profile flexible instrumentation that targets impinging tissue in the spinal canal, lateral recess, and foramen (figure 7). This cadaver study demonstrated that the io - flex system allows for decompression of the spinal canal with limited resection of structural posterior elements and with selective resection of compressing structures in the lateral recess and foraminal regions . Although the clinical relevance of these study findings is unknown, the io - flex system has been utilized in two clinical studies with data published prior to this current study.13 a postmarket pilot study of nine patients with one - year follow - up demonstrated a median 73% reduction in pain, a 50% improvement in back function, 72% and 31% improvements in zurich claudication questionnaire physical function and symptom severity, respectively, and a 36% improvement in health - related quality of life . A retrospective study of 67 patients treated with the io - flex system for lss reported no reoperations or cases of neurologic impairment through approximately one year post - treatment . Thus far, the cumulative data on the io - flex system support safety and clinical utility, although additional study is required . Although laminectomy is the traditional treatment of choice for patients with recalcitrant lss, long - term outcomes are mixed.14,15 laminectomy is often associated with postoperative pain, disability, and dysfunction, due to extensive resection of muscle, ligament, and bone . Resection of excessive bone from the posterior elements may contribute to subsequent lumbar instability and increased intradiscal pressure by establishing an alternate path of axial loading, transferring forces to the adjacent annulus and anterior longitudinal ligament.16 consequently, disc degeneration may be accelerated following this procedure . Johnsson et al reported that 44% of patients presented with subsequent degenerative spondylolisthesis after decompressive lumbar laminectomy.17 furthermore, sanderson and getty reported an average loss of 1.3 mm of disc height after partial undercutting facetectomy.18 progression of stenotic symptoms following laminectomy is quite common19 and is due, in part, to inadequate decompression,20,21 local tissue trauma, and subsequent adhesion formation.22,23 inadequate decompression of lateral recess stenosis has been shown to be responsible for 25%56% of failed back surgery syndrome cases.2426 ultimately, 11%23% of laminectomy patients undergo reoperation within 10 years.27,28 results from this cadaver study confirmed that traditional decompression has a limited effect in improving lateral recess and foraminal area, especially in stenotic specimens . Decompression surgery using the io - flex system may reduce muscle trauma by allowing bilateral decompression through a single access point and, in theory, may result in a lower degree of destabilization, as seen in traditional decompression surgery . Microdecompression procedures have recently been advocated due to a perceived lower risk of iatrogenic insult . However, these procedures require great technical skill and surgical experience, and evidence for these treatments is limited . In addition, while these procedures can be done via a less invasive exposure, fixed - angle tools still limit the ability to perform facet - sparing bilateral decompression of the lateral recess and foramen . Lumbar extension narrows the spinal canal and lateral recess by approximately 15% compared with a neutral posture,29 and therefore exacerbates lss symptoms . Interspinous spacers limit back extension at the affected level by distracting the spinous processes of the degenerated segment, thereby unloading the posterior annulus fibrosus and facet joints . Despite promising mid - term outcomes, loss of radiographic correction after only 1.5 years is a well established phenomenon with these devices,30 and high clinical failure rates have been reported.31 furthermore, interspinous spacers are only appropriate for patients with mild - to - moderate intermittent neurogenic claudication that is relieved by lumbar flexion and they do not directly address the underlying anatomical cause of pain, such as bony neuroforaminal encroachment and buckled ligamentum flavum . Because lss is a progressive condition, interspinous spacers likely represent only a temporary solution to this disease . Overall, decompression using the io - flex system may have potential advantages over micro - decompression and interspinous spacers, including use of a simple minimally invasive approach and procedure, and the ability to treat patients with severe lss symptoms . First, surgeons were not blinded to the procedures being performed in this study and, therefore, the possibility of intervention bias exists . However, all surgeons were well experienced and used a similar surgical technique, thus minimizing potential bias . Second, despite the noted advantages of the io - flex system in this cadaver study, the results do not necessarily translate into superior clinical outcomes compared with hl, especially given the variable relationship of lss severity and clinical symptoms.32,33 additional prospective human clinical studies are required to elucidate further the safety and effectiveness of this procedure . The first study is evaluating the clinical performance of the io - flex system in patients with one or two levels of lss (nct01067014), and the second study (stride) is evaluating the io - flex system in patients with lss and stable grade 1 spondylolisthesis (nct01338766). The minimally invasive io - flex system yields significantly better decompression of the lateral recess and foraminal area in cadaveric specimens with lss, without extensive removal of the stabilizing posterior spinal elements, including the facet joint, compared with traditional hl.
Phyllodes tumor (pt) is a rare fibroepithelial neoplasm accounting for less than 1% of all breast tumors . Benign pt (bpt) has hypercellular stromal fragments organized in leaflike patterns around benign epithelial / myoepithelial lined spaces . Standardized cytologic diagnostic criteria of bpt includes increased stroma over epithelium, highly cellular stromal fragments, presence of phyllodes fragments (i.e., abundant leaflike stromal fragments), myxoid stroma, and numerous single bare nuclei of spindle fibroblastic type . Diagnostic pitfalls of pt in fine - needle aspiration cytology (fnac) may be due to sampling error, high cellularity, ductal hyperplasia, paucity of stromal component, and occasional dissociation of epithelial cells . Occasionally, only epithelial components of pt is represented in smears and may be misdiagnosed as epithelial neoplasm, particularly tubular adenoma and carcinoma . Here, we present a case of bpt diagnosed by excisional biopsy, but its fnac features were misleading due to predominance of the epithelial component showing a tubular adenoma - like pattern . A 19-year - old female presented with an ill - defined mass in the left breast about (2.5 2) cm above the areola in the breast tissue . On fnac, smear was cellular comprising of the predominance of benign ductal epithelial cells showing prominent tubular adenoma - like arrangement [figure 1] and resetting arrangement [figure 2a]. Large branching sheets [figure 2b] and papillary clusters of ductal cells without a wavy or folded shape were seen as well . Because of diversity in cytological findings, various differentials were thought such as tubular adenoma, adenosis, papilloma, and fibroadenoma . Finally, it was cytologically reported as benign proliferative breast lesion with atypical / indeterminate features (c3 category) as per the cytologic categorization by the national coordinating committee for breast screening and the uk national breast screening program . As c3 category is a grey area in breast fnac diagnosis, further investigation such as excision biopsy duct epithelial cell clusters showing tubular adenoma - like pattern in fnac (h&e stain, 200) (a) duct epithelial cells in sheets showing rosette - like arrangement in fnac (h&e stain, 200) (b) large branching sheets of bland epithelial cells (h&e stain, 200) (c) duct epithelial cells showing focal apocrine metaplasia (h&e stain, 400) excision biopsy specimen grossly consisted of two grey white soft tissue masses measuring 3.5 cm 3 cm 1 cm and 2 cm 2 cm 1 cm . Cut section showed grey white areas . On histology, a well - circumscribed biphasic neoplasm containing leaf - like, epithelial - lined papillary - like projections penetrating into cystic spaces with cellular stroma was seen . Though, cytological features of pt have been well characterized, the cytologic diagnosis of pt remains difficult . This tumor is said to be underdiagnosed by the pathologists and undertreated by the surgeons . Thus, the preoperative diagnosis of this tumor becomes very important to allow correct surgical planning and to avoid reoperation (wide local excision with at least 1 cm margin is currently the treatment of choice for bpt). Pt occurs in women aged 40 - 50 years, however, in asian countries pts may occur at a younger age (average age, 25 - 30 years). Cytologic distinction of bpt from fibroadenoma is possible by noting large epithelial clusters longer than 1 mm, with a wavy or folded shape, in contrast to the small or medium - sized clusters with tubular, blunt - branching, or monolayered contours of fibroadenoma . Krishnamurthy et al . Have observed that hypercellular stromal fragments with the presence of a significant proportion of long spindle nuclei (> 30%) is strongly indicative of pt, whereas the stromal cell nuclei in fibroadenoma tends to be short and oval and long spindle nuclei accounts for 10 - 30% . However, in our case, all the abovementioned features were absent, which made cytologic diagnosis difficult . Standardized criteria for the diagnosis of bpt include at least two large stromal fragments, hypercellular fragments and moderate - to - large number of dissociated stromal cells . Predominance of epithelial elements over stromal elements and monotonous population of cells with tubular adenoma - like and rosetting pattern as seen in this case can mimic epithelial neoplasm . Large branching sheets of bland epithelial cells and focal apocrine metaplasia, seen here as well, closely mimics fibroadenoma . Presence of papillary - like clusters and acinar pattern could be the reasons to consider papilloma and adenosis as other differential diagnosis . We conclude that if there is significant epithelial proliferation in bpt, a false diagnosis of epithelial neoplasm in fnac is possible and it is an important diagnostic pitfall . So, very careful strict criteria should be used such as advised repeat fnac, cell block preparation, biopsy, or correlation with clinical and radiologic findings to improve preoperative diagnosis of bpt . This will help in correct planning of surgical treatment, i.e., wide excision with 1 cm margin.
In the past few years, there has been increasing evidence for the association between periodontitis and cardiovascular diseases . Periodontitis is a chronic inflammatory disease and progresses by bacterial colonization around the tooth, which stimulates immunomodulatory response affecting the periodontium, resulting in bone and tooth loss . Periodontal disease could be a risk factor for the development of angina which has become a major medical problem, with increased mortality rate among the indian population . Smoking is one of the significant risk factors for periodontitis affecting the prevalence, extent, and severity of the disease . Few studies have proven that 15% of systemically healthy population can show elevated levels of antiphospholipid antibodies (apa). In several infectious conditions, increased levels of these antibodies are seen . These apas and its prothrombotic activity are the major hallmark of the pathogenesis of antiphospholipid syndrome (aps). Anticardiolipin antibody (acla) is the primary antibody commonly seen in patients with aps directed against b2-glycoprotein - i - dependent (b2gpi) phospholipid . When b2gpi binds to cardiolipin, an anionic lipid present in nucleated cell membranes and inner mitochondrial membranes can be recognized by acla . The aim of the current study is to evaluate the presence of aclas in tobacco users and non - tobacco users with severe chronic periodontal disease . Quantitative analysis of igg, igm aclas in smokers and non - smokers with severe chronic periodontitis . Quantitative analysis of igg, igm aclas in smokers and non - smokers with severe chronic periodontitis . Based on the armitage classification, 2000, 40 severe periodontitis (mean clinical attachment loss greater than 2.5 mm) male patients were selected for the study, with the age range of 3565 years and good general health, who visited the department of periodontology srm kattankulathur dental college, chennai . Duration of the study was approximately 4 months (july 2015 to november 2015). The participants were classified in to smokers (20 subjects) and non - smokers (20 subjects). Study samples were selected based on statistician's recommendations (for 95% confidence interval, p <0.01). Age 35 to 65 years; only males: smokers, those who have smoked more than 100 cigarettes in their lifetime and are currently smoking, and non - smokers . Alcohol consumption, malignancy, autoimmune disorders, diabetes, myocardial infarction, hypertension, stroke . Patients were informed orally about the procedure, and those who agreed, participated in the study by signing the consent form . Patients included in the study were screened by single periodontist using a mouth mirror and william's periodontal probe using direct and indirect illumination in both groups . All patients underwent periodontal evaluation and hematological and biochemical analysis . All subjects provided informed consent for use of their samples . Probing depth: probing depth was measured from the gingival margin to the base of the pocket using a calibrated a william's periodontal probe . Clinical attachment level: clinical attachment level was measured from the cementoenamel junction to the base of the pocket using a calibrated william's periodontal probe . All 40 participants showed mean clinical attachment loss more than 2.5 mm (armitage classification . 2 ml of venous blood sample was obtained by venepunture of the cubital vein in the ante cubical fossa using a 2 ml sterile disposable syringe with a 23 gauge needle . The blood was then transferred to an empty sterile vacutainer and then transported to the clinical laboratory for analysis of acla igg, igm . Elisa kits from sweden diagnostics kit, varelisa igm cardiolipin antibodies, and varelisa 2-glycoprotein 1 (igg) antibodies were used to asses igg and igm acl and igg anti-2gpi . As per the manufacturer's instructions, positive results were considered if the test result was greater than 15 units / ml . Age 35 to 65 years; only males: smokers, those who have smoked more than 100 cigarettes in their lifetime and are currently smoking, and non - smokers . Alcohol consumption, malignancy, autoimmune disorders, diabetes, myocardial infarction, hypertension, stroke . Patients were informed orally about the procedure, and those who agreed, participated in the study by signing the consent form . Patients included in the study were screened by single periodontist using a mouth mirror and william's periodontal probe using direct and indirect illumination in both groups . All patients underwent periodontal evaluation and hematological and biochemical analysis . All subjects provided informed consent for use of their samples . Probing depth: probing depth was measured from the gingival margin to the base of the pocket using a calibrated a william's periodontal probe . Clinical attachment level: clinical attachment level was measured from the cementoenamel junction to the base of the pocket using a calibrated william's periodontal probe . All 40 participants showed mean clinical attachment loss more than 2.5 mm (armitage classification . 2 ml of venous blood sample was obtained by venepunture of the cubital vein in the ante cubical fossa using a 2 ml sterile disposable syringe with a 23 gauge needle . The blood was then transferred to an empty sterile vacutainer and then transported to the clinical laboratory for analysis of acla igg, igm . Elisa kits from sweden diagnostics kit, varelisa igm cardiolipin antibodies, and varelisa 2-glycoprotein 1 (igg) antibodies were used to asses igg and igm acl and igg anti-2gpi . As per the manufacturer's instructions, positive results were considered if the test result was greater than 15 units / ml . Results of the current study showed that smokers [table 1] with severe periodontitis exhibited marked increase in acla igg, igm compared to non - smokers . The results were statistically significant (p <0.001) in smokers in severe periodontitis subjects when compared to non - smokers . Anticardiolipin antibodies igg, igm levels in smokers and non - smokers with severe periodontitis results were analysed using independent student's t - test [table 2]. It was noted that igg, igm levels were increased in smokers compared to non - smokers . Igg levels were significantly increased (mean difference of 9.14000) compared to igm levels (mean difference of 8.52050) in severe smokers . Increase in igg levels show that inflammatory by - products due to smoking were consistently high corresponded to duration . The principal new finding in this study was that severe chronic periodontitis patients showed elevated igg, igm anticardiolipin levels in smokers compared to non - smokers . These results indicate that smokers would be at great risk for systemic heart diseases and brain strokes . Previous studies have shown that igg in patients with periodontitis, who also have elevated serum concentrations of acla, stimulates increased production of the key cytokine monocyte chemo attractant protein -1 (mcp-1), which triggers atherogenesis . The results of the current study correlates and suggests that smokers would have elevated mcp-1, which could predispose both periodontal disease as well as atherogenesis . Previous study by schenkein reported that smoking influenced serum cell adhesion molecule levels and underscored the necessity of demonstrating that concentrations of these inflammatory markers, igg, igm, in periodontitis patients are independent of smoking . Heavy smokers in our present study had increased cell adhesion molecules, which could trigger inflammation in arteries . A study by doruk erkan et al . Reported that patients with aps are more prone to develop arterial and venous thrombi, and women with aps frequently experience recurrent spontaneous abortion . George et al . Showed that periodontal infections are associated with preterm labor, low birth weight, atherosclerosis, endothelial dysfunction, and myocardial infarction; although, cause and effect relationships are doubtful . The autoantibodies reactive with phospholipids which target 2gpi are hypothesized to cause the disease by several pathways, including activation of endothelial cells, inducement of oxidant - mediated injury, or interference with the natural anticoagulant function of 2gpi . This pathogenic mechanism elicited through these studies support that our subjects with tobacco users may initiate prothrombotic reactions and cyclic phenomenon . Blank et al . And schenkein et al . Have proven that anticardiolipin can be stimulated by bacterial pathogens such as porphyromonas gingivalis, hemophilus influenza or neisseria gonorrheae, and cytomegalovirus, which have peptide sequences similar to the tlrvyk peptide of 2gpi . Three peptides sequence that are present in the arg - gingipain protease of the periodontal pathogen porphyromonas gingivalis is similar to the tlrvyk peptide of 2gpi and can induce cross - reactive autoantibodies patients with periodontitis . Our previous study was to compare and correlate the levels of aclas in healthy, mild, moderate, and severe periodontitis patients . We found that patients with increased aclas have deeper pockets with more amount of attachment loss compared to healthy group . Severe periodontitis patients showed statistically significant elevated igg and igm acla (p <0.0001) compared to other group as well as control groups . In 2008, karnoutsos et al . Conducted a study to determine the correlation between periodontal infection and increased concentration of anti - phosphorylcholine and aclas in serum patients with periodontitis they found that patients with elevated anti - phosphorylcholine and aclas demonstrated increased pocket depth and greater mean attachment loss, compared to patients with normal levels of both antibodies . These results could be associated with an increased risk of stroke and atherosclerosis in patients with severe periodontitis . A study by gustafson et al . Evaluated the association between smoking and antiphosplipid antibodies in systemic lupus erythematosus and inferred that smoking was associated with the most pathogenic antiphospholipid antibodies . Another study done by califano et al . To determine the reactivity of igg concentration with recombinant porphyromonas gingivalis hemagglutinin in 117 chronic periodontitis and 90 generalized aggressive periodontitis patients found that igg exhibited reactivity with the organism in both chronic periodontitis and generalized aggressive periodontitis patients . Based on these observations, smoking could raise aclas in severe periodontitis . In the present study, smokers with severe chronic periodontitis showed increase in anticardiolipin igg igm than that in non - smokers . Exact cause for the increase in anticardiolipin in smokers with periodontitis is not known; hence, further research is necessary to establish the same . Limitations of this study were small sample size; hence, studies with large sample size with smokers are needed for better conclusive results . This warrants further longitudinal studies with large sample size to investigate the relationship between coronary heart disease and smoking with severe periodontitis.
Ununited fractures of clavicle are occasionally seen in adults, but are rarely found in children . Post - traumatic non - union of the clavicle is a rare complication in adulthood with a frequency of about 1% . This condition is also exceptional in children, despite the frequency of clavicular fracture at any given age . Clavicular fractures usually occur at the junction of the medial two third with the lateral one third of the bone and usually heal by conservative treatment within three weeks . Surgery is required in about one in 100 cases in which there is remaining deformity . An 8-year - old male child presented with a right - sided clavicular fracture for one year . There was a history of fall while playing and the patient sustained injury to the right shoulder, which led to the fracture of the clavicle . Both swelling and severe pain were present the patient had received treatment from an orthopedic surgeon with a figure 8 bandage for three weeks; however, the pain was not relieved . At one - year follow - up, the upper part of the chest, the bony prominence in the clavicular region and the overlying skin appeared normal . All of the arterial pulses were present in the affected limb (i.e., axillary, brachial and radial). Diagnosis was made following a radiograph of the right shoulder, which showed old, non - union of the clavicle at the junction of the medial two third with the outer one third . The patient was treated conservatively with analgesics and shoulder exercise as the movements of the shoulder were within normal range . X - ray of right shoulder revealed old, non union of clavicle at the junction of medial two 3 with outer one 3 and here was roundening of both ends with no callous formation . A 26-year - old male patient presented with a history of fracture of the right clavicle six years ago . He received treatment from an orthopedic surgeon in patna with figure 8 bandages and a shoulder sling for three weeks . The patient now complained of pain in the right shoulder with limitation of movement for one year . On local examination, there was no swelling or deformity and shoulder joint movements were within normal limits, up to 90% . There had been restriction of the movements of the shoulder joint, up to 10 degrees on internal and external rotation . A radiograph of the shoulder joint revealed an old, non - united fracture of the mid one third of the clavicle on the right side with no callus formation (fig . The patient was treated conservatively with analgesics and physiotherapy exercises . At a six - month follow - up visit, x - ray revealed old non - united fracture of mid one 3 clavicle of right side and there was no callous formation . An 8-year - old male child presented with a right - sided clavicular fracture for one year . There was a history of fall while playing and the patient sustained injury to the right shoulder, which led to the fracture of the clavicle . Both swelling and severe pain were present the patient had received treatment from an orthopedic surgeon with a figure 8 bandage for three weeks; however, the pain was not relieved . At one - year follow - up, the upper part of the chest, the bony prominence in the clavicular region and the overlying skin appeared normal . All of the arterial pulses were present in the affected limb (i.e., axillary, brachial and radial). Diagnosis was made following a radiograph of the right shoulder, which showed old, non - union of the clavicle at the junction of the medial two third with the outer one third . The patient was treated conservatively with analgesics and shoulder exercise as the movements of the shoulder were within normal range . X - ray of right shoulder revealed old, non union of clavicle at the junction of medial two 3 with outer one 3 and here was roundening of both ends with no callous formation . A 26-year - old male patient presented with a history of fracture of the right clavicle six years ago . He received treatment from an orthopedic surgeon in patna with figure 8 bandages and a shoulder sling for three weeks . The patient now complained of pain in the right shoulder with limitation of movement for one year . On local examination, there was no swelling or deformity and shoulder joint movements were within normal limits, up to 90% . There had been restriction of the movements of the shoulder joint, up to 10 degrees on internal and external rotation . A radiograph of the shoulder joint revealed an old, non - united fracture of the mid one third of the clavicle on the right side with no callus formation (fig . The patient was treated conservatively with analgesics and physiotherapy exercises . At a six - month follow - up visit, x - ray revealed old non - united fracture of mid one 3 clavicle of right side and there was no callous formation . An 8-year - old male child presented with a right - sided clavicular fracture for one year . There was a history of fall while playing and the patient sustained injury to the right shoulder, which led to the fracture of the clavicle . Both swelling and severe pain were present the patient had received treatment from an orthopedic surgeon with a figure 8 bandage for three weeks; however, the pain was not relieved . At one - year follow - up, the upper part of the chest, the bony prominence in the clavicular region and the overlying skin appeared normal . All of the arterial pulses were present in the affected limb (i.e., axillary, brachial and radial). Diagnosis was made following a radiograph of the right shoulder, which showed old, non - union of the clavicle at the junction of the medial two third with the outer one third . The patient was treated conservatively with analgesics and shoulder exercise as the movements of the shoulder were within normal range . X - ray of right shoulder revealed old, non union of clavicle at the junction of medial two 3 with outer one 3 and here was roundening of both ends with no callous formation . A 26-year - old male patient presented with a history of fracture of the right clavicle six years ago . He received treatment from an orthopedic surgeon in patna with figure 8 bandages and a shoulder sling for three weeks . The patient now complained of pain in the right shoulder with limitation of movement for one year . On local examination, there was no swelling or deformity and shoulder joint movements were within normal limits, up to 90% . There had been restriction of the movements of the shoulder joint, up to 10 degrees on internal and external rotation . A radiograph of the shoulder joint revealed an old, non - united fracture of the mid one third of the clavicle on the right side with no callus formation (fig . The patient was treated conservatively with analgesics and physiotherapy exercises . At a six - month follow - up visit, x - ray revealed old non - united fracture of mid one 3 clavicle of right side and there was no callous formation . Clavicular non - union is rarely asymptomatic and often results in disability from pain at the site of non - union, altered shoulder mechanics, or a compression lesion involving the underlying brachial plexus or vascular structures . Fractures of the clavicle are usually in the medial two third of the bone, which may result from a fall and subsequent outstretched hand during the fall . The lateral fragment is displaced forward and downward by the weight of the limb, while the medial fragment is held at a higher level by the sternocleidomastoid muscle . The essential treatment is to support the weight of the limb by a sling tied over the opposite shoulder . The fractures are almost always clinically united within three weeks . About one in 100 fractures of the clavicle require primary surgical treatment . Sir robert peel, who established the police force of great britain, died of a fractured clavicle which ruptured the subclavian vein . Peel was attended by sir benjamin brodie who wrote, the hemorrhage itself was the consequence of the subclavian vein having been lacerated by splinters of the fractured bone . If deformity persists at the bony ends of the clavicle after several months, surgical smoothening of these ends is indicated by a short incision in the line of the skin creases . This causes less deformity than the scarring resulting from more extensive operative procedures that may be required for primary open reduction with internal fixation . In addition, major surgical procedures may carry the risk of additional surgery for non - union . The non - union rate of fractures of the lateral end of the clavicle can rise to 37% when a nonsurgical treatment protocol is initially adopted . Reported results for the nonsurgical treatment of fractures of the clavicle have been uniformly positive; a combined series of over 3000 fractures showed a rate of non - union of 0.4% . Occult fracture has been well documented in the hip and the scaphoid and failure to recognize this type of fracture could lead to serious consequences . While clavicular fracture is often viewed as benign, it is important for patients to be aware that any fracture may impact expected time of recovery . In addition, complications such as non - union do occur and inadequate initial immobilization is a common cause . Patients who have suffered a clavicular fracture often recover well in spite of the risk of non - union; fatal complications that may occur following vascular injuries are extremely rare . Fracture location and the type of immobilization have little effect on the final result or prognosis . Careful attention should be paid when obtaining a detailed history and physical examinations, as traumatic arthritis at either clavicular joint may mimic the symptoms of non - union . The explicable evidence of osseous non - union on radiographs may be minor and may not correlate with the clinical symptoms . A patient with an atrophic pattern of non - union may become asymptomatic with time . Surgeons should be cautious when operating on the non - union merely due to its presence, although asymptomatic . If a surgical procedure is planned, possible outcomes should be communicated to the patient, including the possibility of additional surgery, if required.
Despite efforts to combat human immunodeficiency virus type 1 (hiv-1) infection through the use of highly effective combination therapies, hiv-1 has continued to spread . By 2009, the number of individuals living with hiv-1 reached a staggering 33.3 million people worldwide . Since the beginning of the epidemic almost 30 years ago, the proportion of hiv-1-infected women in this population has risen steadily, stabilizing recently at approximately 52% . Women in sub - saharan africa are at particular risk, as evidenced by the greater prevalence of women (approximately 60%) in the hiv-1-infected population and the predominant transmission of hiv-1 through unprotected heterosexual intercourse . This route of transmission continues to prevail because effective methods of prevention, such as the male condom, are inconsistently used due to various social, cultural, economic, and religious issues . To address the critical need for an effective and acceptable means to check the spread of hiv-1, numerous investigators around the world are working toward the development of microbicides, which are formulated chemical entities that can be applied vaginally or rectally to prevent or eliminate hiv-1 transmission [25]. The ideal microbicide would be highly active against hiv-1 but may also have activity against other sexually transmitted disease pathogens . For optimal acceptability, a formulated microbicide should be colorless, tasteless, and odorless to allow for discretion in its use and should also be inexpensive and easy to apply [3, 6]. Importantly, a topical formulation containing an active pharmaceutical ingredient (api) must be safe for topical vaginal and/or rectal use, causing no adverse changes in epithelial barrier functions and no local inflammation that might increase susceptibility to hiv-1 infection . Although a microbicide is not yet available for worldwide use, the reverse transcriptase inhibitor tenofovir was recently shown to be a safe, effective microbicide api in women at risk for hiv-1 infection . Our efforts have been directed toward the development of biguanide - based compounds for use in microbicides effective against hiv-1 . Polybiguanides (pbgs) are polycationic in nature and are made up of repeating biguanide units that are separated by hydrocarbon linkers of varying lengths . Pbgs have been used safely for more than 30 years for vaginal disinfection (chlorhexidine digluconate), antimalarial therapy (proguanil), and treatment of type 2 diabetes (metformin). They are also used as the active ingredient in contact lens disinfectants, mouth rinses, and wound disinfectants [816]. Our studies of pbg compounds as microbicide apis have encompassed activity, safety, and mechanism of action . Early experiments with a small number of pbgs indicated that polyethylene hexamethylene biguanide (pehmb) was an effective hiv-1 inhibitor characterized by minimal in vitro cytotoxicity . Expanded studies of structure - activity relationships among different pbg compounds demonstrated clear relationships between molecule structure and biological activity . Furthermore, the results of these studies suggested that pehmb acted against x4 and r5 strains of hiv-1 as a viral entry inhibitor by interacting with the host cell rather than the virus . In a series of experiments focused on confirming this mechanism of action against hiv-1, pehmb (later designated nb325) was shown to inhibit hiv-1 infection by interacting specifically with the second extracellular loop of cxcr4, which serves as the hiv-1 coreceptor . Additional investigations demonstrated that this interaction and the inhibitory effect on hiv-1 infection were persistent, providing protection from infection even after removal of the compound from the culture medium . The low in vitro cytotoxicity of pehmb (nb325) suggested that this compound would also be characterized as safe in a swiss webster mouse model used to assess cervicovaginal toxicity associated with compound exposure . Previous studies using this murine model of microbicide toxicity (i) confirmed the cervicovaginal toxicity and inflammation associated with topical application of nonoxynol-9 (n-9) in women and (ii) corroborated differences in acceptability between two different formulations of the microbicide api c31 g . In previous mouse model experiments relevant to the present studies, in vivo exposure to unformulated 1% pehmb resulted in minimal epithelial damage and negligible local inflammation these results were consistent with those from in vitro studies of compound cytotoxicity, which demonstrated that pehmb was more than 350-fold less cytotoxic than n-9 [8, 20]. The objectives of the present study were to assess the safety of nb325 formulated at two concentrations (0.5% and 1%) and to investigate time - dependent effects of application on cervicovaginal integrity . Experiments were focused on the acute time points of 10 min, 2 h, and 4 h postapplication, because these exposure durations were previously shown to provide important information for gauging acute epithelial toxicity following n-9 or c31 g exposure [2023]. The extended time points of 8 h and 24 h were also included to provide a full range of observations over a 24 h period . Histological analyses of exposed tissues indicated little to no toxicity after exposure to 0.5% nb325 and minimal toxicity after exposure to 1% nb325 for both unformulated and formulated compound . Interestingly, changes in epithelial integrity that were observed following exposure to nb325 were noted in the vagina rather than in the cervix, where n-9 toxicity was readily apparent in previous studies . These studies provide further evidence of the topical safety of biguanide - based compounds as microbicide apis . Nb325 was synthesized as previously described for polyhexamethylene biguanide and pehmb [8, 18, 24]. The molecular mass of nb325 ranged from 900 to 1,900 da with a median molecular mass of approximately 1,400 da . Gel formulation of nb325 was performed by the international partnership for microbicides using a hydroxyethyl cellulose (hec) based gel, otherwise known as the universal placebo . These experiments, which were similar to previously performed studies [20, 21, 23], used 5- to 6-week - old female outbred swiss webster mice (cfw) (charles river laboratories, wilmington, mass). Prior to compound or formulation application, the mice were synchronized using a 0.2 ml subcutaneous injection of depo - provera (pharmacia and upjohn company, bridgewater, nj) at 7 and 3 days before the start of each experiment . The depo - provera was diluted in ringer's lactated saline solution (baxter, deerfield, ill) to allow administration of 3 mg / animal . Mice were anesthetized with a formulation of ketamine / xylazine (100200 mg / kg and 510 ng / kg, respectively) before the intravaginal application of nb325 . Mice treated with saline alone or 1% n-9 (diluted in saline) were included as controls to evaluate the morphology of normal or damaged cervicovaginal mucosal tissue, respectively . After treatment, mice were sacrificed humanely, and the cervicovaginal tracts were surgically excised and prepared for histological examination . Mice were sacrificed after exposure for 10 min, 2 h, or 4 h and after longer exposures for 8 h or 24 h. the durations of acute exposure were established previously to characterize the appearance of epithelial damage following a single microbicide application . The longer time points were shown to be useful for characterizing the time course of epithelial repair and resolution of tissue inflammation . All excised cervicovaginal tissues were formalin - fixed and embedded in paraffin using previously described procedures [20, 21, 23]. Paraffin - embedded samples were stained with hematoxylin and eosin (h&e) for gross morphological analyses, and representative fields from each treatment group were documented in photomicrographs . Although clinical studies of microbicide safety have included assessments of female reproductive tract health and integrity following application, these studies were often conducted at postexposure durations in excess of 24 h. as a result, information regarding the acute effects of microbicide exposure has been lacking . Our in vivo studies of n-9 using a mouse model of cervicovaginal toxicity indicated the need to include acute and extended exposures in these experiments in order to observe both short- and long - term effects of topical application . As controls in these experiments, mice were exposed to either saline or unformulated n-9 (1%) to provide illustrations of normal or damaged cervicovaginal epithelial tissues, respectively . Saline exposure for 2 h caused no damage to either the vaginal or the cervical epithelial tissue (figure 1); identical results were obtained after exposures of 10 min, 4 h, 8 h, or 24 h (data not shown). As in previous studies, n-9 exposure resulted in severe cervical epithelial shedding and breaks in the epithelia that were greatest at 2 h postexposure (figure 2(b)) and were still evident at 4 h and 8 h after n-9 application (figures 2(d), 2(f)). N-9-associated cervical damage was resolved by 24 h postexposure, as these tissues were similar in appearance to tissues from saline - exposed mice (data not shown). The vaginal epithelium was unaffected by n-9 exposure (figures 2(a), 2(c), 2(e)). Initial nb325 mouse model studies involved the vaginal application of unformulated nb325 at concentrations of 0.5% or 1% . At its lower concentration (0.5%), unformulated nb325 caused little or no postexposure cervicovaginal epithelial damage (data not shown). Although epithelial cell sloughing and breaks in the epithelium were observed sporadically in the vagina and cervix, respectively, these minor indications of toxicity were not apparent by 24 h postexposure (data not shown). Exposure to 1% unformulated nb325 resulted in a slightly greater level of toxicity compared to 0.5% nb325 . At 10 min postapplication, nb325 exposure resulted in no damage to the cervicovaginal tract (figures 3(a), 3(b)). In the vagina and cervix, the tissue appeared the same as the placebo control, the vaginal epithelium retained the keratin layer, the cervix had no cells shed into the lumen, and mucus production was suggested by the observation that mucus - producing cells were present in the epithelial layer . By 2 h and 4 h postapplication, the cervix was still unaffected (figures 3(d), 3(f)), whereas some toxicity was apparent in the vaginal epithelium (figures 3(c), 3(e)). At 2 h after application, the vagina began showing signs of toxicity in the form of cells appearing to loosen from the upper layers of the stratified squamous tissue and thinning of the epithelial layer . By 4 h after application, cells were actively shed from the vaginal epithelium and were present in the lumen . At the extended exposure durations of 8 h and 24 h, little to no toxicity was observed in the vagina (figures 4(a), 4(c)), although at 24 h some shed tissue was still apparent in the lumen . At 8 h postexposure, the cervical epithelium was generally intact, with evidence of a small number of cells present in the lumen (figure 4(b)). By 24 h postexposure, however, these luminal cells were no longer present, and the tissue appeared normal (figure 4(d)). To explore the potential cervicovaginal safety of the formulated compound, nb325 was incorporated into a hec - based gel, otherwise known as the universal placebo, at final concentrations of 0.5% and 1% . Vaginal and cervical epithelial tissues from mice exposed to the gel without nb325 (placebo) were indistinguishable from saline - exposed tissues; there was no observable epithelial damage at any time point (figure 5 and data not shown). The effects of exposure to nb325 formulated at 0.5% were similar to those observed after exposure to unformulated nb325 at the same concentration (data not shown). In the vagina, indications of minor toxicity were observed at 8 h postexposure in the form of epithelial layer thinning, indicating that shedding may have occurred at some point between the 4 h and 8 h time points . By 24 h postexposure, epithelial damage was no longer apparent; tissues were similar in appearance to the controls . The cervix appeared to be unaffected by any length of exposure to 0.5% formulated nb325 . Similar experiments were performed to examine the effects of a formulation containing a higher concentration (1%) of nb325 . At 10 min following application, minimal damage to the vagina was observed (figure 6(a)), whereas the cervix appeared normal and showed indications of mucus production (figure 6(b)). By 2 h after application, signs of toxicity in the vaginal tract were apparent, indicated by the presence of sloughed cells in the lumen and thinning of the epithelial layer (figure 6(c)). At 4 h after application, an increase in vaginal tissue damage relative to the 2-h exposure was observed (figure 6(e)). By 8 h and 24 h postexposure, however, vaginal epithelia appeared normal, with no observable residual tissue damage (figures 7(a), 7(c)). Although cervical epithelial tissues exposed to formulated 1% nb325 were generally intact, some cell infiltration beneath the basal layer of the cervical epithelium was visible at 2 h and 4 h postexposure (figures 6(d), 6(f)). Interestingly, the sporadic appearance of mild cervical epithelial damage was noted at 8 h after application (figure 7(b)). Loose appearance of cells along the epithelial layer, suggesting the presence of epithelial cells in the process of shedding . At 24 h, however, the cervix appeared normal and intact (figure 7(d)). The vaginal and cervical epithelia within the female reproductive tract provide physical barriers that impede pathogen access and play host to cells that mount immunologic responses to those pathogens . The integrity of this physical and immunologic barrier is crucial to maintaining the health of the cervicovaginal environment; disruption of this barrier could result in an increased risk of infection by sexually transmitted disease pathogens . With regard to hiv-1, breaks in these natural barriers can provide a direct route that the virus can take to reach susceptible cells and establish infection . Thus, assessment of cervicovaginal epithelial integrity following exposure to a candidate microbicide is an important part of demonstrating the safety of a topical vaginal formulation and its api . However, a complete evaluation of in vivo safety must also encompass other approaches, such as investigations of inflammatory cell infiltration [20, 21], assays of cytokine expression and release, and experiments to demonstrate increased susceptibility to infection following topical microbicide exposure . The present mouse model studies, which were performed to provide an initial evaluation of formulated nb325 safety, offer the basis for several conclusions . First, the results of experiments involving unformulated nb325 suggest a much greater level of safety relative to n-9, which has been shown in these and other studies to cause severe damage to cervicovaginal epithelial tissues . These studies corroborate and expand on previous results that demonstrated the minimal effect of unformulated pehmb on epithelial integrity and the absence of cd45-positive immune cell infiltration following exposure . The present studies indicate a similar level of safety for unformulated nb325, which was produced through the application of refinements to the synthetic methods used to produce pehmb [17, 18]. Second, these results also indicate a relative level of in vivo safety for nb325 formulated in an hec - based gel . The effects of formulated nb325 on vaginal epithelial integrity are in sharp contrast to the severe and widespread cervical epithelial damage and tissue sloughing (as well as induction of immune cell infiltration) caused by the application of unformulated and formulated n-9 (conceptrol). In fact, the results of mouse experiments involving formulated nb325 are comparable to the results of similar experiments using 1% formulated c31 g, which was shown in several independent clinical trials (as the topical agent savvy) to be safe and acceptable for human application [2729]. However, because the 1% formulated nb325 appeared to be slightly more toxic in vivo relative to the formulation containing 0.5% nb325, the concentration of nb325 in future microbicide formulations will need to be considered further . The indication of concentration - dependent effects on epithelial integrity also hints at the bioavailability of nb325 in the hec - based formulation . Although nb325 bioavailability was not addressed in the present studies, preliminary experiments involving hiv-1 infection of indicator cells in the presence of formulated nb325 indeed demonstrated antiviral activity attributable to nb325 (data not shown). However, the antiviral activity of nb325 in this transwell - based experimental system was partly obscured by the barrier effect of the viscous hec - based gel, indicating a limitation of this method for measuring drug availability and biological activity and the need for additional investigations in this direction . The third conclusion concerns the location of the relatively limited epithelial damage caused by unformulated and formulated nb325 . When epithelial damage was observed as a consequence of nb325 exposure, it was found predominantly in the vagina and observed infrequently in the cervix . In contrast, previous investigations demonstrated that the severe epithelial damage caused by n-9 was confined to the cervix; the vaginal epithelium appeared to be resistant to any damage caused by a single application of n-9 [17, 20]. Similarly, losses in epithelial integrity associated with c31 g exposure were found exclusively in the cervix . These differences may be related both to the chemical nature of each compound and the respective microenvironments of the vagina and cervix . Nb325 is an aqueous, nonsurfactant compound with a net positive charge at physiological ph, whereas n-9 and c31 g are both surfactants . Within the cervix, mucus released from the epithelial surface may interact with nb325 and protect the columnar epithelial cells from any toxicity associated with nb325 . This layer of cervical mucus may not afford the same protection against surfactants such as n-9 or c31 g . Conversely, the absence of mucus in the vagina may leave the vaginal epithelium vulnerable to the potential effects of nb325 . However, the structure of the stratified squamous epithelium in the vagina may be more impervious to the surfactant properties of n-9 or c31 g . The present studies and results (i) stress the importance of considering cervicovaginal environmental factors in designing and evaluating candidate microbicides and (ii) highlight the potential impact of api and formulation chemistry on microbicide safety . Microbicides shown to be safe but ineffective in clinical trials (ushercell, carraguard, and pro2000) may have failed due to electrostatic interactions between their polyanionic apis and charged seminal plasma components that interfere with their antiviral activities [30, 31]. Because nb325 is polycationic indeed, cationic peptides endogenous to semen have been shown to provide an inherent antiviral activity against hiv-1 . The effects of both male and female reproductive tract secretions on nb325 activity will be addressed as part of the preclinical development of this compound . In vivo experiments involving a mouse model of cervicovaginal toxicity provide support for the safe use of a gel formulation containing the biguanide - based antiviral compound nb325 as a topical vaginal microbicide . However, indications of minor dose - dependent effects on epithelial integrity and regional differences in the effects of nb325 on the cervicovaginal tract indicate the need for further preclinical studies to arrive at an optimal api concentration and formulation.
Sarcoidosis is a chronic multisystemic disease of unknown origin; it is characterized by an accumulation of noncaseating epithelioid granulomas . This pathology affects most commonly the lung, skin, lymph nodes, eyes, and rarely the nervous system . The relationship between the occurrence of sarcoidosis and interferon alfa (ifn-) therapy in viral hepatitis c was suggested in earlier reports. [14] this was also supported by the spontaneous resolution of sarcoidosis after cessation of ifn- treatment for hepatitis c. reported clinical manifestations include cutaneous sarcoidotic lesions, pulmonary nodules, and peripheral neuropathy . In this paper, we report the second case in the literature of fatal central nervous system sarcoidosis secondary to ifn- and ribavirin treatment . We aim to underline the importance of screening for sarcoidosis before and during the follow - up of hepatitis c virus (hcv) patients undergoing antiviral therapy . Since march 2002, a 47-year - old woman without any history of sarcoidosis was regularly monitored in consultation for chronic viral hepatitis c (genotype 1). In april 2007, iu / l compared to normal values of 40 iu / l; the serum hcv rna was 6.210 copies / ml . However, the physical and abdominal ultrasound examinations did not show any abnormalities . Because of the presence of biological cytolysis, a percutaneous liver biopsy was performed and revealed severe hepatic fibrosis (metavir score a2/f3). Then, the antiviral treatment was started, and the patient received once a week the pegylated inf-2a at the rate of 180 g that injected subcutaneously, and ribavirin 400 mg was administrated orally twice a day . The biological response was good, and the transaminases were standardized after 2 weeks of treatment . Six weeks after the beginning of the treatment, the patient noticed weight loss of 5 kg associated with dyspnea and progressive appearance of skin small firm nodules on both her upper and lower extremities . Chest x - ray was normal . However, thoracic computed tomography (ct) scan revealed pulmonary nodules associated with bilateral mediastinal lymphadenopathies, suggesting tuberculosis, lymphoma, and/or sarcoidosis . Afterward, bronchoalveolar lavage fluid was performed and showed an increased number of lymphocytes with a normal amount of eosinophils and neutrophils . The histological study of transbronchial lung biopsy revealed a patchy distribution of mild interstitial and perivascular fibrosis, without distinctive granulomas or significant inflammatory cell infiltrations . Finally, a biopsy of the skin nodules was performed and found a noncaseating epithelioid granuloma formation strongly suggestive of sarcoidosis [figure 1]. The serum angiotensin - converting enzyme was significantly elevated (130 u / l for the normal value <40 u / l); the diagnosis of sarcoidosis was retained and oral corticotherapy was started at the dose of 60 mg daily . No caseating epithelioid granuloma formation composed of macrophages, lymphocytes, and fibroblasts four days later, the patient suddenly presented a heaviness of the right upper limb predominant distally, associated with a right central facial paralysis and aphasia; however, she was lethargic and her ophthalmologic examination was normal . The cerebral magnetic resonance imaging (mri) showed a gyriform and nodular left frontal and parietal subcortical enhancement associated with an important perilesional edema [figures 2a b]. Inf- and ribavirin therapies were discontinued, and intravenous bolus methyl prednisolone was then started at the rate of 10 mg / kg / day for consecutive 3 days; this was followed by prednisone (1 mg / kg / day). The neurological state of the patient worsened rapidly and the patient died a week later . Cerebral mri in postgadolinium axial t1-weighted image (a) and flair image (b), showing a gyriform and nodular left frontal and parietal subcortical enhancement associated with an important perilesional edema more than 170 million people worldwide are infected with chronic viral hepatitis c. current antiviral treatments are effective in eradicating the virus in up to 60% of patients . Pegylated ifn- plus ribavirin was found to be superior to all other protocols for sustained eradication of the hcv, especially in individuals with more resistant viral genotypes 1, 4, 5, and 6 . Although several reports have suggested an association between ifn therapy and sarcoidosis, this association was rarely described in the literature . In 1987, abdi et al . Had described the first case of pulmonary sarcoidosis in a patient who received ifn- treatment for renal cell cancer . Since then, various cases have been published suggesting a relationship between sarcoidosis and ifn treatment in patients with a variety of diseases, including renal cell carcinoma, hematological malignancies, and viral hepatitis . So far, more than 30 cases of sarcoidosis occurring in the context of chronic hepatitis c treated by ifn- have been reported in the literature. [24] reported clinical manifestations include cutaneous sarcoidotic lesions, pulmonary nodules, and peripheral neuropathy . In this paper, we report the second case in the literature of severe central nervous system sarcoidosis secondary to ifn- treatment . To the best of our knowledge, only one case of neurosarcoidosis associated with ifn therapy has been reported in the literature . Most of the cases described in the literature occurred in patients who have received a treatment combining ifn- or pegylated ifn- and ribavirin . Indeed, it is well recognized that ifn- is an immunomodulator that has not only direct antiviral activity but also powerful stimulation of the immune activities, especially on t - helper (th1) immune response[810] which is strongly involved in the pathogenesis of sarcoidosis . Furthermore, granulomas in sarcoidosis are associated with an abundance of cd4 t lymphocytes and mononuclear phagocytes, which are being considered a result of cytokine stimulation and immunologic dysregulation . Importantly, in cases of chronic hepatitis c, inf could induce and reactivate sarcoidosis, and inf - based combination antiviral regimens cannot eliminate the occurrence of sarcoidosis . In contrast, ribavirin, an antiviral agent that increases the anti - hcv effect of inf in chronic hepatitis c, would also enhance th1 cytokine response while inhibiting th2 cytokine response . Furthermore, there was no case report of sarcoidosis in patients treated using ribavirin only . This suggests that the combination of inf- and ribavirin enhances the immune response, consequently predisposing the patient to sarcoidosis . The mean time to the onset of the disease after starting inf- therapy is 4 months . In our case, manifestations of sarcoidosis occurred earlier, associating rapid deterioration of clinical symptoms, which lead to death in 10 weeks . Most patients with inf- associated sarcoidosis had spontaneous resolution of the disease without immunosuppressive treatment . Indeed, inf- treatment was discontinued in several cases and subsequent resolution of sarcoidosis within months has followed . In contrast, the treatment protocol was not modified in other cases, and the sarcoidosis resolved several months later, either during or after the achievement of treatment . Indeed, steroids that are the main treatment for systemic sarcoidosis increase the hcv load in both in vitro and in vivo . The prognosis of peripheral neurosarcoidosis is better as compared to the central nervous system involvement, which leads to higher disability and mortality . In addition, neurosarcoidosis of the cns usually occur in the early stages of the disease, while peripheral nervous system and skeletal muscle sarcoidosis are typically seen in the chronic stages of the disease . This would suggest that our patient has recent neurosarcoidosis after introducing inf- and ribavirin therapy . Based on this case report, inf- associated sarcoidosis might also include cns involvement and might lead to death . Considering earlier reports of heterogeneous spectra of sarcoidosis manifestations, clinicians should be aware of associated potential complication while evaluating the benefit / risk ratio of the treatment in patients with chronic hepatitis c infection . Therefore, patients suggested such treatments are recommended to be monitored for sarcoidosis before and during ifn therapy.
Stroke is one of the leading causes of disability and mortality in many developed countries . Approximately 10% of the deaths in the world are related to stroke and it is estimated that the incidence of stroke will increase during the next 20 years . Dietary factors are associated with the risk of stroke, for example, by the impact on blood pressure, resistance to insulin, systematic inflammation, thrombosis, and oxidation . A diet rich in calcium, magnesium, and potassium may decrease the risk of stroke, whereas increasing intake of sodium can lead to higher blood pressure and risk of stroke, but the effect of the intake of iron is not completely clear . Due to the fact that minerals are essential elements for human body, some researchers have investigated the association between stroke and dietary intake of minerals, but the results are inconsistent . So, we aimed to investigate the effect of dietary intake of minerals in patients with stroke . Surveys for this case control study were performed in the alzahra hospital in iran from april 2010 through march 2011 we recruited subjects from two wards of this hospital; the incident stroke patients were referred from the inpatient wards of the neurology department and the control group from the normal population . The control group had neither history nor clinical evidence indicating a previous stroke, and their treatment at the outpatient department was not related to any cardiovascular disease, malignant tumor, or diabetes . We also excluded patients who had been on long - term modification of diet for medical reasons . Finally, 46 men (aged 56 18 years) and 23 women (aged 52 7 years) with stroke were included in this study . Information on typical consumption of food and demographic and lifestyle characteristics was collected in the interview . When patients were unable to answer, we asked for their next of kin to obtain answers . We used a food frequency questionnaire (19) that included 168 items covering foods commonly consumed in iran . We obtained the quantity of minerals for each food item from the food composition table . We used the software of fpii to assay collected information and t - test for comparison between groups . The study population consisted of 129 subjects, 69 patients with acute stroke, 46 men (aged 56 18 years) and 23 women (aged 52 7 years) and 60 controls (30 men and 30 women, 45 5 years of age). Intake of energy and micronutrients in stroke subjects and controls are shown in table 2 . Intake of sodium in male and female patient with stroke was significantly higher than the control group . (p <0.05, p <0.001 in men and female respectively). Comparison of mean intake of vitamins and minerals in the study subjects with the recommended dietary allowance (rda) are shown in tables 3 and 4 . In male patients, intake of iron was 21/5 7/5 mg / day and in healthy men, intake of iron was 14/5 8/5 mg / day, whereas rda is 8 mg / day; therefore, intake of iron in the case group was significantly higher than the control group (p <0/05), but intake of iron in case and control women was not significantly different . Intake of zinc in men with stroke was 17/2 8/5 mg / day and in healthy men was 13/3 7/3, and rda for zinc was 11 mg / day; so, intake of zinc in men with stroke was significantly higher than healthy men (p <0/05). But intake of zinc in women with stroke was 7/2 3/4 mg / day and rda is 8 mg / day, but intake of zinc in healthy women was 5/2 4/1 mg / day; so, this difference was not significant . However, intake of calcium in male patients was 1352 440 mg / day and rda is 1000 mg / day; whereas intake of calcium in healthy men was 972 335 mg / day; so, this difference was not significant . Mean of anthropometric measurements in patients with stroke measurement of consumption of energy and micronutrients in stroke patients comparison of mean consumption of vitamins and minerals in male stroke patients and rda comparison of mean consumption of vitamins and minerals in female stroke patients with rda in this study, we found that men and women with stroke had a diet higher in sodium, the positive association between the intake of sodium and stroke was independent of the intake of potassium, and this was observed similarly for nonoverweight and overweight persons . Findings from previous studies that have examined the relationship between the intake of sodium and risk of stroke have been inconsistent . Our findings were supported by results of some previous studies; two studies of americans and japanese researchers reported that the intake of sodium was related to an increased risk of stroke and mortality and it was reported that about a daily intake of 100 mmol sodium maybe associated with 32% higher incidence of stroke among overweight americans . Japanese men with an intake of 306 mmol sodium had a twof old increased risk of stroke compared with people with a daily sodium intake of 174 mmol . Reported that a daily intake of 100 mmol sodium was related to 83% higher mortality from stroke . In addition, they found a strong positive association between intake of sodium and mortality from stroke for persons with either a body mass index (bmi) <25 or a bmi 25 . Reported that sodium was not significantly associated with risk of any stroke subtypes after potential confounders were controlled for . A prospective study from finland and another from japan showed no significant association between intake of sodium and incidence of stroke . However, another japanese study reported a significant association among men and women with a high intake of sodium (median daily intake of 7194 mg among men and 6478 mg among women). In addition, the national health and nutrition examination survey epidemiologic follow - up reported a positive association among overweight persons . High intake of sodium has been related to high blood pressure . In this study, we reported that men with stroke had a high - iron diet and we supposed that high intake of iron can increase blood pressure as a main cause of stroke, but we did not find this result in women . In a previous study, researchers reported that increased intake of iron may elevate the risk of stroke . They hypothesize that high intake of iron could increase the risk of atherosclerotic cardiovascular disease . In contrast, a previous cross - sectional study involving four countries showed a significant inverse association between intake of iron and blood pressure, whereas a recent study that was a randomized clinical trial did not observe any effect of reducing iron stores among phlebotomy on the risk of stroke and myocardial infraction after six years of intervention . So, more investigation is needed to diagnose the effect of iron on risk of stroke . In our study, we observed no association between intake of calcium and stroke . Susanna et al . Reported that intake of calcium was not significantly associated with risk of stroke after potential confounders were controlled for . In addition, the health professionals follow - up study observed no association between intake of calcium and stroke . The nurses health study reported an inverse association between intake of calcium, especially dairy calcium, and risk of stroke . Likewise, in a cohort of japanese men and women, intake of dairy calcium was inversely associated with stroke mortality . In a previous study that was a randomized trial including 36,282 postmenopausal women, intake of calcium and vitamin d supplementation neither decreased nor increased the risk of stroke over a seven - year period . Intake of calcium was positively associated with the risk of intracerebral hemorrhage in eight prospective studies of calcium intake in relation to stroke incidence or mortality; four reported an inverse association between stroke and intake of dairy calcium but not nondairy calcium, and no association was found for total intake of calcium from both dairy and nondairy foods . The reason for the inconsistent results for the association of calcium intake with stroke maybe due to the difference in the range of exposure or the lack of adjustment for potential confounders . In this study, a high intake of sodium was associated with a significantly increased risk of stroke and findings from this study did not report a protective effect of calcium on risk for stroke . The limitations of the present study are first, the estimated intake of sodium from the present questionnaire study was 50% lower than that estimated from dietary records . Second, we estimated intake of sodium with the food frequency questionnaire, whereas urinary measurement is a better tool than a food frequency questionnaire . Third, the diet was associated with a self - administered questionnaire that may have led to some errors in the measurement of dietary intake.
The 2011 summer was the second warmest on record in the continental united states, and 41 states of the lower 48 had an above - normal, much - above - normal, or record warmest july [national oceanic and atmospheric administration (noaa) 2011, 2012a]. Such extreme heat can harm human health and increase risk of heat stroke (bouchama and knochel 2002) and more common health problems such as respiratory and cardiovascular hospitalizations and deaths (anderson et al . 2010; jones et al . 1982; naughton et al . 2002; weisskopf et al . 2002). Heat - related health impacts can be especially severe for certain groups including those living in urban areas (fischer et . Al 2012) and areas with low air conditioning prevalence (oneill et al . 2005) and for those who are isolated, of lower socioeconomic background (hajat and kosatky 2010), elderly, or belonging to some specific ethnic groups (white - newsome et . These heat - related health impacts vary by region (mcgeehin and mirabelli 2001) and community (anderson et al . 2013) and can change over time (davis et al . 2003). To save more lives, increased capacity to deal with current and future heat - related health threats is needed (hess et al . Monitoring and evaluation of heat preparedness and response can help policy makers set priorities and refine strategies (huang et al . 2011), but little is known about local heat preparations and responses on a national scale in the united states . Studies on local heat preparedness and response have identified heat - planning activities that include crafting heat emergency plans (bernard and mcgeehin 2004), increasing communication about heat - coping strategies (meehan et al . 2002), and using heatwave early warning systems that alert local populations of health - threatening weather conditions (kalkstein et al . Previous u.s . Surveys of environmental health directors and other local officials asked about perceptions of climate - related health risks and existing barriers to responding and planning for heat waves (see supplemental material, table s1). Local health officials in california felt that climate change posed a large risk to public health, but lack of information and resources to manage the risks, and lack of coordination among different agencies, remained major challenges in preparing and responding to climate change (bedsworth 2009). Other surveys revealed that climate change was not a top priority [environmental defense fund (edf) 2008], and new partnerships and financial resources were needed to support local actions (oneill et al . To increase the knowledge of broad patterns in local heat preparedness and response to extreme heat in the united states, we conducted a survey of 586 counties within 30 u.s . States (the district of columbia is included in this number) soon after the extremely hot summer of 2011 . States were selected based on either record - breaking or -tying temperatures during the summer of 2011 based on the national climatic data center s u.s . Record temperature database (alaska, arkansas, colorado, connecticut, district of columbia, iowa, kansas, maryland, missouri, montana, new hampshire, new jersey, new mexico, oklahoma, pennsylvania, texas, vermont, wisconsin, west virginia) (noaa 2012b), membership in the center for disease control and prevention climate resilient states and cities initiative (arizona, california, maine, michigan, minnesota, north carolina, new york, oregon) [centers for disease control and prevention (cdc) 2012], or previous experience of a significant heat wave (illinois, indiana, ohio) (cdc 2000, 2003). The survey was limited to these 30 states because of time and resource constraints and challenges in identifying appropriate personnel . Within the surveyed states (see supplemental material, figure s1), we used internet searches and phone calls to identify the most knowledgeable contact at each local health division, based on the state governance structure . We contacted county health departments and the health department in the most populous city in each of the survey states . Where possible, at least three contacts were identified with a title or role related to health officer, public health officer, emergency preparedness coordinator, and/or administrative assistant . Once all local health department contact information was compiled, 20 health divisions were randomly selected from each state to receive a survey, along with the most populous city . Developing the adaptation survey . A 38-question survey was developed by reviewing previous climate change and public health surveys and incorporating best survey practices identified from publications (dillman et al . 2009) and a professional survey developer (jsi research and training institute; http://www.jsi.com/jsiinternet/). The survey comprised multiple - choice and open - ended questions that addressed geographic boundaries, the general budget for the department, and whether specific policies and procedures were in place for extreme weather events, with an emphasis on heat . We also asked about heat - related preparation (existence of heat plans and heat definitions in 2011) and heat - related response for nine broad categories detailed in the statistical analysis section . To ensure that survey questions and choices for responses were appropriate, these were pretested with local public health department officials and public health practitioners outside the anticipated survey pool . The final survey tool was prepared using survey monkey (web - based survey tool; http://www.surveymonkey.com/), but was also available in paper format . The final survey was deployed in october 2011 to the randomly selected health departments via email, mail, and fax . Jsi research and training institute, an independent health research firm, handled survey deployment and response management . We used a three - tier survey approach to get as many responses as possible from agencies representing each county . We first surveyed local public health departments (lhds) using our original long - survey form . After three reminders, the nonrespondent lhds were sent a truncated version of the survey that asked four main questions from the original survey: do you have a heat plan? If you do have a heat plan, how long has it been in place? How did you protect your community during the heat event? Would you complete the longer survey? For those lhds that did not respond to the long or short version of the survey, the original long survey was sent to county emergency response agencies and/or personnel . We followed up with the same short survey for nonrespondent emergency response personnel as well . Multiple respondents were invited within some counties, often because of delay by initial contact, resulting in eventual responses by both initial and back - up contacts . In total, 1,062 individual respondents were invited, representing 602 local (both county and city) health departments and 460 emergency response departments . All survey responses were exported from surveymonkey to r v2.15.2 (www.r-project.org/) for analysis . Statistical analysis . We generated summaries of survey responses related to two factors: heat preparedness and response . Broadly, we investigated heat preparedness based on whether, in 2011, counties a) had existing heat plans (from any agency health departments, emergency response programs, etc .) And b) had existing heat wave definitions . We investigated heat response based on survey answers related to nine broad responses: a) communication about heat risks; b) outreach or education to the public on heat; c) collaboration with other organizations, including police and fire departments, social organizations, and medical professionals; d) opening of cooling centers; e) activation of a heat plan; f) assistance with relocation during electrical outages; g) financial assistance in response to the heat; h) hiring new staff in response to heat; and i) providing transportation . For each county, we also calculated an index of county heat response: the number of responses out of these nine broad responses performed in 2011 . This index could take values between 0 (no responses performed) to 9 (all nine responses performed) and was used in regional summaries and summaries relating county characteristics to county heat response index levels . For each survey response, the percent of counties responding affirmatively to a question was calculated based on counties providing nonmissing responses for the specific question . If multiple officials from the same county responded to the survey, an affirmative response was defined as at least one person in the county reporting use of the preparation or response . We also investigated geographic diversity by generating regional summaries of heat preparedness and heat response using regional divisions (northeast, midwest, south, and west) based on the cdc wide - ranging online data for epidemiologic research (wonder) (cdc 2013) database s regional classifications . To assess whether heat preparedness in 2011 was associated with county characteristics, we fit logistic regression models of county heat plan status in 2011 (0, county lacked a heat plan; 1, county had a heat plan), regressed separately on four county characteristics: county population, county s typical july weather (see description below), percent of county population 65 years of age, and percent of county population in poverty (percent of population below poverty as defined by the american community survey thresholds (u.s . The absolute measure of temperature for each county (i.e., typical july weather) was determined as the average of county measurements of daily maximum temperature from 2001 through 2010 . These weather data were taken from the cdc s wonder database (cdc 2013), available for all study counties except those in alaska, which were excluded from this stage of analysis . We focused on weather data in the decade before the summer of 2011 (i.e., 20012010) to adequately capture county - level prior heat experience . The county - level demographic data were collected from the american community survey s 5-year estimates for the years 20062010 (u.s . County population was modeled using a log10 transformation, whereas all other variables were untransformed . Based on these logistic regression models, we estimated the probability of a county having a heat plan in place in 2011 at the 25th and 75th percentile values for each county characteristic and calculated the odds ratio (or) for an increase in this (iqr). We also investigated whether the heat response in 2011 (index 09) was associated with county characteristics, how extreme the weather was during the 2011 summer, and the presence of a heat plan . To do this, we calculated means of this index across the counties by quantile bins of each of these county characteristics . County - level demographic data for this analysis were the same as previously described . To measure how extreme the 2011 summer was in each county, we calculated the deviation above normal temperature (i.e., the difference between the average of daily maximum temperatures in july 2011 in the county and the county s average of daily maximum july temperature in 20012010) (cdc 2013), in degrees fahrenheit . 190 responded (32%). In 2011, 40% of 188 counties with nonmissing responses had existing heat plans, and 30% of 185 responding counties had heat wave definitions (table 1). Of responding counties with heat plans (n = 72), less than half had created the plan recently (12% within 1 year of the summer of 2011; 33% within 13 years) (see supplemental material, table s2). A few counties had long - standing heat plans, with plans created 10 years before 2011 (12%). Of counties responding (n = 71), most had last updated their heat plan within the last year (73%) (see supplemental material, table s2). Summaries of county - level preparedness and response to the extreme heat of 2011 in 30 u.s . States . Of counties with nonmissing responses (n = 185) fifty - four counties included details describing their heat wave definitions; of these, 15 (28%) used definitions based on the u.s . National weather service or noaa heat wave definitions (noaa 2009), four (7%) used state - wide definitions, and two (4%) used definitions based on city - specific heat health watch systems (see supplemental material, table s2). For several other counties, heat wave definitions were tied to specific temperatures (examples: in the 90s; 98f day and 89f night for a 18-hr period; 3 consecutive days over 101f); other definitions accounted for air moisture either explicitly or through the use of heat index as a trigger (temperatures above 95f with humidity above 50%; heat index above 115f). Other heat wave definitions were more qualitative, and identified heat wave as temperatures above normal, particularly when temperatures caused health concerns . On average, midwestern, northeastern, and southern counties in this study were more prepared for the extreme heat of 2011, with 48% (n = 64), 46% (n = 26), and 44% (n = 55) of surveyed counties in these regions, respectively, having existing heat plans in 2011 (figure 1). Western counties were, on average, less prepared, with 22% (n = 40) of surveyed counties reporting heat plans in 2011 . (a) percent of counties in each region that had an existing plan in 2011 . (b) average heat response index for each region in 2011 (possible values between 0 and 9, with higher index values indicating more extensive heat response). Heat plans were more likely in more populous counties (table 2, figure 2). Based on a logistic regression of heat plan status on county population (n = 185), 34% of counties with populations of 22,000 (25th percentile across all county populations) were expected to have heat plans, versus 48% of counties with populations of 161,000 (75th percentile across all county populations) (table 2). The or for having a heat plan for an iqr increase in county population was 1.83 (95% ci: 1.23, 2.72) (table 2). However, analysis of or and temperature quantiles suggests the relationship between heat plan status and average july temperature may be nonlinear and not statistically significant, as counties with hottest july temperatures did not have the highest prevalence of heat plans (table 2, figure 2). Heat plan status was inversely associated with the percentage of the population 65 years of age (or for an iqr increase in percent of population 65 years of age: 0.76; 95% ci: 0.52, 1.12) and with county poverty rates (or for an iqr increase in poverty status: 0.79; 95% ci: 0.55, 1.14), although again neither estimate was statistically significant (table 2). Association between county characteristics and the probability of having an existing heat plan in 2011, based on a simple logistic regression of heat plan status on each community characteristic . County characteristics: distributions and associations with heat preparedness in 2011 (n = 185). (a) distributions of county population, july climate (average of daily july maximum temperature values, 20012010), percent poverty, and percent of population 65 years . (b) percent of counties with heat plans in 2011 in each quantile bin for county characteristics . All counties were divided into five bins based on the county characteristics, with breaks between bins at the 20th, 40th, 60th, and 80th percentiles of the characteristic; black vertical lines show divisions between bins as well as minimum and maximum values; points are positioned on the x - axis at the median characteristic value for the counties within the bin . Response to the extreme heat of 2011 . Based on nonmissing survey responses, for which sample numbers are provided in table 1, the most frequent county responses to the 2011 extreme heat was communication about heat risks (73%) and outreach or education to the public (64%) (table 1). Counties also reported collaboration with other organizations (46%), opening cooling centers (40%), or activating existing heat plans (24%). Few counties reported assisting with relocation during electrical outages (4%), providing financial assistance (3%), providing transportation (2%), or hiring new staff in response to the heat (1%). Survey responses for the next several paragraphs are captured in supplemental material, table s2 . Based on nonmissing responses (n = 180), the most common methods of communication about heat risks were public service announcements (53%) and websites (48%). Several counties also reported communicating about heat through social media (30%), flyers and posters (18%), and email messages (17%). Few counties reported communicating through joint events with other groups (8%), telephone hotlines (6%), door - to - door campaigns (4%), or telephone calls (1%). Of counties responding (n = 177), many reported providing outreach or education to people working with the elderly (37%), people with certain medical conditions (37%), people with low incomes (29%), and health care providers (25%). Fewer counties reported providing outreach to people who are mobility challenged (15%), people working with the homeless (12%), people living in high - rise apartment buildings (10%), and people with nervous system disorders (9%). Some counties wrote in other forms of education or outreach, including outreach geared toward children through schools and child care centers; outreach to people working outdoors; and outreach to agencies serving the mentally ill . Of 145 counties with responses, 46% reported collaborating with at least one other organization to respond to the extreme heat of 2011 . Collaborations were common among all four types of organizations presented in the survey: among counties with nonmissing responses, 36% reported collaborating with medical professionals, 28% with social / civic organizations, 25% with fire departments, and 23% with police departments . Some counties gave examples of the social or civic organizations with which they collaborated, including departments of social services, the red cross, united way, salvation army, meals on wheels, rotary, local shelters, and local libraries . Most of the counties that opened cooling centers opened five or fewer centers (83%) and most opened the cooling centers for 5 days of the summer (58%). However, some counties reported opening more cooling centers or operating cooling centers for longer: 12% of counties reported opening> 10 cooling centers, and 5% of counties reported keeping cooling centers open all summer . Of the counties that activated heat plans during the summer of 2011, other counties activated heat plans for longer periods, and one county kept its heat plan activated the entire summer . In responding counties (n = 46), the decision to activate a heat plan was most commonly prompted at least in part by an internal decision (46%), and some counties also based their decision on a predefined trigger in a heat wave early warning system (41%), suggestions from an emergency preparedness team (30%), directives or suggestions from the state health department (28%), or a noticeable spike in heat - related sickness and deaths (22%). Seven counties (4%) of 177 reported assisting with relocation during electrical outages related to the heat of 2011 . Seventy - three of these counties reported that relocation was irrelevant because their county experienced no electrical outages during the 2011 summer . Six (3%) of 177 counties reported providing any type of financial assistance in response to the heat . Assistance was provided to help pay for utility bills in five counties and to help pay for food / water and for air conditioning / fans in one county each . Four counties (2%) reported providing transportation as a response to the heat of 2011 (n = 179). This transportation was provided to people who called and requested transportation during the heat as well as residents of a specific apartment or neighborhood . One county reported hiring new staff in response to the heat (n = 167). Based on responses about these nine broad categories of heat response (table 1), adequate data existed to calculate a response index (county - specific sum of how many of the nine responses were undertaken in 2011) for 117 counties . Across these counties, this response index ranged between 0 and 7, with a median of 2 (the highest potential value of this index is 9). When counties were divided by region, counties in the northeast and midwest had, on average, more extensive responses to the 2011 heat, with average heat response indices of 3.9 (n = 24) and 3.0 (n = 50) respectively (i.e., on average counties performed between three and four of nine considered heat responses) (figure 1). Counties in the south and west had lower average heat response indices [south: 2.6 (n = 35); west: 1.2 (n = 30)]. Heat response was greater if a county had a heat plan in 2011 (figure 3). Heat response was also associated with county population, with larger counties more likely to perform more extensive heat response than smaller counties . The heat response index was also higher in counties where july 2011 temperatures were more extreme compared with average july temperatures in the previous decade, and in counties with a lower proportion of residents 65 years of age or with a lower proportion of residents in poverty (figure 3). County characteristics: distributions and associations with heat response in 2011 (n = 117). (a) distributions of heat plan status, county population, difference between july 2011 and july climate (average of daily july maximum temperatures in 2011 average of daily july maximum temperature values, 20012010), percent poverty, and percent of population 65 years of age . (b) average heat response index in 2011 in each quantile bin for that county characteristics . All counties were divided into five bins based on the county characteristics, with breaks between bins at the 20th, 40th, 60th, and 80th percentiles of the characteristic; black vertical lines show divisions between bins as well as minimum and maximum values; points are positioned on the x - axis at the median characteristic value for the counties within the bin . The survey also asked counties whether they had evaluated their own response to the heat of 2011 . Twelve of the 176 responding counties (7%) reported that they had evaluated their heat response . However, the response index for these counties ranged from 1 to 5, suggesting more variation between these counties in response than was identified by their self - analysis . This study provides a current, national review of heat adaptation at the county level shortly after the summer of 2011, one of the hottest u.s . We collected these data shortly after the hot summer to reduce recall bias and provide a better picture of on - the - ground adaptation . The size of our study population and number of respondents were large compared to previous surveys (190 responding counties in 30 u.s . States), and we had a response rate (32%) comparable with most previous surveys (see supplemental material, table s1). Our survey selection was randomized and, coupled with the response rate and state participation, represented a diversity of u.s . Most counties were likely underprepared for the extreme heat of 2011: most lacked either heat plans (only 40% of counties who responded to questions about heat plans reported having them) or heat wave definitions (only 30% of counties with nonmissing answers reported having heat wave definitions). (2010) similarly found that most of their survey respondents had not established a plan to deal with extreme heat . Public health departments believed that lack of human and financial resources could be a key constraint preventing climate change from being incorporated into public health preparedness, but the survey did not address specific costs associated with climate change adaptation (edf 2008). Our survey collected information about how heat preparedness and response was financed, which will be part of a future analysis . Counties with heat plans performed, on average, between three and four of the nine broad responses we considered in this analysis, compared with, on average, between one and two responses performed by counties without heat plans (figure 1). Further, heat plans may be critical to ensure the effectiveness of specific heat responses . For example, although heat wave early warning systems can save lives, their success can be limited by not having clear decision - making protocols among the relevant institutions and end users or their advocates (kovats and ebi 2006); such protocols could be established through heat planning . Only weak evidence was found to suggest that counties where summers are typically hotter (based on averages of daily july maximum temperature measurements for 20012010) were more likely to have established heat plans in preparation for extreme heat by 2011 than were milder counties . The health impacts of heat are not necessarily more severe in hot regions of the united states than in milder regions (anderson and bell 2009; anderson et al . 2013), which suggests a level of adaptation to typical summer temperatures within a community . The most common county responses to heat were communicating about heat risks and providing outreach or education to the public . Many counties reported using newer technologies to communicate about heat, including websites, social media, and emails . Although websites and internet access are powerful and cost - effective communication tools, health inequalities underpinned by differential access to health services may be further reinforced by disparities in access to the internet (e.g., ethnicity, education, and economic resources). In other words, the less educated have fewer resources and potentially less access to health care, and could be left out of the communication loop (gilmour 2007), particularly when communication efforts rely on newer technologies . This can become especially dangerous for isolated populations who may not hear about heat risks . Some underused methods, based on our survey, including telephone calling and door - to - door campaigns, could be more useful to reach these isolated populations . Additionally, preexisting beliefs about personal resilience and personal heat adaptation behaviors need to be accounted for in communication to the general public, potentially through local health departments or collaborators, such as the medical community (astrom et al . Although many of our responding counties reached out to the elderly and those with medical concerns or low income, fewer counties (25% of responding counties) reached out to those working with the homeless or those with mobility challenges or nervous system disorders, or those living in high - rise residences . Outreach to these vulnerable populations may represent a missed opportunity to limit heat impacts in many u.s . Encouragingly, our survey found that many counties worked with organizations such as medical professionals, the fire department, the police department, and social organizations such as the red cross, rotary, and meals on wheels to respond to the heat . Planning and programming for heat - health protection will likely be most effective if performed in a bottom - up and community - specific manner and as a collaborative effort among multiple levels of government and local stakeholders (e.g., community / health centers, hospitals, clinics, volunteer groups, transit officials, schools, emergency services) (yardley et al . 2011). The need for the collaborations between health and various other agencies to help identify these populations is essential . For example, some organizations have even started developing registries for those who need to be checked on during extreme heat events . A good example of such a registry is from the city of toronto (2012). It was rare for counties to perform some of the responses included in the survey, like assisting with relocation during electrical outages (only seven counties), providing financial assistance (only six counties), providing transportation (only four counties), and hiring new staff (only one county) (table 1). For example, heat wave early warning systems coupled with direct interventions such as buddy systems and home visits, used, for example, in philadelphia, pennsylvania, could potentially play an important role in reducing heat related deaths (kalkstein et al . Setting up such programs from scratch may be costly to establish and maintain in terms of time and effort and therefore less common compared with outreach and communication strategies . Counties might want to explore policy changes or resource sharing among local stakeholders (e.g., private industry, nonprofits) to provide these more costly services to the local community . In our study, heat response was lower in counties with a higher percentage of people 65 years of age, where efforts to improve heat preparedness and response may provide even greater benefits than in counties with younger populations . Many older americans live in regions that could be hard hit by extreme events associated with climate change, including heat waves, and the u.s . Population is projected to include 88.5 million americans 65 years of age by 2050 (gamble et al . Respondents also noted that formal evaluations of their heat plans and response were not being conducted . Evaluation results can support policy that could potentially save more lives as more funding goes toward these types of public health preventive activities . Study limitations and future research directions . Because selection was randomized, areas with high populations of low - income, minority populations and recognized tribal areas were not a large segment of the survey response pool . Another challenge was that some states had more centralized health systems that were managed at the county level, whereas others had health services distributed across health districts not defined by county lines (i.e., alaska, connecticut, iowa, and maine). Some respondents were uncertain concerning heat preparedness and response within their county . Finally, we had little information about counties that were invited to participate in our survey but did not respond, so our analysis cannot be used to infer heat preparedness and response in other counties in these states or in states that were not surveyed . Although this survey provides a description of the current status of heat preparedness and response within> 100 u.s . Counties, more research is necessary to evaluate and quantify the effectiveness of the heat responses described in this article, particularly in terms of preventing negative heat - related health impacts (i.e., heat - related morbidity and mortality). Few studies have evaluated the associations between the responses (adaptation practices) we present here and heat - related morbidity / mortality . However, researchers have documented reductions in the risks for heat - related health incidences related to heat response plans and other related efforts (michellozzi et al . Buildings and infrastructure, the availability of social services, the impact of heat and heat island mitigation activities, and community support networks are just a few of the factors requiring further exploration at the county level . Some particular adaptation strategies that can be considered include having persons with special medical needs register with their local emergency management agency to ensure they will receive necessary services or evacuation assistance, coordinating service providers, creating vulnerability mapping to assist with planning, and developing strategies to reduce the urban heat island effect (ebi and semenza 2008; wilhelmi and hayden 2010). The various structures for planning and heat response throughout the united states are diverse and complex, but our study shows evidence of a lack of action and planning, regardless of community size . In this study, unlike others, we asked questions specifically to understand exactly how counties plan and respond, filling a current gap in this area of research . To get a workable baseline we hope that this method will be used in future studies and serve as a platform to expose existing idiosyncrasies and make climate change preparedness and response a unique challenge for each and every community . Given that heat waves are expected to become more frequent and severe under climate change (intergovernmental panel on climate change 2012), it is critical to enhance local education and planning for extreme heat events to enhance collaborations and reach beyond the more recognized vulnerable populations (i.e., the elderly) to other less recognized populations (i.e., those with mobility challenges, the homeless) during heat events . Information clearinghouses [such as that offered by the georgetown climate change collaborative (georgetown climate center 2013)] can help foster the exchange of information on best practices and resources for outreach, education, and emergency planning . In the coming years, extreme heat in the united states will continue to affect rural and urban, low - income and wealthy, and prepared and unprepared communities . Thus, planning and implementation of heat adaptation programs, which are not widespread in the united states, have great potential for reducing the toll of heat on health in this nation.
The study group comprised 15 patients of both sexes, with age ranging from 13 to 65 years, attending the outpatient department . Only those periapical lesions which appeared as radiolucent on the radiographs were included in the study group . Those periapical lesions which appeared as radiopaque on the radiographs were eliminated from the study group . All the subjects underwent ultrasonographic examination using ge logic 400 mr3 color doppler machine having multifrequency linear transducer using 711 mhz frequency at sheethal diagnostics, davangere, india . All the examinations including real - time and color doppler imaging were performed in the periapical area extraorally . The ultrasonographic images were analyzed based on the following principles: cystic lesion: a hypoechoic well - contoured cavity surrounded by reinforced bone walls, filled with fluid, and with no evidence of internal vascularization on color doppler examination.granuloma: a poorly defined hypoechoic area, showing rich vascular supply on color doppler examination.mixed lesion: predominantly hypoechoic area with focal anechoic area, showing vascularity in some areas on color doppler examination . Cystic lesion: a hypoechoic well - contoured cavity surrounded by reinforced bone walls, filled with fluid, and with no evidence of internal vascularization on color doppler examination . Granuloma: a poorly defined hypoechoic area, showing rich vascular supply on color doppler examination . Mixed lesion: predominantly hypoechoic area with focal anechoic area, showing vascularity in some areas on color doppler examination . The results of ultrasound were correlated with radiographic and histological findings and were statistically analyzed . All the subjects underwent ultrasonographic examination using ge logic 400 mr3 color doppler machine having multifrequency linear transducer using 711 mhz frequency at sheethal diagnostics, davangere, india . All the examinations including real - time and color doppler imaging were performed in the periapical area extraorally . The ultrasonographic images were analyzed based on the following principles: cystic lesion: a hypoechoic well - contoured cavity surrounded by reinforced bone walls, filled with fluid, and with no evidence of internal vascularization on color doppler examination.granuloma: a poorly defined hypoechoic area, showing rich vascular supply on color doppler examination.mixed lesion: predominantly hypoechoic area with focal anechoic area, showing vascularity in some areas on color doppler examination . Cystic lesion: a hypoechoic well - contoured cavity surrounded by reinforced bone walls, filled with fluid, and with no evidence of internal vascularization on color doppler examination . Granuloma: a poorly defined hypoechoic area, showing rich vascular supply on color doppler examination . Mixed lesion: predominantly hypoechoic area with focal anechoic area, showing vascularity in some areas on color doppler examination . The results of ultrasound were correlated with radiographic and histological findings and were statistically analyzed . Out of the 15 subjects, 12 (80%) were diagnosed as periapical cyst ultrasonographically, whereas 14 (93.3%) were diagnosed as periapical cyst histopathologically and the correlation between ultrasonographic and histopathologic diagnoses was found to be in 12 (85.7%) subjects [table 1 and graph 1]. Correlation of histologic features with ultrasonographic findings correlation of histologic features with ultrasonographic findings out of the 15 subjects, 2 (13.3%) were diagnosed as periapical granuloma ultrasonographically, whereas 1 (6.7%) was diagnosed as periapical granuloma histopathologically and the correlation between ultrasonographic and histopathologic diagnoses was found to be in 1 (50%) subject . Out of the 15 subjects, 1 (6.7%) was diagnosed as periapical abscess ultrasonographically, whereas no subjects were diagnosed as periapical abscess histopathologically and there was no correlation between ultrasonographic and histopathologic diagnoses in this subject . The correlation between histopathologic and ultrasonographic diagnoses was found to be in 13 (86.7%) subjects . Comparing the results of ultrasonography with the histopathologic features, the sensitivity, specificity, positive predictive value, and negative predictive value of ultrasonography to diagnose periapical lesions were calculated . In our study, tp denotes true positive = 12 subjects, tn denotes true negative = 1 subject, fp refers to false positive = 2 subjects, fn is false negative = 0 subjects, and n is total number of patients = 15 subjects [table 2 and graph 2]. Out of the 15 subjects, 12 (80%) were diagnosed as periapical cyst ultrasonographically, whereas 14 (93.3%) were diagnosed as periapical cyst histopathologically and the correlation between ultrasonographic and histopathologic diagnoses was found to be in 12 (85.7%) subjects [table 1 and graph 1]. Correlation of histologic features with ultrasonographic findings correlation of histologic features with ultrasonographic findings out of the 15 subjects, 2 (13.3%) were diagnosed as periapical granuloma ultrasonographically, whereas 1 (6.7%) was diagnosed as periapical granuloma histopathologically and the correlation between ultrasonographic and histopathologic diagnoses was found to be in 1 (50%) subject . Out of the 15 subjects, 1 (6.7%) was diagnosed as periapical abscess ultrasonographically, whereas no subjects were diagnosed as periapical abscess histopathologically and there was no correlation between ultrasonographic and histopathologic diagnoses in this subject . The correlation between histopathologic and ultrasonographic diagnoses was found to be in 13 (86.7%) subjects . Comparing the results of ultrasonography with the histopathologic features, the sensitivity, specificity, positive predictive value, and negative predictive value of ultrasonography to diagnose periapical lesions were calculated . In our study, tp denotes true positive = 12 subjects, tn denotes true negative = 1 subject, fp refers to false positive = 2 subjects, fn is false negative = 0 subjects, and n is total number of patients = 15 subjects [table 2 and graph 2]. Diagnostic validity of ultrasonography diagnostic validity of ultrasonography out of the 15 subjects in the present study, ultrasonographic examination showed 12 (80%) periapical cysts whereas histopathologic diagnosis revealed 14 (93.3%) periapical cysts . One of the periapical cysts (2 subject) was misdiagnosed ultrasonographically as periapical granuloma . This misinterpretation was because of the content of the lesion which showed high density equivalent to a solid lesion, even though with lack of vascular supply on color doppler examination . One of the periapical cysts (11 subject) was misdiagnosed ultrasonographically as periapical abscess [figure 1]. This lesion showed both hypoechoic and anechoic regions, i.e., mixed echogenic patterns with numerous scattered internal echoes and varying density values within the lesion suggestive of periapical abscess . Similar to our observations, dib et al . In their study of 72 intraosseous lesions of the jaw the reason for this could be due to the presence of thick cortical bone covering the lesion, the occurrence of infected cysts and solid areas within the cystic lesion . The cystic lesions on the ultrasound imaging appeared as anechoic lesion in six cases, turbid fluid in one case, scattered internal echoes in one case, and posterior enhancement in three cases [figures 2 and 3]. Ultrasonograph showing well - defined hypoechoic lesion with vascularity ultrasonograph showing well - defined anechoic lesion ultrasonograph showing well - defined anechoic lesion the internal echoes present in infected cysts and odontogenic keratocyst could be due to dense particles (cholesterol clefts in the infected cysts) in the cystic fluid and keratin in odontogenic keratocyst . The density values varied within the lesion in case of infected cysts and odontogenic keratocyst, indicative of difference in the contents within the lesion, which reflects the disease process . Out of the 15 subjects in the present study, ultrasonographic examination showed 2 (13.3%) periapical granuloma among which 1 (50%) was diagnosed histopathologically as periapical granuloma and the other as periapical cyst . This observation is similar to the observations made by cotti et al . And gundappa et al, where they had accurately diagnosed all the periapical granulomas (100%) ultrasonographically . Thus, the observations from the present study infer that the diagnostic validity of ultrasonography is similar to that made by dib et al, where they have reported 92.8% accuracy and less compared to that made by cotti et al, and gundappa et al . Where they have reported 100% accuracy . In all the subjects, it pictorially represents the anatomic location of the periapical lesion in relation to the particular involved tooth and it provides size of the lesion [figures 5 and 6], whereas ultrasound was able to accurately identify the underlying disease process reasonably accurately and the size measurements in three dimensions using the ultrasound software . Panoramic radiograph showing unilocular radiolucent lesion occlusal radiograph showing unilocular radiolucent lesion intraoral periapical radiograph showing radiolucent lesion a major concern over the performance of ultrasonography was the difficulty in attributing the lesions to a specific area of the maxillary bone because the dental landmarks (i.e. Roots) were not specifically visualized . This made it difficult to orient the lesion in the different regions of the mouth without using a reference radiograph . Out of the 15 subjects in the present study, ultrasonographic examination showed 12 (80%) periapical cysts whereas histopathologic diagnosis revealed 14 (93.3%) periapical cysts . One of the periapical cysts (2 subject) was misdiagnosed ultrasonographically as periapical granuloma . This misinterpretation was because of the content of the lesion which showed high density equivalent to a solid lesion, even though with lack of vascular supply on color doppler examination . One of the periapical cysts (11 subject) was misdiagnosed ultrasonographically as periapical abscess [figure 1]. This lesion showed both hypoechoic and anechoic regions, i.e., mixed echogenic patterns with numerous scattered internal echoes and varying density values within the lesion suggestive of periapical abscess . Similar to our observations, dib et al . In their study of 72 intraosseous lesions of the jaw the reason for this could be due to the presence of thick cortical bone covering the lesion, the occurrence of infected cysts and solid areas within the cystic lesion . The cystic lesions on the ultrasound imaging appeared as anechoic lesion in six cases, turbid fluid in one case, scattered internal echoes in one case, and posterior enhancement in three cases [figures 2 and 3]. Ultrasonograph showing well - defined hypoechoic lesion with vascularity ultrasonograph showing well - defined anechoic lesion ultrasonograph showing well - defined anechoic lesion the internal echoes present in infected cysts and odontogenic keratocyst could be due to dense particles (cholesterol clefts in the infected cysts) in the cystic fluid and keratin in odontogenic keratocyst . The density values varied within the lesion in case of infected cysts and odontogenic keratocyst, indicative of difference in the contents within the lesion, which reflects the disease process . Out of the 15 subjects in the present study, ultrasonographic examination showed 2 (13.3%) periapical granuloma among which 1 (50%) was diagnosed histopathologically as periapical granuloma and the other as periapical cyst . This observation is similar to the observations made by cotti et al . And gundappa et al, where they had accurately diagnosed all the periapical granulomas (100%) ultrasonographically . Thus, the observations from the present study infer that the diagnostic validity of ultrasonography is similar to that made by dib et al, where they have reported 92.8% accuracy and less compared to that made by cotti et al, and gundappa et al . Where they have reported 100% accuracy . In all the subjects, conventional radiography provided the preliminary information about the lesion [figure 4]. It pictorially represents the anatomic location of the periapical lesion in relation to the particular involved tooth and it provides size of the lesion [figures 5 and 6], whereas ultrasound was able to accurately identify the underlying disease process reasonably accurately and the size measurements in three dimensions using the ultrasound software . Panoramic radiograph showing unilocular radiolucent lesion occlusal radiograph showing unilocular radiolucent lesion intraoral periapical radiograph showing radiolucent lesion a major concern over the performance of ultrasonography was the difficulty in attributing the lesions to a specific area of the maxillary bone because the dental landmarks (i.e. Roots) were not specifically visualized . This made it difficult to orient the lesion in the different regions of the mouth without using a reference radiograph . With its potential usefulness to differentiate the periapical lesions, ultrasonography can be considered as a better imaging modality with improved efficacy when compared to conventional radiography . In comparision to histopathologic diagnosis, ultrasonography showed less accuracy and thus can be considered to be a supplementary tool in the differential diagnosis of periapical lesions . Further research can be directed in larger samples and in different types of periapical lesions to assess the diagnostic validity of ultrasonography.
In africa, pelvic inflammatory disease (pid) and its sequelae are a predominant cause of gynecologic morbidity [1, 2]. These include tubal factor infertility, ectopic pregnancy, chronic pelvic pain, and recurrent pelvic infections [3, 4]. Hiv-1 seroprevalence in women with pid is consistently 27 times greater than measured in matched populations without pid [57]; both infections are most commonly acquired through unprotected sexual activity . Prompt diagnosis and treatment of women with upper genital tract infections is important in reducing morbidity, but it is complicated by lack of a sensitive and specific clinical and laboratory diagnostic test . Laparoscopy is the gold standard for the diagnosis of salpingitis, but is not practical for routine clinical practice . Kiviat et al . Evaluated women with clinical pid; evidence of endometritis as defined by 1 plasma cell (pc) and 5 polymorphonuclear lymphocytes (pmn) per high - powered field (hpf) was 92% sensitive and 87% specific compared with visual findings of salpingitis determined by laparoscopy . Using the same diagnostic criteria in a study of acute salpingitis in kenya, plasma cell endometritis as defined by 1 pc / hpf was identified in 49% of women with salpingitis: this increased with disease severity and hiv - infection . Studies on hiv-1-infected women have found an increased prevalence of plasma cell endometritis even in the absence of clinical disease [10, 11]. Thus, we conducted this analysis to determine the optimum endometrial histopathological criteria for predicting salpingitis in a population with a high hiv-1 seroprevalence . We anticipate that these data will help to plan future clinical trials, increase the understanding of the pathogenesis of upper genital tract infection among hiv-1 infected women, and in certain circumstances provide a tool to confirm the clinical diagnosis of pid . Briefly, between april 2000 and july 2002, women aged 1840 admitted to kenyatta national hospital (knh) acute gynecology ward with a complaint of lower abdominal / pelvic pain for 2 weeks or less plus one or more of the following signs or symptoms: temperature 38c, dysuria, and complaint of abnormal vaginal discharge were eligible for enrollment . After induction of anesthesia, an endometrial biopsy was obtained with a pipelle suction curette (unimar, inc ., wilton, conn). At laparoscopy, samples from peritoneal fluid, tubal ostia, and pyosalpinx / tubo - ovarian abscess (toa) were obtained for n. gonorrhoeae and c. trachomatis pcr . Using the jacobson and westrom criteria, the severity of acute salpingitis was graded as (1) mild (tubal erythema or edema, mobile tubes, and with or without spontaneous exudate), (2) moderate (marked tubal erythema and edema, limited tubal mobility, questionable or no tubal patency, and gross exudate), and (3) severe (pyosalpinx or toa). Women desiring permanent sterilization underwent laparoscopic tubal ligation preceded by an endometrial biopsy obtained using a pipelle suction curette . Hiv-1-seropositive controls were enrolled from an hiv care and treatment clinic at the center for respiratory disease research at the kenya medical research institute . Subjects had no clinical evidence of pid . Enrollment and study procedures of the hiv-1-seropositive control group are detailed elsewhere . After informed consent was obtained, an endometrial pipelle biopsy was obtained in the research clinic . Samples from the cervix, endometrium, fallopian tube, and abscess were examined by pcr (roche molecular diagnostics, pleasanton, calif, usa) for n. gonorrhoeae and c. trachomatis . Endometrial specimens were fixed in 10% buffered formalin, processed, and stained with hematoxylin, eosin, and methyl green pyronin . Pmns in glands and pcs in stroma were counted per high - power field . One pathologist (nk) who was blinded to the patients' diagnosis read the slides . Serum was tested for hiv antibodies by elisa (detect hiv, biochem immunosystems, montreal, canada) with positive results confirmed by a second elisa (recombigen, cambridge biotech, ireland). Univariate analyses used chi - square and fisher's exact tests for categorical data and student's t - test for continuous variables . Logistic regression was done for multivariate analysis . One hundred and sixty women were enrolled with clinical pid, 140 (88%) had laparoscopically confirmed salpingitis, 125 (89%) of whom had an endometrial biopsy specimen: 56 (45%) had mild, 31 (25%) had moderate, and 38 (30%) had severe disease based on laparoscopic criteria . Nineteen women had other diagnoses at laparoscopy including appendicular abscess (n = 2), endometriosis (n = 1), ovarian cyst (n = 12), frozen pelvis (n = 1), pelvic tuberculosis (n = 1), cancer of the sigmoid volvulus with abscess (n = 1), and ovarian torsion (n = 1). Asymptomatic women (n = 20) desiring permanent sterilization underwent laparoscopic tubal ligation and served as hiv-1-negative controls . A single control subject had a sticky exudate emanating from the fallopian tubes and was excluded from the analysis leaving 19 hiv-1-seronegative controls . Forty - five asymptomatic hiv-1-seropositive controls were enrolled from an hiv care clinic; one woman had c. trachomatis detected . Forty - eight (38%) of the women with salpingitis were hiv - seropositive . Women with salpingitis were younger, less likely to be married, and less likely to have ever used contraception (table 1). As expected, none of the hiv-1-seronegative controls had signs or symptoms consistent with pid, and none were infected with n. gonorrhoeae or c. trachomatis . However, t. vaginalis was detected in a similar proportion of salpingitis cases (23%) and hiv - seronegative controls (21%) (table 1). Of the 125 women with salpingitis, endometrial biopsies from 107 (86%) were evaluated histological . Inadequate biopsies corresponded to endometrial specimens demonstrating sloughing, frank pus, and lack of tissue . In general, more severe disease as demonstrated by higher clinical severity score (css) (15.5 versus 13, p <.03) and severity of salpingitis based on laparoscopic findings (p - trend <.04) was associated with unevaluable endometrial biopsy results (table 2). Similarly, history of depomedroxyprogesterone acetate (dmpa) was associated with an increased likelihood of obtaining an unevaluable biopsy (p = .02). Hiv - infected women were more likely to have an unevaluable endometrial biopsy (57% versus 36%, p <.05) than hiv - uninfected women . Although not significant, participants with an inadequate endometrial histological specimen had a higher prevalence of gonorrhea compared to those with an adequate biopsy (23% versus 12% p <0.23) (table 2). In multivariate analysis, after controlling for factors found significant in univariate analysis, the use of dmpa at any time (adjusted or = 3.1, 95% ci 1.18.5), hiv-1 infection for women with mild (aor = 4.6, 95% ci 1.118.3) but not moderate salpingitis (aor = 0.89, ci 0.155.3), or severe salpingitis (aor = 2.63, ci 0.6810.2) was associated with an increased odds of an unevaluable endometrial biopsy . In addition, 12 (63%) of 19 specimens from hiv-1-seronegative subjects were evaluable for histopathology . We reviewed the distribution of pmn and pc by hiv-1 serostatus and severity of salpingitis . Women with severe salpingitis regardless of hiv-1 serostatus had the highest frequency of pmn and pc per high - power field . Only two patients with hiv-1 infection and salpingitis did not have pmn found in the endometrium . Although pmn density did not increase with severity of salpingitis among women with hiv-1 infection (p - trend = .49), this association was significant for hiv-1-uninfected women with salpingitis (p - trend = .05). In contrast, the frequency of pcs increased with severity of salpingitis among those with hiv-1 infection (p - trend = .04), but not among hiv-1 uninfected (p - trend = .14). Furthermore, hiv-1 infection was associated with a higher frequency of pcs / hpf (p - trend <.001), and presence of lymphoid follicles (p <.04). Only 2 (6%) of 34 hiv-1-infected women with salpingitis did not have any plasma cells present in the endometrium versus 23 (41%) of 56 hiv-1-uninfected women with salpingitis . We next set out to determine the sensitivity, specificity, and positive predictive value of four histopathologic criteria for diagnosis of endometritis in comparison to the laparoscopic diagnosis of salpingitis . The four rules evaluated included: (a) 3 pmn and 1 pc per high - power field, (b) 1 pmn and 1 pc per high - power field, (c) 1 pmn per high - power field, and (d) 1 pc per high - power field . Women with moderate and severe disease were grouped together and compared to women with mild salpingitis and to the two control groups . Table 3 outlines the comparison between the laparoscopic diagnosis for mild and moderate / severe salpingitis and the four histological rules stratified by hiv-1 serostatus . Because the diagnosis of the moderate and severe disease requires more objective evidence of tubal inflammation (e.g., pus from tubes, pyosalpinx, abscess, and fresh adhesions) than mild disease, we chose to gauge the sensitivity of each histological rule using laparoscopic evidence of moderate / severe salpingitis as the gold standard . Rule a, although less sensitive than rules b through d for women with moderate / severe salpingitis (hiv - seropositive = 74% versus 63%; hiv - seronegative; 93% versus 75%), was the most specific, demonstrating endometritis in 25% and 7% of hiv-1-seronegative and hiv-1-seropositive controls, respectively, in comparison to 58%67% and 38%62% for rules b through d . Among the 19 women enrolled with a clinical diagnosis of pid, but who did not have salpingitis on laparoscopy, rule a had the least false positive, while rule d, at least one plasma cell, scored the highest false - positive rate . This study had three key findings: (1) 3 pmn and 1 pc per hpf as a histologic criteria for the diagnosis of moderate to severe salpingitis, while performing better than the other criteria, appears to have limited utility even more so for cases of mild salpingitis; (2) endometrial specimens were often unevaluable for histopathology, and unevaluable specimens were more likely in subjects with severe salpingitis and hiv - infection, and thus may affect the utility of endometrial histopathology to confirm the clinical diagnosis of pid in similar settings; (3) the pmn response increased with disease severity for hiv-1 seronegative but not hiv-1 seropositive women with salpingitis . Since kiviat et al . Published their paper, histologic endometritis has been used as a surrogate marker for salpingitis, especially in the study of mild to moderate pid . Even though the criteria for histologic endometritis had never been validated in hiv-1-infected populations, several studies of pid were conducted in high hiv-1 seroprevalence settings [6, 7, 9, 12, 14]. 1990, criteria for 5 pmns and 1 pc for the diagnosis of pid . In the kiviat et al . Cohort, n. gonorrhoeae and/or c. trachomatis was found in 49% of the patient population; in comparison, the cohort in our study had a high hiv-1 prevalence and a combined gonorrhea and/or chlamydia prevalence of 18% (table 1). Similar to another report, only 72% of endometrial biopsies in our study were evaluable . The increased frequency of unevaluable endometrial biopsies in women with severe salpingitis, likely due to increased endometrial sloughing and presence of pus, and hiv-1 infection further limits the utility of endometrial histopathology as a diagnostic tool for studies of pid in similar populations . The low sensitivity of histologic endometritis for mild salpingitis amongst women symptomatic for pid was unexpected . Studies of endometritis in populations of asymptomatic women have consistently demonstrated a relatively high prevalence of endometritis [10, 15, 16] which led authors to describe endometritis as an intermediate infection to pid . . Studied hiv-1-infected women presenting to a family planning clinic and found endometritis in 38% of participants . This is a higher prevalence than what we found in hiv-1-negative women (16%) and a lower prevalence than what we found in hiv-1-seropositive women (57%) with mild salpingitis using less stringent criteria for endometritis . An alternative explanation may result from the subjectivity of the laparoscopic criteria for mild salpingitis that leads to misclassification of cases . The distribution of pmns and pcs in the endometrium of women with salpingitis was affected by hiv-1 serostatus and disease severity . Pmns are only found in the healthy endometrium during menses, and form part of the endometrial immune response, they are also the first line immune defense against bacterial infections . The increased density of pmn with severe disease in hiv-1-uninfected but not in hiv-1-infected women with salpingitis is not well understood . Consistent with other studies [7, 9, 10, 14], we found increased pc endometritis with hiv-1 infection . This could represent hiv-1 infection in the genital tract; chronic plasma cell endometritis [11, 14]; or the presence of opportunistic infections . Reported an association between hsv-2 seropositivity and plasma cell endometritis; notably hsv-2 is extremely prevalent among hiv-1-infected persons (kais 2007 . Evaluated 20 endometrial biopsy samples from women with asymptomatic histologic endometritis and failed to detect herpes simplex virus by pcr, and cytomegalovirus was detected equally in women with and without histological endometritis . One limitation of this study is that hiv-1-infected controls did not undergo laparoscopic evaluation . Therefore, unlikely we cannot firmly exclude subclinical salpingitis from this population as we can for the hiv-1-seronegative controls . Furthermore, we did not attempt to detect suspected etiologies of endometritis such as bacteria other than n. gonorrhoeae and c. trachomatis including m. genitalium and potential etiologies such as cytomegalovirus and herpes simplex virus infection . Such data might help to elucidate the reason for the different findings among hiv-1-seropositive and hiv-1-seronegative women with salpingitis in regards to endometrial histopathology . This study raises some important questions regarding pid and its sequelae . With increased access to highly active antiretroviral therapy (haart), hiv-1-infected population data from uganda plus others have demonstrated reduced fertility in hiv-1-infected women regardless of disease stage . Further research is required to determine if women using haart return to normal fertility or not . Lastly, although endometrial histopathology serves as a reasonable surrogate for salpingitis in hiv-1-uninfected populations, its utility in populations with a high hiv-1 seroprevalence appears to be limited . Discovery of a sensitive and specific biomarker or set of biomarkers for salpingitis could facilitate further research on pid and its sequelae in such settings.
More than 170 million persons worldwide are infected with the hepatitis c virus (hcv), and the number of deaths caused by hcv - related liver diseases is more than 35 thousand per year.1 in addition, hcv infection is associated with an increased incidence of hepatocellular carcinoma.2 to reduce the risk of developing hepatocellular carcinoma and hcv - related liver diseases, a treatment based on interferon (ifn) is commonly used for patients with chronic hepatitis c.3 two types of pegylated interferon (peg - ifns) (alfa-2a and alfa-2b) are used, which are pegylated to improve their pharmacokinetic effects and pharmacodynamic actions, and which have a higher rate of sustained virologic response (svr) and lower frequency of incidence of adverse reactions than ifn.4 thus far, the general recommendation for the treatment of patients with chronic hepatitis c involves combination therapy with peg - ifn (alfa-2a or alfa-2b) and ribavirin (rbv).3 however, previous studies that have compared the effectiveness and safety of treatment with peg - ifn alfa-2a and alfa-2b for patients with chronic hepatitis c have yielded conflicting results.58 the largest randomized controlled trial (rct) to date was a comparison in 2009 of svr and safety in the treatment of 3,070 patients with chronic hcv genotype 1 among three groups (low - dose peg - ifn alfa-2b plus rbv; standard - dose peg - ifn alfa-2b plus rbv; and peg - ifn alfa-2a plus rbv).9 that study excluded patients with hepatitis b virus (hbv) or human immunodeficiency virus (hiv) infection, as well as other liver diseases, and found that the rates of svr and adverse event profiles among the three groups were similar . On the other hand, some meta - analyses of rcts comparing peg - ifn alfa-2a and alfa-2b suggested a higher svr rate in the alfa-2a group than in the alfa-2b group.5,6 however, several limitations involving the study quality were pointed out, including a small sample size as well as inadequate blinding and randomization.5 a large retrospective cohort study was conducted to compare the svr rate obtained with peg - ifn alfa-2a and alfa-2b for the treatment of patients with chronic hepatitis c in germany.10 a total of 3,414 patients were identified as a study cohort from the database . This study allowed the inclusion of patients who had a comorbid hbv or hiv infection and any hcv genotype, and used matched pair analysis, using the baseline patient characteristics to compare the rates of svr with peg - ifn alfa-2a and alfa-2b . Although a higher svr rate was found for alfa-2a than for alfa-2b, the possibility of residual uncontrolled confounding was acknowledged, such as details of the therapeutic profiles, for eg, the duration of therapy in the two groups were different, even after subject matching.10 thus far, a definitive conclusion has yet to be reached regarding the superiority of one peg - ifn over the other . In this study, we aimed to compare the rates of svr with peg - ifn alfa-2a and alfa-2b in combination with rbv for chronic hepatitis c, using a large database of hepatitis cases to improve the generalizability of these results . Because hepatitis is becoming one of the most prevalent major infectious diseases, the basic act on measures against hepatitis in japan was established to conquer hepatitis, by providing high quality medical care for patients with a hepatitis viral infection.11 as part of this measure, the japanese interferon database, which is comprised of more than 16,000 patient records regarding ifn treatment for chronic hepatitis and/or cirrhosis, collected retrospectively from throughout japan, was developed . This database contains diagnosis, ifn treatment data (type of ifn, with or without rbv), results of laboratory tests (date of test, hcv rna, type of hcv genotype, aspartate aminotransferase, alanine aminotransferase [alt], and platelet count), adverse drug reactions, and outcomes (svr, and completed or discontinued treatment), which have been recorded in a standardized report form by practitioners . Serum hcv rna levels were quantitated by cobas amplicor hcv monitor v2.0 (roche molecular systems, pleasanton, ca, usa) or cobas taqman hcv test (roche molecular systems). This study was approved by the institutional review board of the national center for global health and medicine . We identified all of the patients with chronic hepatitis c from the japanese interferon database who had received a combination treatment of peg - ifn (alfa-2a or alfa-2b) and rbv between december 2009 and april 2013 . Patients meeting any of the following criteria were excluded from this study: comorbid cirrhosis or hbv infection; without rbv use; dual therapy with peg - ifn alfa-2a and alfa-2b; any missing data items (sex, age, diagnosis, and/or comorbid condition, ie, liver diseases); and age under 16 years . Descriptive statistics were calculated as absolute numbers, percentages, and means (standard deviation [sd]) for each group . Patient baseline characteristics were compared between peg - ifn alfa-2a and alfa-2b, using a t - test for age, the wilcoxon rank - sum test for duration of therapy, the mantel haenszel chi - square statistic for genotype, and the chi - square test for the other categorical variables . We also compared adverse events in the patients withdrawn from study, according to hcv genotype, using the fisher s exact test . To explore the association of patient svr with chronic hepatitis c, we used multivariate logistic regression to calculate the adjusted odds ratio (or) and the associated 95% confidence intervals (cis). We included covariates to adjust for age, sex, platelet counts, alt level, hcv viral load, genotype, and frequency of treatment . Interaction analyses were performed with the logistic regression model, to test for interactions between peg - ifn and level of treatment experience or hcv genotype . The results indicated that there were no significant interactions (between type of peg - ifn and treatment experience [p=0.229] or between type of peg - ifn and hcv genotype [p=0.069]). Because hepatitis is becoming one of the most prevalent major infectious diseases, the basic act on measures against hepatitis in japan was established to conquer hepatitis, by providing high quality medical care for patients with a hepatitis viral infection.11 as part of this measure, the japanese interferon database, which is comprised of more than 16,000 patient records regarding ifn treatment for chronic hepatitis and/or cirrhosis, collected retrospectively from throughout japan, was developed . This database contains diagnosis, ifn treatment data (type of ifn, with or without rbv), results of laboratory tests (date of test, hcv rna, type of hcv genotype, aspartate aminotransferase, alanine aminotransferase [alt], and platelet count), adverse drug reactions, and outcomes (svr, and completed or discontinued treatment), which have been recorded in a standardized report form by practitioners . Serum hcv rna levels were quantitated by cobas amplicor hcv monitor v2.0 (roche molecular systems, pleasanton, ca, usa) or cobas taqman hcv test (roche molecular systems). This study was approved by the institutional review board of the national center for global health and medicine . We identified all of the patients with chronic hepatitis c from the japanese interferon database who had received a combination treatment of peg - ifn (alfa-2a or alfa-2b) and rbv between december 2009 and april 2013 . Patients meeting any of the following criteria were excluded from this study: comorbid cirrhosis or hbv infection; without rbv use; dual therapy with peg - ifn alfa-2a and alfa-2b; any missing data items (sex, age, diagnosis, and/or comorbid condition, ie, liver diseases); and age under 16 years . Descriptive statistics were calculated as absolute numbers, percentages, and means (standard deviation [sd]) for each group . Patient baseline characteristics were compared between peg - ifn alfa-2a and alfa-2b, using a t - test for age, the wilcoxon rank - sum test for duration of therapy, the mantel haenszel chi - square statistic for genotype, and the chi - square test for the other categorical variables . We also compared adverse events in the patients withdrawn from study, according to hcv genotype, using the fisher s exact test . To explore the association of patient svr with chronic hepatitis c, we used multivariate logistic regression to calculate the adjusted odds ratio (or) and the associated 95% confidence intervals (cis). We included covariates to adjust for age, sex, platelet counts, alt level, hcv viral load, genotype, and frequency of treatment . Interaction analyses were performed with the logistic regression model, to test for interactions between peg - ifn and level of treatment experience or hcv genotype . The results indicated that there were no significant interactions (between type of peg - ifn and treatment experience [p=0.229] or between type of peg - ifn and hcv genotype [p=0.069]). Between december 2009 and april 2013, a total of 16,349 patients were recorded in the japanese interferon database . Of these, 3,643 subjects were excluded for the following reasons: comorbid condition of cirrhosis in 569 subjects and hbv infection in 300 subjects; without rbv use in 2,414 subjects; dual therapy of peg - ifn alfa-2a and alfa-2b in 225 subjects; missing data (any of sex, age, diagnose, and/or comorbid condition [liver diseases]) in 261 subjects; and under 16 years of age in three subjects (figure 1)., 3,578 subjects (1,710 [48%] males; 1,868 [52%] females) were taking peg - ifn alfa-2a, with a mean age (sd) of 59.1 (10.0) years; and 9,128 subjects (4,652 [51%] males; 4,476 [49%] females) were taking peg - ifn alfa-2b, with a mean age (standard deviation) of 57.2 (10.9) years (table 1). Several differences were found between the two groups, as follows: the mean age was higher in the alfa-2a group than in the alfa-2b group; the proportion of male patients was higher in the alfa-2b group; the proportion of patients with an hcv genotype1 infection in the peg - ifn alfa-2a group (2,874 [83%]) was higher than in alfa-2b group (5,564 [62%]); and, a higher proportion of initial treatment patients was obtained in the alfa-2b group (2,181 [62%]) than in the alfa-2a (7,086 [79%]). The svr rate in patients receiving peg - ifn alfa-2b was higher than that in patients receiving alfa-2a (crude or=1.31; 95% ci: 1.23 to 1.44). After adjustment for potential confounders (including age, sex, platelet count, alanine aminotransferase [alt] level, hcv viral load, genotype, and treatment experience), no significant difference was found in svr rates between the peg - ifn alfa-2a group and the peg - ifn alfa-2b group (adjusted or=0.96; 95% ci: 0.88 to 1.05) (table 2). No significant differences were found between the peg - ifn alfa-2b and alfa-2a groups, in terms of adjusted svr rate, in patients receiving their initial treatment (crude or=1.27; 95% ci: 1.14 to 1.40; adjusted or=0.90; 95% ci: 0.80 to 1.01), patients receiving retreatment (crude or=1.15; 95% ci: 1.00 to 1.33; adjusted or=1.09; 95% ci: 0.93 to 1.27), and patients with hcv genotype 1 infections (crude or=1.00; 95% ci: 0.91 to 1.09; adjusted or=0.95; 95% ci: 0.86 to 1.05). The adjusted svr rate for peg - ifn alfa-2b in patients with hcv genotype 2 or 3 was superior to that of peg - ifn alfa-2a (crude or=1.36; 95% ci: 1.10 to 1.69; adjusted or=1.35; 95% ci, 1.08 to 1.69) (figure 2). During the study, significant differences between the two treatments were observed in patients with hcv genotype 1 . Ninety - six patients withdrew from treatment due to throm - bocytopenia (46 [1.6%] in the peg - ifn alfa-2a group and 50 [0.9%] in the alfa-2b group [p=0.005]), and 57 patients withdrew due to retinopathy (13 [0.5%] in the alfa-2a group and 44 [0.8%] in alfa-2b group [p=0.091]) (table 3). In contrast were the patients with genotype 2 or 3, who displayed anemia (ten [1.6%] in the alfa-2a group and 25 [0.7%] in the alfa-2b group [p=0.034]), thrombocytopenia (eight [1.3%] in the alfa-2a group and 14 [0.4%] in alfa-2b group [p=0.012]), and retinopathy (five [0.8%] in the alfa-2a group and five [0.1%] in the alfa-2b group [p=0.010]) (table 4). Between december 2009 and april 2013, a total of 16,349 patients were recorded in the japanese interferon database . Of these, 3,643 subjects were excluded for the following reasons: comorbid condition of cirrhosis in 569 subjects and hbv infection in 300 subjects; without rbv use in 2,414 subjects; dual therapy of peg - ifn alfa-2a and alfa-2b in 225 subjects; missing data (any of sex, age, diagnose, and/or comorbid condition [liver diseases]) in 261 subjects; and under 16 years of age in three subjects (figure 1)., 3,578 subjects (1,710 [48%] males; 1,868 [52%] females) were taking peg - ifn alfa-2a, with a mean age (sd) of 59.1 (10.0) years; and 9,128 subjects (4,652 [51%] males; 4,476 [49%] females) were taking peg - ifn alfa-2b, with a mean age (standard deviation) of 57.2 (10.9) years (table 1). Several differences were found between the two groups, as follows: the mean age was higher in the alfa-2a group than in the alfa-2b group; the proportion of male patients was higher in the alfa-2b group; the proportion of patients with an hcv genotype1 infection in the peg - ifn alfa-2a group (2,874 [83%]) was higher than in alfa-2b group (5,564 [62%]); and, a higher proportion of initial treatment patients was obtained in the alfa-2b group (2,181 [62%]) than in the alfa-2a (7,086 [79%]). The svr rate in patients receiving peg - ifn alfa-2b was higher than that in patients receiving alfa-2a (crude or=1.31; 95% ci: 1.23 to 1.44). After adjustment for potential confounders (including age, sex, platelet count, alanine aminotransferase [alt] level, hcv viral load, genotype, and treatment experience), no significant difference was found in svr rates between the peg - ifn alfa-2a group and the peg - ifn alfa-2b group (adjusted or=0.96; 95% ci: 0.88 to 1.05) (table 2). We also performed subgroup analyses, according to treatment experience and genotype . No significant differences were found between the peg - ifn alfa-2b and alfa-2a groups, in terms of adjusted svr rate, in patients receiving their initial treatment (crude or=1.27; 95% ci: 1.14 to 1.40; adjusted or=0.90; 95% ci: 0.80 to 1.01), patients receiving retreatment (crude or=1.15; 95% ci: 1.00 to 1.33; adjusted or=1.09; 95% ci: 0.93 to 1.27), and patients with hcv genotype 1 infections (crude or=1.00; 95% ci: 0.91 to 1.09; adjusted or=0.95; 95% ci: 0.86 to 1.05). The adjusted svr rate for peg - ifn alfa-2b in patients with hcv genotype 2 or 3 was superior to that of peg - ifn alfa-2a (crude or=1.36; 95% ci: 1.10 to 1.69; adjusted or=1.35; 95% ci, 1.08 to 1.69) (figure 2). During the study, significant differences between the two treatments were observed in patients with hcv genotype 1 . Ninety - six patients withdrew from treatment due to throm - bocytopenia (46 [1.6%] in the peg - ifn alfa-2a group and 50 [0.9%] in the alfa-2b group [p=0.005]), and 57 patients withdrew due to retinopathy (13 [0.5%] in the alfa-2a group and 44 [0.8%] in alfa-2b group [p=0.091]) (table 3). In contrast were the patients with genotype 2 or 3, who displayed anemia (ten [1.6%] in the alfa-2a group and 25 [0.7%] in the alfa-2b group [p=0.034]), thrombocytopenia (eight [1.3%] in the alfa-2a group and 14 [0.4%] in alfa-2b group [p=0.012]), and retinopathy (five [0.8%] in the alfa-2a group and five [0.1%] in the alfa-2b group [p=0.010]) (table 4). We conducted a retrospective cohort study to compare the svr rate in patients with chronic hepatitis c treated with peg - ifn alfa-2a and alfa-2b in combination with rbv, using data from the large japanese interferon database to improve the generalizability of the results . We employed multivariate logistic regression analysis to adjust for potential confounders, as the demographic characteristics at baseline differed between the two groups . We found similar svr rates in patients with chronic hepatitis c who received either peg - ifn alfa-2a or alfa-2b in combination with rbv . Our findings were consistent with previous results from the largest reported rct, which involved 3,070 patients.9,12,13 on the other hand, some other previous studies,10,14 including the largest retrospective cohort study, which involved 3,414 subjects, presented results that conflicted with our findings . As mentioned above, several limitations of these previous studies have been pointed out (eg, small sample size, inadequate blinding or randomization, difference in treatment regimen, and different study populations).5,15 to enhance generalizability, we conducted a study with a larger sample size (12,706 subjects) than used in previous studies.57,9,10 we found no difference in the adjusted svr rate between the two treatment groups, even though the sample size was large enough to detect such a difference . In addition, our results were obtained from a realistic medical setting, using data recorded according to the actual treatments applied to patients with hepatitis, by practitioners . Previously, few studies had been performed that reflected a realistic medical setting for treatment of patients with chronic hepatitis c because most of the previous studies were rcts.57 we also performed two subgroup analyses, according to treatment experience and genotype, because the largest reported rct9 had included only patients receiving their initial treatment and patients with hcv genotype 1 . Similar to our other results, we also found no difference in the adjusted svr rate between treatment with peg - ifn alfa-2a and alfa-2b from the subgroup analyses, except for the subgroup of genotypes 2 and 3 . In this subgroup, alfa-2b had higher a svr rate, although the interaction was not significant . While the present study suggests that peg - ifn alfa-2b may be the preferable treatment for patients with hcv genotypes 2 and 3, the results of several rcts9,16,17 showed no difference between treatment with peg - ifn alfa-2a or alfa-2b in patients with genotype 1 or with genotypes 14 . In a study comparing the pharmacokinetics and pharmacodynamics of the two drugs,18 peg - ifn alfa-2b displayed a greater effect of biological activity by reducing hcv - rna and upregulating ifn - related response genes . However, the duration of this effect may be affected by the time period between dosing because the volume of distribution of peg - ifn alfa-2b is large, and it is metabolized faster than peg - ifn alfa-2a.8 due to its higher molecular weight and branched pegylation, the extravascular volume of distribution of peg - ifn alfa-2a is smaller . Thus, its duration in the body is longer than that of peg - ifn alfa-2b . In general, the amount of a drug in the body tends to decrease as body weight increases; however, in this study, the amount of peg - ifn alfa-2b in the body was constant regardless of body weight because the dosage was adjusted for body weight.18 in the present study, peg - ifn alfa-2b was more effective than peg - ifn alfa-2a for the treatment of patients with genotypes 2 and 3 . This may be because the patients treated with peg - ifn alfa-2b received a dosage based on body weight, whereas patients treated with peg - ifn alfa-2a were treated with a fixed dosing regimen, without accounting for weight . However, information regarding the patients body weight and dosage of peg - ifn was not included in this database . We found that proportions of adverse events, such as thrombocytopenia, retinopathy, and anemia, were higher in patients treated with peg - ifn alfa-2a . In a previous study comparing pharmacokinetics and pharmacodynamics,18 both leukocytes and neutrophils were significantly decreased in patients receiving peg - ifn alfa-2a versus those receiving peg - ifn alfa-2b . Additionally, the incidences of hematological abnormalities tend to be higher in peg - ifn alfa-2a treated patients.18 in contrast, rcts comparing peg - ifn alfa-2a and alfa-2b, including a japanese study19 and the largest study ever conducted,9 showed no difference in hematological abnormalities following termination of treatment . In the rcts, the subjects may have been selected from patients in good condition or from those who were expected to adhere to the study treatment . Our data suggests that hematological abnormalities, such as thrombocytopenia, may develop in actual medical care situations, because we used data obtained from the database of medical records from patients receiving ifn treatment . The strengths of our study include the large sample size, the use of data collected from actual medical settings, and the inclusion of patients with all types of hcv genotype infections and patients with or without previous treatment experience . First, there were residual confounders, which could persist due to unmeasured or imprecisely measured potential confounding factors . Secondly, there was a reporting bias because the data were recorded by practitioners using a standardized report form . Additional studies are needed to validate the data, using an alternative existing database source (eg, electronic medical records database or claims database). Thirdly, the viral load levels might have been misclassified because the measurement methods for hcv rna in this database differed depending on the time of therapy . Finally, further studies are required to assess the rate of svr between the two medications in combination with simeprevir, a new agent for the treatment of chronic hepatitis c, because the current standard therapy for chronic hepatitis c is triple therapy with peg - ifn (alfa-2a or alfa-2b), rbv, and simeprevir.20,21 there was no significant difference in the svr rates and safety profile between chronic hepatitis c patients treated with the peg - ifn alfa-2a and alfa-2b.
Molecular structure descriptors or topological indices are used for modelling information of molecules, including toxicologic, chemical, and other properties of chemical compounds in theoretical chemistry . Topological indices play a very important role in mathematical chemistry, especially in the quantitative structure - property relationship (qspr) and quantitative structure activity relationship (qsar). Many topological indices have been introduced and investigated by mathematicians, chemists, and biologists, which contain energy, the laplacian - energy - like invariant [25], the kirchhoff index [613], and so forth . The energy of the graph is an important invariant of the adjacency spectrum and is the sum of the absolute values of all the eigenvalues of a graph g, which is studied in chemistry and used to approximate the total electron energy of a molecule . During researching the character of the conjugated carbon oxides e is closely related to the energy releasing from the formation progress of the conjugated carbon oxides and could be approximately calculated by hckel molecular orbital theory . And in the method of hmo, the calculation of e can be attributed to the sum of the absolute values of all the eigenvalues of its molecular graph [1420]. Compared with adjacency matrix, the definition of laplacian matrix added to all vertices degrees . As mohar said, cvetkovi and simi [2123] pointed out that, as molecular structure descriptors, the laplacian - energy - like invariant not only well describes the properties of most of the descriptors which are indicated, such as entropy, molar volume, and molar refractivity, but also is able to describe some more difficult properties, such as boiling point and rub points . Due to the fact that laplacian - energy - like invariant has a significant physical and chemical background [24, 25], it has received wide attention to research it from many mathematical and chemical workers . All the graphs discussed in this paper are simple, finite, and undirected . For a graph g, the vertex set and edge set of g will be denoted by v(g) = {v 1, v 2,, v n} and e(g) = {e 1, e 2,, e m}, respectively . The adjacency matrix and the diagonal matrix of g are, respectively, a(g) and d(g); then the matrix l(g) = d(g) a(g) is called the laplacian matrix of the graph g [27, 28]. The characteristic polynomials and laplacian polynomials of the graph g are g() = det(i a(g)) and g() = det(i l(g)). Both a(g) and l(g) are symmetric matrices; their eigenvalues are real numbers [30, 31]. Thus, we can order the eigenvalues of the graph g as 1 2 n, and the laplacian eigenvalues are 1 2 n [32, 33]. If g is a connected graph, then i> 0, i = 1,2,, n 1, n = 0 [3436]. The energy of a graph g is the sum of the absolute values of all the eigenvalues of g; that is,(1)eg=i=1ni . The energy of a graph g is the sum of the absolute values of all the eigenvalues of g; that is,(1)eg=i=1ni . Let g be a graph of order n. the laplacian - energy - like invariant of g, denoted by lel(g), is defined as(2)lelg=i=1ni . Let g be a graph of order n. the laplacian - energy - like invariant of g, denoted by lel(g), is defined as(2)lelg=i=1ni . Definition 3 (see). For two matrices a = (a i, j)mn, b = (b i, j)st, the tensor product of a and b, denoted by a b, is defined as(3)a11ba12ba1nbam1bam2bamnb . For two matrices a = (a i, j)mn, b = (b i, j)st, the tensor product of a and b, denoted by a b, is defined as(3)a11ba12ba1nbam1bam2bamnb . Let {g n} be a sequence of finite simple graphs with bounded average degree such that (4)limnvgn=,limnlelgnvgn = h0 . N} be a sequence of spanning subgraphs of {g n} such that (5)limnvvhn: dhnv = dgnvvgn=1;then (6)limnlelhnvgn = h . That is, g n and h n have the same asymptotic laplacian - energy - like invariant . Let {g n} be a sequence of finite simple graphs with bounded average degree such that (4)limnvgn=,limnlelgnvgn = h0 . N} be a sequence of spanning subgraphs of {g n} such that (5)limnvvhn: dhnv = dgnvvgn=1;then (6)limnlelhnvgn = h . That is, g n and h n have the same asymptotic laplacian - energy - like invariant . In what follows, we will explore the laplacian - energy - like invariants formulas of the modified hexagonal lattice, modified union jack lattice, and honeycomb lattice . The modified hexagon lattice with toroidal boundary condition is denoted by mh(n 1, n 2). Let i = 2i / n 1, j = 2j / n 2 . I = 2i / n 1, j = 2j / n 2 . Proofwith the proper labelling of the vertices of the modified hexagonal lattice, its laplacian matrix is (8)where i n1, i n2 are the unit matrices and m n is tensor product of matrices m and n. consider(9)bn2=01000001000000110000n2n2 . The matrix l(mh(n 1, n 2)) can be defined as follows: (10)lmhtn1,n2in2cn1bn2pn1bn2tpn1t = in26in1bn1bn1tbn2in1+bn1tbn2tin1+bn1 ., g} be a cyclic group of order n. obviously,: g b n can express the group . The cyclic group of order n has linear values of n i (i = 0,1,, n 1), i(g) = n, where n are said n - times unit roots.therefore, there is a reversible matrix (11)qn=nijn0i, jn1,such that (12)qn1bnqn = diag1,n,,nn1dn . It is not difficult to find that i n2 (6i n1 d n1 d n1) d n2 (i n1 + d n1) d n2 (i n1 + d n1) is a diagonal matrix whose diagonal elements are (16)6n1in1in2jn2jn1in2j+n1in2j=62cos2in12cos2jn22cos2in1cos2jn2,where 0 i n 1 1 and 0 j n 2 1.this means that the eigenvalues of the matrix l are = 6 2cos i 2cos j 2cos(i j), 0 i n 1 1, and 0 j n 2 1, where i = 2i / n 1 and j = 2j / n 2.by formula (2), the laplacian - energy - like invariant is (17)lelmhtn1,n2=i=0n11 j=0n2162cosi2cosj2cosij . So (18)limn1 limn2lelmhtn1,n2n1n2=limn1 limn21n1n2i=0n11 j=0n2162cosi2cosj2cosij=0162cos2x2cos2y2cos2xydx dy=1420262cosx2cosy2cosxydx dy2.3705 . With the proper labelling of the vertices of the modified hexagonal lattice, its laplacian matrix is (8)where i n1, i n2 are the unit matrices and m n is tensor product of matrices m and n. consider(9)bn2=01000001000000110000n2n2 . The matrix l(mh(n 1, n 2)) can be defined as follows: (10)lmhtn1,n2in2cn1bn2pn1bn2tpn1t = in26in1bn1bn1tbn2in1+bn1tbn2tin1+bn1 ., g} be a cyclic group of order n. obviously,: g b n can express the group . The cyclic group of order n has linear values of n i (i = 0,1,, n 1), i(g) = n, where n are said n - times unit roots . Therefore, there is a reversible matrix (11)qn=nijn0i, jn1,such that (12)qn1bnqn = diag1,n,,nn1dn . It is not difficult to find that i n2 (6i n1 d n1 d n1) d n2 (i n1 + d n1) d n2 (i n1 + d n1) is a diagonal matrix whose diagonal elements are (16)6n1in1in2jn2jn1in2j+n1in2j=62cos2in12cos2jn22cos2in1cos2jn2,where 0 i n 1 1 and 0 j n 2 1 . This means that the eigenvalues of the matrix l are = 6 2cos i 2cos j 2cos(i j), 0 i n 1 1, and 0 j n 2 1, where i = 2i / n 1 and j = 2j / n 2 . By formula (2), the laplacian - energy - like invariant is (17)lelmhtn1,n2=i=0n11 j=0n2162cosi2cosj2cosij . Such computations would be possible on a computer with high memory and processing speed, we used mac pro with processor 2 2.93 ghz 6-core intel xeon (24 hyperthreads in total) and memory 24 gb 1333 mhz ddr3 to obtain the results . The numerical integration value in last line is calculated with the software matlab . As such computations would be possible on a computer with high memory and processing speed, we used mac pro with processor 2 2.93 ghz 6-core intel xeon (24 hyperthreads in total) and memory 24 gb 1333 mhz ddr3 to obtain the results . By theorems 4 and 5 theorem 7 . For the modified hexagonal lattices mh (n 1, n 2), mh (n 1, n 2), and mh (n 1, n 2) with toroidal, cylindrical, and free boundary conditions, then,(19)1 limn1 limn2lelmhtn1,n2n1n2=limn1 limn2lelmhcn1,n2n1n2=limn1 limn2lelmhfn1,n2n1n22.3705,2 lelmhtn1,n2=lelmhcn1,n2=lelmhfn1,n22.3705n1n2 . For the modified hexagonal lattices mh (n 1, n 2), mh (n 1, n 2), and mh (n 1, n 2) with toroidal, cylindrical, and free boundary conditions, then,(19)1 limn1 limn2lelmhtn1,n2n1n2=limn1 limn2lelmhcn1,n2n1n2=limn1 limn2lelmhfn1,n2n1n22.3705,2 lelmhtn1,n2=lelmhcn1,n2=lelmhfn1,n22.3705n1n2 . The modified union jack lattice with toroidal boundary condition is denoted by s (n 1, n 2). Let i = 2i / n 1; j = 2j / n 2 . Then (20)1 lelstn1,n2=i=0n11 j=0n2182cosi2cosj4cosicosj,2 limn1 limn2lelstn1,n2n1n2=1420282cosx2cosy4cosxcosy dx dy2.7586 . I = 2i / n 1; j = 2j / n 2 . Then (20)1 lelstn1,n2=i=0n11 j=0n2182cosi2cosj4cosicosj,2 limn1 limn2lelstn1,n2n1n2=1420282cosx2cosy4cosxcosy dx dy2.7586 . Proofwith a proper labelling of the vertices of the modified union jack lattice, its laplacian matrix can be represented as (21)lstn1,n2=gu000ututgu0000utg000000gu0000utguu000utgn2n2,g=810001181000018000000810000181100018n1n1,u=110001111000011000000110000111100011n1n1 . Let(23)qn=nijn0i, jn1,such that(24)qn1bnqn = diag1,n,,nn1dn . Actually, (25)bnt = bn1,qnt = qn1;consequently, (26)qn1bntqn = diag1,1,,nn1dn1 . It is not difficult to find that (28)in28in1dn1dn11dn2in1+dn1+dn11dn21in1+dn11+dn1is a diagonal matrix whose diagonal elements are (29)8n1in1in2jn2jn1in2j+n1in2j+n1in2j+n1in2j=82cos2in12cos2jn24cos2in1cos2jn2,where 0 i n 1 1 and 0 j n 2 1.this means that the eigenvalues of the matrix l are = 8 2cos i 2cos j 4cos i j, 0 i n 1 1, and 0 j n 2 1, where i = 2i / n 1 and j = 2j / n 2.by formula (2), the laplacian - energy - like invariant is (30)lelstn1,n2=i=0n11 j=0n2182cosi2cosj4cosicosj . So (31)limn1 limn2lelstn1,n2n1n2=limn1 limn21n1n2i=0n11 j=0n2182cosi2cosj4cosicosj=0182cos2x2cos2y4cos2xcos2y dx dy=1420282cosx2cosy4cosxcosy dx dy2.7586 . With a proper labelling of the vertices of the modified union jack lattice let(23)qn=nijn0i, jn1,such that(24)qn1bnqn = diag1,n,,nn1dn . Actually, (25)bnt = bn1,qnt = qn1;consequently, (26)qn1bntqn = diag1,1,,nn1dn1 . It is not difficult to find that (28)in28in1dn1dn11dn2in1+dn1+dn11dn21in1+dn11+dn1is a diagonal matrix whose diagonal elements are (29)8n1in1in2jn2jn1in2j+n1in2j+n1in2j+n1in2j=82cos2in12cos2jn24cos2in1cos2jn2,where 0 i n 1 1 and 0 j n 2 1 . This means that the eigenvalues of the matrix l are = 8 2cos i 2cos j 4cos i j, 0 i n 1 1, and 0 j n 2 1, where i = 2i / n 1 and j = 2j / n 2 . By formula (2), the laplacian - energy - like invariant is (30)lelstn1,n2=i=0n11 j=0n2182cosi2cosj4cosicosj . So (31)limn1 limn2lelstn1,n2n1n2=limn1 limn21n1n2i=0n11 j=0n2182cosi2cosj4cosicosj=0182cos2x2cos2y4cos2xcos2y dx dy=1420282cosx2cosy4cosxcosy dx dy2.7586 . By theorems 4 and 8, it is not difficult to arrive at the following theorem . Theorem 9 . For the modified union jack lattices s (n 1, n 2), s (n 1, n 2), and s (n 1, n 2) with toroidal, cylindrical, and free boundary conditions, then, (32)1 limn1 limn2lelstn1,n2n1n2=limn1 limn2lelscn1,n2n1n2=limn1 limn2lelsfn1,n2n1n22.7586,2 lelstn1,n2=lelscn1,n2=lelsfn1,n22.7586n1n2 . For the modified union jack lattices s (n 1, n 2), s (n 1, n 2), and s (n 1, n 2) with toroidal, cylindrical, and free boundary conditions, then, (32)1 limn1 limn2lelstn1,n2n1n2=limn1 limn2lelscn1,n2n1n2=limn1 limn2lelsfn1,n2n1n22.7586,2 lelstn1,n2=lelscn1,n2=lelsfn1,n22.7586n1n2 . The honeycomb lattice with toroidal boundary condition, denoted by hc(n 1, n 2), can be constructed by starting with an m n square lattice and adding two diagonal edges to each square . Let i = 2i / n 1 and j = 2j / n 2 . Then (33)1 lelhctn1,n2=i=0n11 j=0n213 + 3 + 2cosi+2cosj+2cosij+i=0n11 j=0n2133 + 2cosi+2cosj+2cosij,2 limn1 limn2lelhctn1,n22n1n2=182023 + 3 + 2cosx+2cosy+2cosxydx dy+1820233 + 2cosx+2cosy+2cosxydx dy1.6357 . Let i = 2i / n 1 and j = 2j / n 2 . Then (33)1 lelhctn1,n2=i=0n11 j=0n213 + 3 + 2cosi+2cosj+2cosij+i=0n11 j=0n2133 + 2cosi+2cosj+2cosij,2 limn1 limn2lelhctn1,n22n1n2=182023 + 3 + 2cosx+2cosy+2cosxydx dy+1820233 + 2cosx+2cosy+2cosxydx dy1.6357 . Proofsimilarly, the laplacian matrix of the honeycomb lattice is lhctn1,n2=3im - f - ft3im, where m = n 1 n 2 and f is an m m matrix . The matrix f can be written in the following form: (34)f = w000iiiw00000iw000000w00000iw00000iwn2n2,w=100001110000011000000100000110100011n1n1,where i represents the unit matrix of n 1 n 1 and i m represents the unit matrix of m m, respectively.based on theorem 5, the matrix f can be written as (35)fin2wn1+bn2tin1=in2in1+bn1t+bn2tin1 . Let (36)qn=nijn0i, jn1,such that (37)qn1bnqn = diag1,n,,nn1dn . Similarly, (38)bnt = bn1,qnt = qn1; hence, (39)qn1bntqn = diag1,1,,nn1dn1 . It is not difficult to find that i n2 (i n1 + d n1) + d n2 i n1 is a diagonal matrix whose diagonal elements are 1 + n1 + n2, so matrix l(hc(n 1, n 2)) can be reduced to the following form: (41)lhctn1,n2=31n1in2j1n1in2j3 . By det(i l(hc(n 1, n 2))) = 0, we can get (42)321n1in2j1n1in2j=3+n1i+n1i+n2j+n2j+n1in2j+n1in2j=3 + 2cos2in1 + 2cos2jn2 + 2coscos2in1cos2jn2 . Therefore, the l(hc(n 1, n 2)) characteristic eigenvalues are (43)=33 + 2cos2in1 + 2cos2jn2 + 2cos2in12jn2,where 0 i n 1 1 and 0 j n 2 1.let i = 2i / n 1 and j = 2j / n 2 . By formula (2), we may obtain the laplacian - energy - like invariant: (44)lelhctn1,n2=i=0n11 j=0n213 + 3 + 2cosi+2cosj+2cosij+i=0n11 j=0n2133 + 2cosi+2cosj+2cosij . By the definition of double integration, we arrive at (45)limn1 limn2lelhctn1,n22n1n2limn1 limn212n1n2i=0n11 j=0n213 + 3 + 2cosi+2cosj+2cosij+limn1 limn212n1n2i=0n11 j=0n2133 + 2cosi+2cosj+2cosij=12013 + 3 + 2cos2x+2cos2y+2cos2xydx dy+120133 + 2cos2x+2cos2y+2cos2xydx dy=182023 + 3 + 2cosx+2cosy+2cosxydx dy+1820233 + 2cosx+2cosy+2cosxydx dy1.6357 . Similarly, the laplacian matrix of the honeycomb lattice is lhctn1,n2=3im - f - ft3im, where m = n 1 n 2 and f is an m m matrix . The matrix f can be written in the following form: (34)f = w000iiiw00000iw000000w00000iw00000iwn2n2,w=100001110000011000000100000110100011n1n1,where i represents the unit matrix of n 1 n 1 and i m represents the unit matrix of m m, respectively . Based on theorem 5, the matrix f can be written as (35)fin2wn1+bn2tin1=in2in1+bn1t+bn2tin1 . Let (36)qn=nijn0i, jn1,such that (37)qn1bnqn = diag1,n,,nn1dn . Similarly, (38)bnt = bn1,qnt = qn1; hence, it is not difficult to find that i n2 (i n1 + d n1) + d n2 i n1 is a diagonal matrix whose diagonal elements are 1 + n1 + n2, so matrix l(hc(n 1, n 2)) can be reduced to the following form: (41)lhctn1,n2=31n1in2j1n1in2j3 . By det(i l(hc(n 1, n 2))) = 0, we can get (42)321n1in2j1n1in2j=3+n1i+n1i+n2j+n2j+n1in2j+n1in2j=3 + 2cos2in1 + 2cos2jn2 + 2coscos2in1cos2jn2 . Therefore, the l(hc(n 1, n 2)) characteristic eigenvalues are (43)=33 + 2cos2in1 + 2cos2jn2 + 2cos2in12jn2,where 0 i n 1 1 and 0 j n 2 1 . I = 2i / n 1 and j = 2j / n 2 . By formula (2), we may obtain the laplacian - energy - like invariant: (44)lelhctn1,n2=i=0n11 j=0n213 + 3 + 2cosi+2cosj+2cosij+i=0n11 j=0n2133 + 2cosi+2cosj+2cosij . By the definition of double integration, we arrive at (45)limn1 limn2lelhctn1,n22n1n2limn1 limn212n1n2i=0n11 j=0n213 + 3 + 2cosi+2cosj+2cosij+limn1 limn212n1n2i=0n11 j=0n2133 + 2cosi+2cosj+2cosij=12013 + 3 + 2cos2x+2cos2y+2cos2xydx dy+120133 + 2cos2x+2cos2y+2cos2xydx dy=182023 + 3 + 2cosx+2cosy+2cosxydx dy+1820233 + 2cosx+2cosy+2cosxydx dy1.6357 . By theorems 4 and 10, hc(n 1, n 2), hc(n 1, n 2), and hc(n 1, n 2) with toroidal, cylindrical, and free boundary conditions, then, (46)1 limn1 limn2lelhctn1,n2n1n2=limn1 limn2lelhccn1,n2n1n2=limn1 limn2lelhcfn1,n2n1n21.6357;2 lelhctn1,n2=lelhccn1,n2=lelhcfn1,n21.6357n1n2 . For the honeycomb lattices hc(n 1, n 2), hc(n 1, n 2), and hc(n 1, n 2) with toroidal, cylindrical, and free boundary conditions, then, (46)1 limn1 limn2lelhctn1,n2n1n2=limn1 limn2lelhccn1,n2n1n2=limn1 limn2lelhcfn1,n2n1n21.6357;2 lelhctn1,n2=lelhccn1,n2=lelhcfn1,n21.6357n1n2 . In this paper, we mainly studied the laplacian - energy - like invariants of the modified hexagonal lattice, modified jack lattice, and honeycomb lattice . The proposed results imply that the asymptotic laplacian - energy - like invariants of those lattices are independent of the three boundary conditions . The problems on the various topological indices of lattices have much important significance in the mathematical theory, chemical energy, statistical physics, and networks science.
Chf is not only the failure of the heart to generate sufficient cardiac output, but is a multisystemic disorder with immune activation leading to increased concentrations of several cytokines . In chf several studies showed increased concentrations of proinflammatory cytokines such as tnf, interleukin (il)-1, il-6, il-18, and cardiotrophin-1 (ct-1) [25]. One of the most examined proinflammatory molecules in chf is tnf. Tnf is a trimeric 17-kda polypeptide mainly produced by monocytes and macrophages . Short - term tnf expression is thought to be an adaptive mechanism; whereas prolonged expression causes left ventricular dysfunction and cardiomyopathy leading to chf propagation . However, tnf influences not only the heart itself but causes endothelial dysfunction and peripheral muscle wasting . Cardiotrophin-1 (ct-1) is a member of the il-6 cytokine family that consists of il-6, il-11, ciliary neurotrophic factor (cntf), cardiotrophin-1 (ct-1), cardiotrophin - like cytokine (clc), leukemia inhibitory factor (lif), neuropoietin (npn), and oncostatin m (osm) and has recently been supplemented by the addition of two newly characterized cytokines: il-27 and il-31 . All these cytokines bind to a specific receptor chain (il-6r, il-11r, or lifr for ct-1, lif, osm). Following cytokine binding the cytokine / receptor complex associates with glycoprotein 130 (gp130) causing tyrosine phosphorylation of gp130 and the signal is transduced via the janus kinase (jak)/signal transducer and activation of transcription 3 (stat3) pathway [810]. Ct-1 is expressed in a time - dependent manner during embryogenesis of organs, is expressed in the heart during life, induces cardiac myocyte hypertrophy, and is able to prevent myocyte apoptosis via a mitogen dependent kinase pathway [8, 11]. Increased ct-1 concentrations were detected in patients with acute myocardial infarction and chronic heart failure (chf). Furthermore, ct-1 plasma concentrations correlate with the severity of left ventricular dysfunction [1114]. However, ct-1 has not only effects on myocytes but also on vasculature by decreasing systemic vascular resistance in an animal model, by induction of acute phase proteins in rat hepatocytes, and by attenuation of endotoxin - induced acute lung injury . There are several studies showing that in chf pbmcs produce tnf [18, 19]. But so far the mechanisms responsible for tnf production in these cells under these circumstances are not determined . In this study we investigated whether ct-1 induces tnf expression in human pbmc of healthy volunteers . Recombinant human ct-1 was purchased from r&d systems (wiesbaden, germany) and dissolved according to the manufacturer's instruction . Actinomycin d, brefeldin a, and parthenolide were purchased from sigma chemicals (deisenhofen, germany). The blocking antibody against ct-1 was purchased from r&d systems (wiesbaden, germany). Human peripheral blood mononuclear cells were obtained from healthy volunteers by ficoll - paque (amersham bioscience, uppsala, sweden) centrifugation . The cells were washed three times with pbs, resuspended in rpmi 1640 supplemented with 10% fetal calf serum, 1% penicillin, streptomycin (all from biochrom ag, berlin, germany), and cultured in plastic dishes at 37c in a humified 5% co2 atmosphere . Cells were cultivated for 24 hour with rpmi 1640 supplemented with 10% fetal calf serum, 1% penicillin, streptomycin . Afterwards, cells were subconfluent and medium was replaced by fresh medium . After 24 hours, over 90% of pbmc were alive tested by trypan blue exclusion . Primary cultures from human vein endothelial cells were purchased from promocell (heidelberg, germany). Cell culture was done according to the manufacturer's manual in endothelial growth medium with 2% fetal calf serum (egm, promocell, heidelberg, germany). Cells were grown to confluence in collagen i coated tissue culture plastic (becton dickinson, franklin lakes, usa). All stimulants, inhibitors and media were without significant endotoxin levels according to the manufacturers' instructions . Pharmacological agents, dissolved in fresh medium, were added to the cells for defined time intervals and concentrations . As a control, approval for this study was given by the ethics committee of the friedrich schiller university of jena, and subjects gave their written informed consent according to the university guidelines . Total rna from cultivated pbmc was extracted according to the rneasy protocol (qiagen, hilden, germany). One g of total rna was reversely transcribed into cdna in a volume of 20 l with avian myeloma leukaemia virus (amv) reverse transcriptase and oligo dt primers (promega, madison, usa) according to the manufacturers manual . Real - time pcr measurement of tnf cdna was performed with the light cycler instrument using the fast start dna master sybr green i kit (roche diagnostics, mannheim, germany). For verification of the correct amplification product, the specific primer for human tnf was purchased from r&d systems . The amplification program for tnf consisted of 1 cycle of 94c with a 4-minute hold followed by 40 cycles of 95c with a 45-second hold, 59c annealing temperature with a 45-second hold, and 72c with a 45-second hold . The specific primer pair for gapdh was: sense primer 5 ggg aag gtg aag gtc gg 3, antisense primer 5 tgg act cca cga cgt act cag 3. the amplification program for gapdh consisted of 1 cycle of 95c with a 30-second hold followed by 30 cycles of 95c with a 5-second hold, 59c annealing temperature with a 10-second hold, and 72c with a 20-second hold . Each reaction (20 l) contained 2 l cdna, 2.5 mm mgcl2, 1 pmol of each primer, and 2 l of fast starter mix (containing buffer, dntps, sybr green dye and taq polymerase). A negative control without cdna was run with every pcr to assess the specificity of the reaction . Pcr efficiency was determined by analysing a dilution series of cdna (external standard curve). The identity of the pcr product was confirmed by comparing its melting temperature (tm) with the tm of amplicons from standards or positive controls . Gapdh was analyzed in parallel to each pcr and the resulting gapdh values were used as standards for presentation of tnf transcripts . Tnf concentrations in the culture supernatants were determined by elisa (quantiglo, r&d systems, wiesbaden, germany) according to the manufacturer's instructions . Nuclear extracts were achieved by the epiquik nuclear extraction kit i (epigentek, ny, usa) according to the manufacturer's manual . Afterwards, protein concentrations of nuclear extracts were determined according to the bradford methode . For determination of nfb 2 g of nuclear proteins were used and further analyzed by gel electrophoretic mobility shift assay (emsa) according to the suppliers manual . Emsa kits and probes were purchased from panomics, redwood city, usa . For intracellular staining peripheral blood was collected in lithium - heparin tubes . 100 l of blood was added to rpmi-1640 medium including brefeldin a (final concentration: 1 g / ml) (sigma, taufkirchen, germany), and incubated for 6 hours time at 37c . Were stained with monoclonal antibodies against the surface antigens cd3 (coulter - immunotech, krefeld, germany), cd4 (caltag, hamburg, germany) cd8 or cd14 (bd - pharmingen, heidelberg, germany) (15 minute, rt), followed, after a washing step, by fixation with 100 l 2% paraformaldehyde for 10 min at rt . After a wash the cells were incubated in 100 l permeabilisation solution (0,1% saponin and 0,1% nan3 in pbs) together with 1 l directly conjugated anti - tnf antibody (bd - pharmingen, heidelberg, germany) for 15 minute at rt . Followed by a wash with permeabilisation solution the cells were resuspended in pbs/2% fcs and fluorescence intensity was analyzed by flow cytometry (facscalibur, becton - dickinson, heidelberg, germany). For analysis regions were defined by forward scatter and side scatter as well as cd3/cd4- or cd3/cd8-lymphocyte populations and cd14 monocyte population . Data were analyzed with cellquest software . Because the amount of the cytokines produced was different in each experiment, the effects on tnf production were normalized to unstimulated cells, which were set as one . Data were analysed by nonparametric methods to avoid assumptions about the distribution of the measured variables . In the first sets of experiments we analysed whether ct-1 is able to induce tnf in pbmc . As shown in figure 1(a), ct-1 induced in a concentration - dependent manner tnf in the supernatant determined by a commercial available elisa . Maximal tnf concentration was found after 3 to 6 hours and declined afterwards reaching nearly control values after 24 hours, indicating that ct-1 causes only a transient tnf release in pbmc . In the next experiments we determined intracellular tnf protein in monocytes, cd4 and cd8 lymphocytes after stimulation with various concentrations of ct-1 in the presence of brefeldin a using immunofluorescent flow cytometry . Intracellular tnf determination in cd4 and cd8 lymphocytes did not show an effect of ct-1 on tnf expression (data not shown). In monocytes we found a concentration - dependent increase of intracellular tnf after ct-1 application (figure 1(b)). These results showed that ct-1 induced tnf in pbcm independent of culture conditions and independent of determination methodes . On tnf mrna level we found maximal mrna after 1 hour . Blocking ct-1 by an antibody against ct-1 inhibited ct-1 induced tnf mrna (data not shown) indicating that tnf induction is specifically caused by ct-1 . With the next experiments we addressed the question whether tnf protein expression is dependent on mrna synthesis and intracellular protein transport . In figure 3 it is shown that both inhibition of mrna synthesis by actinomycin d and intracellular protein transport by brefeldin a were able to abolish ct-1 induced tnf protein induction in the supernatant . As shown in figure 4 ct-1 caused a concentration - dependent nfb translocation to the nucleus determined by emsa reaching maximal translocation with 100 ng / ml ct-1 . In the next sets of experiments we used emsa to verify that nfb activation was responsible for ct-1 induced tnf expression in pbmc . Human umbilical vein endothelial cells (huvecs) stimulated with tnf were used as a control . Unstimulated cells did not show significant nfb protein in the nucleus; whereas ct-1 caused translocation of nfb into the nucleus . Parthenolide, an inhibitor of nfb activation, was able to inhibit nfb translocation to the nucleus (figure 5). Nfb translocation is essential for tnf expression as shown in figure 6(a) and 6(b). Because parthenolide was able to inhibit tnf expression both on protein and mrna level we conclude that ct-1 not only was responsible for nfb translocation to the nucleus but this translocation was responsible for tnf expression . Using flow cytometry we found in monocytes an increase of intracellular tnf after ct-1 application which could be inhibited by parthenolide (figure 6(c)). These results show that ct-1 induced tnf in pbcm independent of culture conditions and independent of determination methodes and nfb seems to be essential for the effect of ct-1 on tnf induction in pbmc . The first result of our study is that ct-1 is able to induce tnf mrna and protein in pbmc of healthy volunteers . Tnf is increased in serum of patients with chf and correlates with the severity of heart failure, cachexia, and clinical outcome . Tnf may be involved in progression of chf because high levels of tnf can induce left ventricular dysfunction, ventricular remodelling, cardiomyopathy, and pulmonary edema [22, 23]. Cultured human pbmc can synthesize and secrete tnf. In heart failure, both the heart itself and activated monocytes are able to secrete tnf [18, 24]. Furthermore, the capacity of pbmc of chf patients to secrete tnf is increased compared to control . Our data are in good agreement with these former studies and in opposite to shimokawa et al . Who found decreased cytokine generation capacity of monocytes in severe heart failure after stimulation with lipopolysaccharide . Besides the hypothesis that in chf the failing heart itself is the main source of tnf it is speculated by other groups that activated monocytes are responsible for increased tnf serum concentrations . Monocytes may be activated by lps from the gut because the barrier function of the gut by cardial edema is disturbed and bacteria can easily translocate from the gut lumen to the blood stream . As a third possibility our data suggest at least in theory a new mechanism for tnf production of pbmc in heart failure . Ct-1 produced by the failing ventricle is able to induce tnf in pbmc without lps . The here presented mechanism might also explain why tnf may be still elevated in chf even after edema were treated successfully with diuretics and the integrity of gut mucosa was restored . Furthermore, our data support the study of petretta et al . Who found that tnf is not produced by the failing heart or the gut in patients with mild to severe heart failure . The second result of our study is the fact that ct-1 activates the nfb system in a concentration - dependent manner in pbmc of healthy volunteers our in vitro data are in line with studies that found an activation of the nfb system in peripheral blood cells in chf . Jankowska et al . Reported an activation of the nfb system in peripheral blood leukocytes in chf patients measured by immuncytochemistry . Found an augmented activation of nfb activation in blood mononuclear cells using electromobility shift assay in patients with chf compared to healthy controls . The exact pathway responsible for nfb activation in chf is still unknown and remains to be determined . But within 3 hours, there is no cytotoxic effect as shown by o'neill et al . In . We also used high ct-1 concentrations compared to concentrations reported in patients with chf by ng et al . . On the other hand a paper published in 2008 reported serum ct-1 concentration in healthy controls and patients with metabolic syndrome of about 100 ng / ml . So far serum concentration of ct-1 in healthy controls and patients is a matter of discussion . But independent of reported ct-1 serum concentration the concentration of ct-1 should be much higher in the myocardium which is the source of ct-1 in chf . Exact intramyocardial ct-1 concentrations are not determined so far, only mrna and immunohistochemical studies showed increased ct-1 in hearts of patients with chf . In our experiments both tnf mrna expression and tnf protein production of pbmc showed a large standard variation . First one explanation for the large standard deviation may be a different genetic susceptibility of pbmc from different persons to stimuli . Second, we used the low basal mrna concentration as the basis of normalization explaining the large standard variation . Third, the fact that the increase of tnf mrna expression after ct-1 application is much higher compared to the increase of protein in the supernatant may be explained methodically . Because in chf many proinflammatory cytokines are elevated and pbmc are activated, it is not easy to study the effect of a single cytokine in pbmc of patients with chf . For this reason we used pbmc from healthy volunteers in culture and stimulated them with recombinant ct-1 . In conclusion, interestingly, in our study lps is not needed for elevated tnf expression in pbmc . Elevated tnf concentrations may be important in the pathogenesis and perpetuation of heart failure by modulating systemic metabolism, causing apoptosis and having a negative inotropic effect . In the light of our results modulating ct-1
We report a case, carbon dioxide (co2) embolism a potentially devastating complication of laparoscopic surgery, during dissection phase and was successfully resuscitated . A 35-years - old female, weighing 42 kg was posted for transperitoneal laparoscopic boari's ureteric reimplantation . Her, physical examination and routine investigations were unremarkable . With routine monitoring, balanced general anesthesia was given . Co2 pneumoperitoneum was created uneventfully using open insufflation technique and sustained with pressure of 14 mm hg . After 2 h, when the surgeon was dissecting the ureters, there was a sudden drop in etco2 (8 mm hg), tachycardia (130/min) followed by multiple ventricular premature beats (vpbs) with blood pressure (bp) 110/72 mm of hg and sao2 - 98% . Surgical bleeding and endotracheal tube blockage were ruled out, and gas embolism was suspected . Injection lignocaine 80 mg intravenously was given for vpbs, and the regular rhythm was restored with the heart rate of 42/min . At this point, the sao2 decreased from 98% to 77% while the breath sounds were normal with no adventitious sounds . Since there was no response to these measures, injection dopamine 20 ug / kg / min and nor - adrenaline 0.36 ug / kg / min were infused . Multiorifice central venous catheter with its tip at the superior vena cava -right atrium (svc - ra) junction was inserted via right internal jugular vein but air could not be aspirated . After 20 min, resuscitative efforts were successful, patient had pulse rate of 120/min, bp 100/54 mm / hg, sao2 100%, etco2 30 mm / hg and ecg showed sinus rhythm with right ventricular strain pattern.at this time the cvp was found to be 10 mm of hg . Two - dimension echocardiography (2-d echo) showed no patent ductus arteriosus or any other congenital defects . She was shifted to intensive care unit with dopamine 5 ug / kg / min . Which was tapered and stopped after h. the patient did not receive hyperbaric oxygen as we did not have the facility . Co2 embolism can occur either during peritoneal insufflationor during the surgical dissection . In this case, possible causes of sudden cardiovascular collapse like hypovolemia, hemorrhage, vaso - vagal reflex, pneumothorax and endotracheal tube blockage were ruled out before considering gas embolism the adhesions caused by previous surgery might have led to vascular injury during dissection and the operating site was above the level of the heart (trendelenburg position) creating a pressure gradient thus the open venous channels, at lower pressure were open to gaseous medium at a higher pressure leading to gas embolism . Either, slow infusion of air (0.01 - 2.00 ml / kg / min) or bolus of gas (25 - 200 ml or 1 - 13 ml / kg), creates a gas paradoxical air embolism was ruled out by a 2-d echo that showed no intra - cardiac channels . Any kind of pulmonary embolism leads to sudden deterioration of the hemodynamic status associated with decreased etco2 levels . Since co2 is a highly soluble gas (54 ml / dl), prompt recovery of the vital signs is usual . Aspiration of gas through central venous catheter is the definitive means of treating gas emboli . In our case, failure to aspirate the gas may suggest that it might have been trapped in the pulmonary vasculature . Tee is the gold standard for detection of pulmonary air embolism, however, it is not indicated routinely for intra - operative monitoring . Sudden decrease in etco2 is an early sensitive but nonspecific sign of venous air embolismprecordial stethoscope enhances specificity of etco2 and mean pulmonary artery pressure values . Immediate treatment of gas embolism requires deflation of pneumoperitonium, placing the patient in durant's position, hyperventilation and administration of 100% oxygen . Aggressive cardiopulmonary resuscitation should be continued till the acute effects of the embolism abate and one should maintain positive circulating volume to prevent further entrainment of the gas . Thus, in laparoscopic surgeries early detection of gas embolism with prompt resuscitation can result in a favorable post - operative outcome.
Since the particle size is a key consideration in nuclear transport, three nps of different sizes (15.0 1.2, 8.5 0.8, and 5.5 0.8 nm, respectively) were synthesized to study the size effect of the nanoparticle on nuclear internalization (see tem images in figure s1). After modification with a dense shell of the das, the hydrodynamic diameters of these nps (termed 15-np, 8.5-np, and 5.5-np), as measured by dynamic light scattering (dls), were 37.8 1.3, 18.2 1.2, and 11.7 1.5 nm, respectively . To prevent mutual interference between tumor targeting and nanodrug capture, a directional modification was performed on the nrs (65 nm 20 nm), which means the aptamer and the cs were separately modified on the end faces and the side face of the nr, rather than being randomly spread . The principle of this selective modification process was based on the preference of ctab to accumulate on the side face of nr, which makes its end faces more reactive to thiolated dnas . Specifically, the aptamer at a relatively low concentration was first used to occupy the nr ends . Subsequently, the cs at a high concentration was added to replace ctab on the nr side face . To make the resulting nanomaterial biocompatible, thiolated peg was used, followed by several washing steps . Via dna hybridization, as characterized by tem (figure 1), nps were exactly immobilized onto the nr sides, leaving the nr ends bald, indicating successful fabrication of the side - assembly nanostructures (termed 15-np / nr, 8.5-np / nr, and 5.5-np / nr). The self - assembly process was further investigated with dls and uv vis absorption spectroscopy . The results from different angles all demonstrated the successful assembly of nps and nrs resulting from dna hybridization (see details in figures s2a and s2b). Tem images of 15-np / nrs, 8.5-np / nrs, and 5.5-np / nrs . 15-nps, 8.5-nps, and 5.5-nps mostly attach onto the nr sides, but not the nr ends, indicating the successful fabrication of the side - assembly nanostructures . The scale bar represents 50 nm . After synthesizing and characterizing these nanoassemblies, we next investigated their nir - response and drug - carrying capability . The efficient photothermal effect of nrs was verified by the rapid temperature rise of the nr medium when irradiated by the nir laser (see details in figure s3a). Then the nir - activated release of nps from nrs was demonstrated by the reduced average size (dls) and the disassembled structure (tem) of the 15-np / nrs after exposure to the laser (figure s3b). To fabricate the nuclear - uptake nanodrug delivery system, doxorubicin (dox), a widely used anticancer drug, was chosen as the model and loaded on the nps by intercalating into the (cgt)6/(acg)6 duplexes of the das (table s1). The drug payload of nps was measured by monitoring the dox fluorescence from the supernatant after nps were centrifuged down . As shown in figure 2a, a gradual decrease of dox fluorescence was observed when increasing the molar ratio of nps . By interpolating from a standard calibration curve, the dox payload of each 15-, 8.5-, and 5.5-np was 450 19, 277 28, and 40 13 molecules, respectively . The stability of np / dox complexes was evaluated via a drug leakage experiment using mini dialysis units . As shown in figure 2b, the release of dox from nps was rather slow, with less than 30% of the entire payload detected in the solution after 60 h, in comparison to the rapid diffusion of free dox, indicating high stability of the np / dox complexes . Furthermore, the nanoasemblies stored at 4 c for 8 weeks remained mostly intact . (a) dox fluorescence spectra of the supernatant after centrifuging to precipitate the das - modified nps, the dox concentration was fixed at 2 m when increasing the 15-np - das / dox mole ratio . (b) dox leakage dynamics from np - dox complexes (dox: 10 m). (c) clsm images of cem cells after incubation with 15-nps, 8.5-nps, and 5.5-nps (which represent the das - modified nps of 15, 8.5, and 5.5 nm, respectively) at 37 c for 10 or 22 h. the green and blue fluorescence arises from the tamra fluorophore labeled on the 3 end of das and hoechst 33342, respectively . The scale bar represents 5 m . Having confirmed the potential of using the np / nr nanoassemblies as nir - responsive drug nanocarriers in buffer solution, we proceeded to test their performance in cells . A nondrug - resistant leukemia cell line, cem, was first used as the cell model . To monitor the cellular uptake and intracellular distribution of nps of different sizes, the das was labeled with a 5-end tamra fluorophore (das - tmr). After modification with das - tmr, the fluorescent particles were incubated with the cem cells at 37 c for different lengths of time . Then confocal laser scanning microscopy (clsm) measurements were performed, and the results are shown in figure 2c . Nps of 15 nm were mainly localized in the cytoplasm, even after 22-h incubation, as indicated by the tamra fluorescence signal outside the nucleus . In contrast, 5.5-nps accumulated in the nucleus after incubation for 10 h, demonstrating rapid nuclear uptake . For 8.5-nps, no obvious signal was observed in the nucleus for 10-h incubation, but the nucleus emitted tamra fluorescence after incubation for 22 h, indicating that 8.5-np - dass can enter the nucleus starting from 10 h. to strike a balance between drug loading capability and nuclear translocation efficiency, the 8.5-np was used as a model nuclear - uptake nanoscaffold of dox in the following study . To achieve active tumor targeting, sgc8, an aptamer that can specifically bind to protein tyrosine kinase 7 (ptk7, kd = 0.8 nm) which is overexpressed on the membrane of cem cells but not ramos cells, the specific binding of sgc8, nr - sgc8, and np / nr - sgc8 to the target cem cells, rather than nontarget ramos cells, was demonstrated by flow cytometry (figure 3a). Also, the specific cellular uptake and cytoplasmic location of nr - sgc8s were visualized with clsm (figure s4). Moreover, the amount of the internalized nps delivered by the nr - sgc8s was higher than that of equivalent free nps (figure s5), which may have resulted from the high payload and the favorable cell - uptake size of the nanoassembly, as well as the promotion of the cell - internalizing aptamer . Specific cell binding and photocontrolled intracellular distribution of np / nr - sgc8s. (a) flow cytometry assay proving the specific binding of sgc8, nr - sgc8, and np / nr - sgc8 to target cem cells not to nontarget ramos cells (lib represents a random library sequence). (b) clsm images of cem cells after treatment with 15-np / nr - sgc8s without (i) and with (ii) nir irradiation or after treatment with 8.5-np / nr - sgc8s without (iii) and with (iv) nir irradiation . From left to right: fluorescence image for np - tmr, nr - cy5, and overlay of the np - tmr, nr - cy5, and hoechst 33342 fluorescence channels plus the bright field channel . The scale bar represents 5 m . The nir - responsive behavior of 8.5-np / nr - sgc8s and 15-np / nr - sgc8s in cem cells was investigated with clsm . Without laser treatment, the 8.5-np signal was observed in the cytoplasm and overlapped with the nr - sgc8 signal (figure 3b). However, upon nir irradiation, 8.5-nps were found in the nuclei, while nr - sgc8s remained in the cytoplasm, indicating that 8.5-nps were released from nrs after exposure to the laser and then diffused into the nuclei . The 15-nps remained in the cytoplasm irrespective of nir irradiation, corresponding well with their inability to undergo nuclear internalization . The photocontrolled nuclear internalization of 8.5-nps was further confirmed by using inductively coupled plasma atomic emission spectrometry (icp - aes, figure s6). On the basis of the fluorescence quenching of dox by intercalating into the gc duplex, the intracellular distribution of dox was investigated by treating cem cells with nonfluorophore - labeled 8.5-np - dox / nr - sgc8s . After nir irradiation and then incubation for another 22 h, the recovered fluorescence of dox was highly accumulated in the nuclei, with a relatively small amount in the cytoplasm, indicating that most dox were released in the nuclei (figure s7). However, without nir treatment, the intranuclear dox fluorescence was rather weak, showing a slow and sustained release of dox in the cytoplasm . As a control, cells were treated with free dox, and the dox signal was found throughout the cells, resulting from concentration - gradient diffusion . Furthermore, the negligible influence of the laser irradiation on the stability of the np - dox complex was confirmed by the small dox signal change from the 8.5-np - dox / nr - sgc8s - treated cem cells before and right after laser exposure (figure s8). These results show great potential of the 8.5-np - dox / nr - sgc8 system for nir - controlled intranuclear drug delivery . The therapeutic effect of this 8.5-np - dox / nr - sgc8 system on cem and ramos cells was tested by mts assay . As shown in figure 4 and figure s9, the nanomaterials themselves and the pure nir irradiation had little negative impact on either cem or ramos cells (the cell viability of both cell lines remained above 95%), indicating excellent biocompatibility of these nanomaterials and the laser . For free dox, a dose - dependent cytotoxicity was observed on both cem and ramos cells . However, when treated with 8.5-np - dox / nr - sgc8s, only cem cells showed dose - dependent cell inactivity, indicating the selective cytotoxicity of dox delivered by this nanoassembly platform . After nir irradiation, a dramatic decrease of cell viability on cem cells was caused by 8.5-np - dox / nr - sgc8s with a nearly 3-fold lower ic50 of 0.36 m compared to that without nir irradiation (1.22 m). To verify whether the enhanced therapeutic efficacy originated from the synergy of dox, photothermal effect, and np / nr nanocomplexes, cem cells were incubated with free dox and non - dox - loaded 8.5-np / nr - scg8c (the dox loading site of das was replaced with a common dna duplex) together and then irradiated with the nir laser . The therapeutic effect of this case was lower than that of the nir - activated 8.5-np - dox / nr - scg8c, indicating that the synergistic effect was not an important consideration in this system (figure s10). Thus, it is reasonable to attribute the enhanced killing efficiency to the released 8.5-np - dox from the nanotruck . To ensure that the nuclear accumulation of nanodrug induced higher cell apoptosis, the nir - activated 15-np - dox / nr - sgc8 system was used as a non - nuclear - uptake control . As shown in figure s11, with nir irradiation, the cytotoxicity of 8.5-np - dox / nr - sgc8s was 24% higher than that of 15-np - dox / nr - scg8c, while no obvious difference was observed in either case without nir irradiation . These results demonstrate that this nuclear - uptake nanodrug delivery system can greatly enhance the therapeutic efficacy on the target cancer cells . Viability of cem cells (a) and ramos cells (b) with different treatments . The error bars represent the standard deviation of three independent experiments . To investigate the ability of this nuclear - uptake nanodrug delivery system to address the mdr problem, k562/d, a drug - resistant cancer cell line with overexpression of p - gp (figure s12), was used, while its specific internalizing aptamer, kk1b10, was used as the targeting ligand to functionalize the nanotruck . The specific binding of kk1b10, nr - kk1b10, and np / nr - kk1b10 with k562/d cells was proven by flow cytometry (figure s13). As shown in figure 5 and figure s14, enhanced killing efficiency was achieved by incubating k562/d cells with 8.5-np - dox / nr - kk1b10s with nir irradiation, while a much lower therapeutic effect of free dox was detected for this cancer cell line . To confirm that the enhanced therapeutic effect originates from the intracellular accumulation of dox by this nir - responsive nanodrug delivery system, flow assay was performed to measure the dox signal in k562/d cells under different treatments . Upon nir irradiation, the dox fluorescence from the sample incubated with 8.5-np - dox / nr - kk1b10s was 2.5-fold higher than that from the sample incubated with free dox (figure s15). In contrast, without laser treatment, the cells incubated with 8.5-np - dox / nr - kk1b10s produced a modest dox signal . These results demonstrate that this nuclear - uptake nanodrug delivery system can recover the chemotherapeutic sensitivity of k562/d to dox by bypassing cell membrane - expressed p - gp . Viability of k562/d cells treated with free dox or nir - activated 8.5-np - dox / nr - kk1b10s . In summary, we have developed a dna - based nanoassembly platform for cancer therapy . Unlike traditional intranuclear transport strategies of nanoparticles, no nuclear localization signal (nls) peptides are required in our design, and the nuclear uptake of nanodrugs is mainly attributed to small particle size, thus avoiding complicated nls modification processes and maintaining the valid occupancy of drug - loading probes on the np surface . By using this photocontrolled, size - transformable nanosystem, nanodrugs can be efficiently transported across the cell membrane and enter the nucleus in a coordinated and harmonious manner . Furthermore, this dna - based nanoassembly platform can accumulate chemotherapeutic drugs in the nuclei, thus greatly enhancing their therapeutic efficacy against drug - resistant cancer cells by effectively bypassing p - gp . This proof - of - concept structure also opens a new door in the use of nanoassemblies for the design of drug delivery systems for biological and clinical research . To comprehensively evaluate the superiority of this nuclear - uptake nanodrug delivery system, further efforts are being made on the testing in tumor - bearing mice models . On the other hand, since the tissue penetration of the nir laser is limited to around ten millimeters, an alternative strategy for activatable dissociation of the nanoassemblies are needed to apply this system to treatment of drug - resistant metastatic tumors . All dna strands were synthesized on an abi 3400 dna synthesizer (applied biosystems, foster city, ca, usa), and the specific sequences are listed in table s1 . Both the synthesis and the deprotection processes were conducted as described by the reagent manufacturers . Then, the dnas were precipitated by high - salt ethanol in a freezer at 20 c for 30 min and collected by centrifugation at 4000 rpm for 30 min . Subsequently, the dna precipitates were dissolved with 400 l of 0.2 m triethylamine / acetate (glen research corp). The purification step was performed by hplc (prostar, varian, walnut creek, ca, usa) with a c18 column (5 m, 250 mm 4.6 mm, alltech) using acetonitrile and 0.1 m triethylammonium acetate (teaa) aqueous solution as the mobile phase . After being dried by a rotary evaporator, the purified dnas were detritylated with 80% acetic acid, precipitated with cold salted ethanol, collected by centrifugation, and dried by vacuum . Finally, the dna products were obtained, and their concentrations were measured with a uv vis spectrometer (cary bio-300, varian). Nps of 5.5 and 8.5 nm were synthesized by a seed - mediated growth method and then washed by centrifugation to remove hexadecyltrimethylammonium bromide (ctab). The modification of nps with the drug - loading / attaching dna strand (das) was conducted following a reported protocol . The thiolated das (0.2 mm, 20 l) was deprotected by 10 mm tris(2-carboxyethyl) phosphine (tcep, neutral ph, thermo scientific) at room temperature for 60 min and then mixed with 1 ml of nps (20 nm). After incubation for 16 h, the mixture was salt - aged by slowly adding 200 l of nacl (1 m) and allowed to incubate for 16 h. then, the excess dnas were twice removed by centrifugation, and the precipitate was resuspended in 250 l of 1 pbs . Silver nanorods were synthesized and washed according to the procedure in our previous report, and the concentration was determined through the longitudinal absorption band of the uv vis spectrum . The selective modification of nr was performed according to a reported protocol with some adjustment . Briefly, nrs were incubated with targeting aptamers (nr / aptamer ratio was 1:100) in 2 mm ctab solution for 12 h. then, the capture strands (cs) were added (nr / cs ratio was 1:500) and allowed to incubate for another 12 h. the adsorbed ctab was further displaced with thiolated peg . After that, the modified nr was salt aged by slowly adding 1 m nacl to give a final 0.3 m concentration of na, and the mixture was held at room temperature for at least 12 h. after washing 5 times by centrifugation, the resultant nrs were resuspended in 1 pbs for further use . To fabricate the self - assembly nanocomplexes, the das - modified nps of 5.5, 8.5, and 15 nm were mixed with the modified nrs at a np / nr ratio of 20:1, 15:1, and 10:1, respectively, and incubated at room temperature for at least 24 h. the mixture was then gently centrifuged to remove unbound nps . For dox loading, nps were modified with das whose 3-end portion can form a (cgt)6/(acg)6 duplex with a corresponding cdna . The dox loading was conducted by mixing dox with np - dass, incubating at room temperature for 30 min, and then centrifuging to collect the np / dox complexes . The number of dox per np was determined by measuring the fluorescence intensity of unbound dox in the supernatant and then interpolating from a standard linear calibration curve . For the dox leakage assay, 150 l of np / dox complexes was added to a mini dialysis unit [3.5 molecular weight cutoff (mwko), thermo scientific], and the equivalent of free dox was used as a control . Each unit was immersed in 3 ml of 1 pbs in a 5 ml beaker with gentle stirring at 450 rpm . At each given time point, a 100-l aliquot of the dialysis solution was collected for dox fluorescence measurement . After that, the collected solution was returned to the corresponding beaker . Cem (human acute lymphoblastic leukemia) and ramos (human burkitt s lymphoma) were purchased from american type culture collection . K562/d (doxorubicin - resistant chronic myelogenous leukemia) was generously provided by dr . Troy a. a. harkness of the department of anatomy and cell biology, college of medicine, university of saskatchewan . Cem, ramos, and k562/d cells were cultured in rpmi 1640, rpmi 1640, and imdm medium, respectively, supplemented with 10% fbs (heat - inactivated; gibco) and 100 iu / ml penicillin cells (5 10 in 100 l of medium) were incubated with free dox, fluorescent nanoparticles, or nanocomplexes at 37 c with 5% co2 for different time lengths . After several centrifugation / washing steps, the cells were suspended in 1 pbs . For photoactivation, cells were irradiated with nir laser (600 mw / cm) for 10 min . Bisbenzimide hoechst 33342 (sigma - aldrich) was used for nuclear staining by incubating with cells at 37 c for 20 min . Fluorescence imaging was performed on a leica tcs sp5 confocal microscope (leica microsystems) with a 63 oil - immersion objective . In most cases, the red color represents the fluorescence of cy5 (em = 670 nm), the pink color represents the fluorescence of dox (em = 600 nm), and the green color represents the fluorescence of tamra (em = 570 nm). Cem cells (1 10) were incubated with 8.5-np / nr - sgc8s or 15-np / nr - sgc8s at 37 c with 5% co2 for 6 h. then the cells were washed with pbs three times, irradiated with nir laser for 0 or 10 min, and incubated at 37 c with 5% co2 for another 22 h. to extract nuclei, the cells were collected by centrifugation, resuspended in 10 mm tris - hcl buffer (ph 7.4) containing 100 mm nacl, 1 mm edta, and 1% triton x-100 at 4 c for 10 min and finally centrifuged at 1000 g for 3 min . After several centrifugation / washing rounds to remove the adsorbed nanoparticles on the nuclear membrane, the collected nuclei were lysed by a lysis solution containing 0.5% triton x-100 and 1 m naoh with sonication . Subsequently, the nanoparticles from the nuclei were digested by incubating with aqua regia at 65 c overnight and diluted in 2% hno3 solution . Nps accumulated in nuclei were measured by quantifying au element by inductively coupled plasma atomic emission spectrometry (icp - aes). To evaluate the cell binding affinity of different nanocarriers, the aptamer was labeled with a fluorescein (fitc). Cells were incubated with free aptamers, nr - aptamers, or 15-np / nr - aptamers at 4 c for 30 min . After removal of unbound materials by several centrifugation / washing steps, the cells were analyzed on a facscan cytometer (accuri c6) by counting 20 000 events . The cell viability under different treatments was determined by celltiter 96 cell proliferation assay (promega). Cells were incubated with free dox, np / nrs, or np - dox / nrs at 37 c with 5% co2 for 2 h and then centrifuged to precipitate . The supernatant (80%) was removed, followed by adding equivalent fresh culture medium (10% fbs). For nir - responsive regulation, cells were irradiated with a nir laser (808 nm, 600 mw / cm) for 10 min, followed by additional incubation to allow further growth for 48 h. then, 80% of the medium was removed and replaced with 20 l of mts reagent diluted in 100 l of rpmi 1640 . The resulting cell samples were incubated at 37 c for 12 h. finally, the absorbance at 490 nm was collected using a tecan safire microplate reader, and cell viability was calculated using the equation provided by the manufacturer.
Public health policy decisions must balance a range of scientific, budgetary, social, and political considerations . Ideally, each of these elements should be considered in a transparent fashion before reaching a decision or implementing a specific policy . While socio - political considerations will always be somewhat subjective, scientific evidence can in theory for example, in the setting of a high - profile outbreak, the probability of making political gains or alleviating public fears is not objectively quantifiable (despite their importance to the decision - making process), but scientific outcomes, such as potential trajectories of the outbreak under different policy decisions, can be estimated quantitatively with appropriate tools using the best available data as inputs, such as the known incubation period . In the realm of infectious diseases, the tools for integrating and translating scientific data into policy - relevant outcomes are often classified in the domain of mathematical models, which are defined here as quantitative frameworks for the analysis of dependent happenings (events where the number affected at one time depends on the number already affected). For example, systems of diagnosis and treatment are represented in mathematical terms such as the rate of movement from an infectious to a treated state . These models have the ability to translate existing scientific evidence into projected outcomes at the population level for both endemic diseases like tuberculosis (tb) and epidemic situations such as the ebola virus disease (evd) outbreak in west africa in 20142015, in a way that is transparent and verifiable or refutable by external observers . These estimates can also help with clinical decision - making at the individual level, to improve patient outcomes . Unfortunately for most public health decisions regarding the control of infectious diseases, such models are seldom constructed and when they are, they often have limited impact upon the decision - making process . This is likely due to several factors, including perceptions that models are too complex to understand or too dependent on assumptions, coupled with a history of insufficient communication between public health practitioners with specific policy questions and modellers with the quantitative tools to address them . Here, the potential role of mathematical modelling in decision - making for health policy in the realm of infectious diseases is explored, and key reasons why mathematical models have historically not fulfilled this potential are evaluated . To do this, the current status of modelling in public health decision - making is first outlined and a case study modelling question described . Details of how to construct a relevant model and how to link it to policy are then given, and some of the potential limitations and challenges of using modelling described . Finally, a framework by which improved collaborations between public health stakeholders and modellers may broadly benefit public health is proposed . The use of structured frameworks for applying evidence to public health decision - making is well established . For example, the world health organization (who) advocates the use of the grade process, which is a framework that connects a public health question to an evidence - based analysis and recommendation . The united states preventive services task force (uspstf) similarly uses decision - making algorithms to assess the level and quality of evidence to support the introduction of specific interventions . However, these frameworks for using scientific evidence to support policy decisions often lack quantitative assessments of how different decisions will impact health at a population level . This is especially true in the realm of infectious diseases, where dynamics of transmission may cause great disparity between the individual - level benefit or harm of an intervention (for example, side effects of a vaccine for a rare disease such as polio that may outweigh an individual s risk of contracting the disease) and its population - level impact (for example, maintaining elimination of polio through herd immunity). As a result, in settings where population - level benefits are unproven, interventions with strong scientific evidence for individual effectiveness may be recommended over those with a potentially dramatic impact for populations . This decision - making process, if uninformed by insight at the population or system level (as provided by models), may perversely result in outcomes that are good for certain people, but bad for the population as a whole . Models can address this knowledge gap by estimating the effects of interventions when the collection of population - level empirical evidence (e.g., from cluster - randomized trials) is infeasible, unethical, or untimely . For example, mathematical models suggested that universal voluntary hiv testing and immediate antiretroviral therapy (art) might dramatically reduce future hiv transmission, even though the individual - level effectiveness of art at higher cd4 + t - cell counts is small, and reduced transmission at the population level is difficult to prove empirically . By projecting population - level effects of potential interventions, the models informed not only key policy decisions but also the design of future clinical trials . Despite the potential impact that model outputs can have on public policy decisions, the use of models by public health decision - makers has traditionally been limited . Many public health and policy decisions must be reached rapidly, in too short a time for new models to be developed, parameterized, and calibrated . Modellers must therefore achieve a balance between anticipating future policy questions (in which case models may ultimately not speak to the specific policy question at hand) and responding to existing questions (in which case models may be constructed too late to inform policy decisions). In addition, as mentioned above, complex models that are poorly presented are unlikely to be used by time - pressured policymakers . Furthermore, it remains unclear in most settings how to weigh evidence from models against other epidemiological and clinical data . As described below these aspects of models are often not well - understood by public health stakeholders, and as a result, model outputs may be seen as difficult to interpret and untrustworthy . A framework by which modellers and decision - makers can work together to more appropriately incorporate evidence from infectious disease models into public health decisions, without over- or underemphasizing the importance of those models, is proposed here . To demonstrate the utility and process by which mathematical models can inform infectious disease policy, the case study of a new molecular diagnostic test for tb is used: the xpert mtb / rif test (xpert). Xpert provides a comparatively rapid, point - of - treatment diagnosis in under two hours, if placed in settings where individuals present for initial tb diagnosis and/or follow - up evaluation . Xpert is also substantially more sensitive than the most widely used diagnostic test for tb worldwide (sputum smear microscopy). However, at over 10 times the cost of sputum smear microscopy (which costs less than $2 fully - loaded per test, compared to about $20 for xpert), scale - up of xpert has the potential to dramatically increase the cost of tb control in high - burden settings . The key policy - related questions around the use of xpert are the following: do the clear individual - level benefits of improved diagnosis translate into population - level effects on transmission, and if so, would scale - up of xpert have sufficient impact to justify the added cost (i.e., would xpert be cost - effective)? These questions can be, and have been, addressed effectively using mathematical modelling . In the case of xpert, an initial modelling study projected the impact on tb - associated morbidity and mortality in six countries of southern africa . This study adopted a regional approach, which allowed the authors to use a single model framework (due to similar epidemics across the six countries) and existing data (which are reported on the national level). A global model would likely have required more model complexity, whereas a sub - national model might have been limited by available data or generalizability . The authors aimed primarily to publish their results in the scientific literature, although the model has subsequently been used in country - level discussions and extended to other regions . The model predicted a relatively large potential population level impact of xpert on tb transmission and mortality, based on the assumption that increased rapid diagnosis would increase treatment rates . In the absence of pre - existing data, this model had to make a number of reasonable simplifying assumptions, including the proportion of individuals with tb who would ultimately be diagnosed by the existing algorithm in the absence of xpert and the speed at which that diagnosis might happen . These results which reflected the best available data and assumptions at the time were used to support policy recommendations to scale up xpert in the region and worldwide . Subsequent clinical trials revealed that xpert did not identify many more patients than were already being started on treatment, due to unexpectedly high levels of empiric treatment practices in tb - endemic settings . Using this new information, the model was then revised to account for empiric treatment practices, and new estimates predicted a much smaller impact of xpert . Several of these models were extended into cost - effectiveness analyses of xpert scale - up . This case study illustrates the ability of models to iteratively incorporate updated data, leading to better estimates and highlighting existing weaknesses in both model assumptions and available data over time . A pertinent translation of individual - level effect to population impact is also displayed . Unfortunately, the case study also demonstrates the challenges in linking model results to policy . Major obstacles exist to the implementation of xpert, especially in trying to use xpert as a true point - of - care test . Nevertheless, despite its impact on population health being initially relatively uncertain, xpert has received strong support from policymaking bodies such as the who, based primarily on systematic reviews of sensitivity and specificity . This recommendation places pressure on many high - burden countries to scale xpert up, and at tremendous expense . Even in light of emerging data and updated model projections that suggest xpert may not improve population - level outcomes, recommendations to implement xpert have become increasingly strong, partially due to political momentum and the known individual - level benefits of xpert . The policy modelling disconnect in recommendations for xpert contrasts, for example, with that for systematic screening for tb in high - risk populations . Systematic screening for tb has been shown in multiple mathematical models to have a potentially dramatic impact at the population level, but the benefits of systematic screening at the individual level are difficult to prove . The recommendation for systematic screening is therefore much less enthusiastic . As a result of this discrepancy between individual - level evidence (which is easier to collect directly but arguably less important to public health) and population - level evidence (which is difficult to collect directly and thus often requires models but is critical to public health decision - making), many countries are pressured to implement xpert rather than systematic screening . This pressure exists despite the evidence from models that systematic screening might have much greater impact on reducing tb at the population level and potentially at a more favourable cost effectiveness ratio . In summary, as shown by the case study of xpert, modelling interventions is often the only way to evaluate the comparative effectiveness (and cost - effectiveness) of interventions at the population level in the short term . In doing so, models may not only help to prioritize those interventions that might have greatest impact at the population level, but may also identify the data elements needed to better inform estimates of such impact for different public health policies . This process which ideally occurs iteratively as new data emerge can lead to better alignment between research efforts and policy priorities . However, major obstacles exist to the implementation of this process, and current practice continues to prioritize infectious disease interventions with more benefit for individuals than for populations . Developing a useful model includes identifying, in sequence (1) a useful question and its epidemiological context, (2) a framework through which that question could be addressed, (3) the parameters required to address the specified question using that framework, and (4) the empirical evidence available to inform meaningful values of those parameters (for examples and further details see publications by vynnycky and white and keeling and rohani). Once a question, framework, parameters, and empirical evidence have been identified, the model can then be used to inform decision - making by projecting the potential outcomes associated with different policy decisions . For example, in the xpert case study, the question of interest was how big would the impact of this new diagnostic test be? The framework utilized was a transmission model that used both natural history and tb control parameters, such as treatment success, informed by country - level tb programme data . For models to be useful to decision - makers in general, useful models are built to answer a key question that should guide the structure and complexity of the model (rather than the model determining the question). One such question might be to evaluate the expected epidemiological and economic impact of different strategies for scaling up xpert for tb diagnosis (e.g., centralized or in individual clinics), or the required bed capacity during the recent evd outbreak . Defining a central question also helps to inform the structure of the model, which should incorporate relevant scientific data (e.g., transmission rates, existing levels of infection control and treatment). The epidemiological setting is also important; for example, a model of implementing xpert should include not only the sensitivity and specificity of the test but also the diagnostic processes, underlying disease prevalence, and clinical algorithms in the chosen setting . A model of tb diagnosis in the usa would need to account for immigration, for instance, whereas a model of tb in sub - saharan africa would require a more detailed description of art scale - up . In some cases first - pass results across a range of settings and interventions; in other cases (or when more precisely calibrated results are needed), separate models will be required for each setting . In general, models allow for an exploration of the system and give a holistic picture of the realm of possible outcomes . As such, uncertainty and sensitivity analyses around the main components of the model are critical . Inputs, such as the proportion of patients accessing different diagnostic services, into uncertainty in model sensitivity analyses aim to attribute portions of this uncertainty to specific parameters . In a one - way sensitivity analysis, for example, a key parameter (such as the tb transmission rate) might be set sequentially to its highest or lowest plausible values, and the model results assessed at each of those points . In the case of xpert, population - level impact has been shown to be very sensitive to existing levels of empirical therapy for tb . Broader consideration of model uncertainty would explore the impact of a range of plausible empirical treatment levels as well as other model parameters (for example, transmission rates) or indeed do a more comprehensive sampling over all parameters . Ultimately, the estimates of any model can only be as accurate as their supporting data, but models can also describe that uncertainty to decision - makers, allowing them to make the most appropriate decisions given existing, imperfect evidence . As such, modelling results with wide confidence intervals that reflect this uncertainty are often valuable to policymakers, as they demonstrate the current state of knowledge . The alternative, where false confidence in predictions is gained via modelling based on strong unsupported assumptions, must be avoided despite the temptation of the clarity of results that such assumptions can provide . Models can inform infectious disease public health policy in at least three ways (see figure 1). Firstly, models can systematically use epidemiological data to better understand the larger systems in which policy decisions must be made . For example, mathematical models of xpert scale - up in africa have suggested that baseline diagnostic patterns affect the incremental benefit of a novel, more sensitive test, thereby suggesting that policymakers should target xpert roll - out to areas with the weakest existing diagnostic systems . Secondly, as described above in the case of universal hiv testing and treatment, models can apply a transparent framework to compare the potential population - level impact of interventions in situations where collecting empirical evidence might be logistically, monetarily, or ethically infeasible . Even when broader empirical studies are feasible, interim policies must nonetheless be set; models can help these policies make maximum use of existing evidence before definitive results are known . For example, modelling estimates for the recent evd outbreak in west africa published in september 2014 predicted that without interventions, liberia and sierra leone would have approximately 550 000 reported ebola cases by january 2015 . The predictions over a shorter timeframe were closer to what actually occurred; for estimates of case numbers by september 2014, the model overestimated the number of cases by only 8.8% in liberia, and underestimated the number of cases by 7.6% in sierra leone . These results, however, demonstrated at the time what most needed to be done to control the outbreak . Wrong but can still be useful . Furthermore, the results made under the assumption of no intervention highlight the impact that outbreak control interventions had on the magnitude of the outbreak and these could later be compared to an alternative model structure that incorporates those interventions to further evaluate the impact of those measures . By highlighting how serious things could be if nothing was done, models emphasized the need for control interventions and the aspects of those interventions that might be most important from an epidemic control perspective . Thirdly, modelling can point to data gaps that, if filled, could better assist decision - making and control of infectious diseases in the future . For example, tb models might find that the comparative impact of xpert scale - up strategies depends strongly on the amount of ongoing tb transmission in a community, which in many places may not be known . These results could motivate further data - gathering activities (e.g., molecular epidemiological characterization of acommunity, or synthesis of existing programmatic data on tb incidence) that could help to improve decision - making related to xpert scale - up in the future . For the spread of rubella, modelling has already led to the collection of new missing data . In each of these cases, mathematical models provide public health decision - makers with key pieces of knowledge that can inform evidence - based decision - making . Furthermore, unlike expert opinion (which often holds sway purely on the basis of reputation or existing dogma), models accomplish this task in a way that is quantitative and open to questioning (or modification) by others in the field . When a model s structure, methods, assumptions, and parameters are laid out in a reproducible manner with direct communication and guidance to those with less methodological expertise, their results should be transparent and accessible rather than being perceived as a while in theory, models are fully transparent, many models are made so complex that few outside the modelling community can fully understand their mechanics . In addition, while models should be tailored to answer a specific policy question of interest, they are often presented in such a fashion that does not speak readily to key policy decisions . Modellers must strive to produce transparent outputs that can directly inform the policymaking process, even when this requires some simplification to be made . An important way to improve transparency is to publish the raw data as well as any modelling code . Whether provided in the appendix of the publication or in online format, this would provide readers with access to the model and the ability to closely review its methods, thus making a more informed determination as to whether the assumptions and data used were sufficient . Open data and code would also allow for the model to be improved and developed iteratively by others in the modelling community to answer other policy - relevant questions . In addition, care must be taken when broadly applying a model specifically structured to answer a certain question . For example, a modelling evaluation of xpert scale - up across southern africa is unlikely to provide useful guidance on where to place xpert machines in the usa, or whether to supplant other funds to pay for xpert testing . However, models can be, and often are, reconstructed as new evidence or considerations come to light . The development begins with models that provide initial insight; such initial models are gradually replaced by more complete models that incorporate updated information . This process of iteratively evaluating modelling output provides a framework in which to place new evidence and improves our understanding of both the problem and the utility of the modelling tool being applied . While the need for such iteration can be seen as a problem with the initial model or input data, it is more appropriately seen as reflecting the natural course of scientific inquiry, in which better data and better tools to utilize those data are continually being developed . Presenting the uncertainty around modelling results, as described above, presents a further challenge as it often reflects limited available data on key parameters . In such cases, modellers should honestly portray this uncertainty rather than providing results that appear more reliable, and policymakers should make decisions based on this uncertainty rather than requesting results that are more precise . Not including such uncertainty may be justified in specific cases such as when the infectious disease situation being modelled (e.g., when considering just a no intervention scenario) is unrealistic and specific policy decisions are not based on this quantitative value but is generally not recommended . There are also significant ethical considerations that must be taken into account when using the results of mathematical models to design public health interventions . These include considering the values and preferences of the target population as well as the available resources . Determinants of health behaviour must be viewed in a social context, including the cultural beliefs, historical patterns, and choices available to the local population . If applied in a vacuum, modeling results may not be relevant to the target population and the estimated impact of a chosen intervention will likely not be realized . How can we improve the utility of modelling for infectious disease relevant to public health decision - making in the future? One important component of any such strategy should involve ongoing collaboration and interaction between infectious disease modellers and public health stakeholders . Such communication and collaboration enables decision - makers to appropriately understand the complexity of model structure and uncertainty in modelling results, and modellers to inform additional refinements or data - gathering efforts to reduce that complexity and uncertainty . In addition, both modellers and public health policymakers must view the results of models within the social context of the target population to consider the ethical impact of applying modelling results to specific settings . Fostering such collaboration will improve the availability of models to public health practitioners, as well as the quality of model structure and relevance of results . It will also improve the likelihood that the perspectives and needs of all critical stakeholders are included . To promote such public health stakeholder collaboration and optimize the role mathematical modelling can have in public health decision - making in the field of infectious diseases, an iterative process for model development is recommended (figure 2). The key stages in this process are: (1) policymakers engage modellers early in the decision - making process and inform them of the key public health questions that are being considered in a given population and/or clinical setting . (2) modellers use this information to construct models that are most likely to effectively address these questions . (3) modellers strive to reduce, or fully justify, the complexity of their models and explain the context and uncertainty of their outputs to decision - makers and others who could openly interrogate their methods . (4) decision - makers seek to incorporate model results into their decision - making process (including decisions for more data gathering) and inform modellers of where model structures, outputs, and uncertainty could be refined for future decision - making . To succeed in this endeavour, it is important that modellers and decision - makers build mutual trust over time; these steps are difficult to complete in a one - off fashion for each new question that arises . Platforms for such interaction, such as conferences and workshops, should be developed and promoted to stimulate discussion and interaction around the important questions and how to address them, including how to share key data . As effective communication is only possible when modellers and stakeholders speak the same language, it is critical that these communities work together to establish long - term collaborative relationships . As an example of such longer - term collaboration between infectious disease modellers and infectious disease policymakers, for example, the tb modelling and analysis consortium (tb mac) brings together quantitative researchers, policymakers, tb programmes, and donors to identify tb control questions that require modelling input . Discussion and interaction between tb control stakeholders is also supported by the tb modelling group at johns hopkins, which holds frequent international conference calls to examine the latest research and areas of interest . Such multidisciplinary collaboration is also being seen in other infectious disease fields, where modelling studies have been used to assist public health interventions and policy - making decisions around hiv, influenza, and evd . While such relationships take time to build, are difficult to incentivize from both the academic and public health perspectives, and may not yield immediate results, increased communication between modellers and public health policymakers is arguably the only viable path towards bringing the wealth of existing epidemiological evidence to bear in making public health decisions for infectious diseases . Infectious disease modelling can provide important and useful data to inform public health policy by improving our knowledge of epidemic disease spread, comparing the impact of potential public health interventions and understanding gaps in existing data used to inform public health decision - making . For models to be most useful, challenges in applying modelling results to public health practice, including the complexity of model structure and uncertainty of model outputs and their relevance to important policy questions, must be understood and considered . While modelling will not provide all the answers for public policy, it can provide useful quantitative evidence when large clinical studies are not possible or are still underway . A framework that can be used to improve the process of applying modelling to public health decision - making for infectious diseases is proposed . Collaboration between public health stakeholders and modellers is essential to heighten the transparency and public health relevance of models, to optimize the use of epidemiological data for decision - making, and to develop policies that incorporate scientific evidence to improve the control of infectious diseases worldwide.
Castleman's disease is a rare b - cell non - clonal lymphoproliferative disorder first described over 50 years ago characterized by its variegated presentation . It can practically affect any region within the body, the most common being the chest in about 70%, the abdomen in about 15% and the remaining occurring within the neck . This condition may resemble other entities causing lymphadenopathy including lymphomas, infectious or inflammatory causes . The predominant histopathological variants include the more common hyaline vascular type, the plasma cell type which is usually associated with poems syndrome, castleman's disease seen with hhv-8 infection usually found in hiv and immunosuppressed patients and an unspecified form of multicentric castleman's disease . Castleman's disease is clinically classified into a unicentric variety, which is usually asymptomatic, or a multicentric variety, which can be associated with systemic inflammatory state manifesting with constitutional symptoms such as fever, night sweats and weight loss, depending on the number of lymph nodes involved [1, 2]. In certain cases, this systemic inflammatory state can be complicated by secondary amyloidosis characterized by deposition of serum amyloid a protein within the extracellular tissue . Castleman's disease has not demonstrated any sex predilection, with unicentric variety peaking in incidence within the second to fourth decades of life, while multicentric variety occurs in older populations . Secondary (aa) amyloidosis has been reported with both unicentric and multicentric variants of castleman's disease . The prognosis of castleman's disease is related to the extent of systemic involvement either directly from the disease itself or from co - existing systemic amyloidosis . Untreated systemic amyloidosis also often has an unrelenting clinical course, leading to multiorgan failure and death . The degree of regression of amyloidosis itself is often contingent upon the variant of castleman's disease, with surgical excision of the primary tumor in a unicentric variant being potentially curative [1, 2]. We report a rare association of unicentric castleman's disease with secondary (aa) amyloidosis . A 51-year - old african - american man who had been born and raised in the united states first presented to the medical clinic with complaints of generalized weakness, fatigue, unintended weight loss, anorexia and progressively worsening abdominal distension of 3 months duration . Physical examination at the time was significant for a firm, indurated right - sided submandibular mass, hepatomegaly and mild epigastric tenderness . An initial set of laboratory studies showed abnormal liver enzymes (alanine transaminase of 61 iu / l, aspartate transaminase of 83 iu / l, markedly elevated alkaline phosphatase of 1,003 iu / l and gamma - glutamyl transferase of 1,879 iu / l with normal bilirubin levels). Additional work - up done for evaluation of abnormal liver enzymes including viral hepatitis panels (hepatitis a, b and c), anti - nuclear antibody, anti - smooth muscle antibody, anti - liver kidney microsomal antibodies and anti - mitochondrial antibody were negative . The patient underwent biopsy of the submandibular mass that revealed features of castleman's disease (fig . 1). A subsequent liver biopsy revealed perisinusoidal deposition of eosinophilic, amorphous material within the extracellular matrix with hepatocyte atrophy, consistent with hepatic amyloidosis (fig . Bone marrow biopsy revealed diffuse extracellular eosinophilic, amorphous material consistent with amyloidosis with increased kappa light chain - restricted plasma cell count (6% of hematopoietic bone marrow cells) (fig . The patient underwent a colonoscopy that revealed no gross mucosal lesions; biopsies were unremarkable . A definitive diagnosis of secondary (aa) reactive amyloidosis with hepatic involvement ascitic fluid analysis revealed an elevated serum - ascites albumin gradient of 1.7 and very low protein of 0.9 g / dl . He was treated with intravenous antibiotics . A computed tomography scan of the abdomen done at the time revealed dilated loops of small bowel consistent with small bowel obstruction, which resolved with conservative management . He was not considered for chemotherapy in view of active infection and was not a liver transplant candidate because of his poor physical condition . The patient was discharged to be readmitted only 2 weeks later with recurrent ascites from decompensated liver disease . His clinical condition rapidly deteriorated with superimposed severe metabolic acidosis resulting from acute renal dysfunction . Castleman's disease often presents in sixth to seventh decades of life with more than half of affected patients being male . Patients with multicentric castleman's disease universally present with peripheral lymphadenopathy as seen in our patient . Only a few cases of secondary (aa) amyloidosis complicating castleman's disease [7, 8, 9] have been reported in the literature . Extensive hepatic involvement from primary systemic amyloidosis is seen in almost two - thirds of the patients [10, 11], but to a lesser degree in secondary (aa) amyloidosis [12, 13]. Hepatic amyloidosis is often asymptomatic, the most common presentations being hepatomegaly in more than two - thirds of those affected and elevated alkaline phosphatase . Hepatic amyloidosis is characterized by parenchymal replacement by amyloid leading to pressure atrophy of the hepatocytes . Ascites, as seen in our patient, is a rare finding and is often a function of decreased cardiac contractility from amyloid infiltration or hypoalbuminemia from nephrotic syndrome . Our patient had a normal cardiac ejection fraction of 71% on echocardiogram, but was profoundly hypoalbuminemic from heavy proteinuria . He was observed to have cholestatic liver function tests with jaundice, which is exceedingly rare and often portends poor outcome [15, 16]. He also likely had collateral gastrointestinal amyloidosis with symptoms like unintended weight loss and was found to have intestinal pseudo - obstruction as well, which likely resulted from enteric neuropathy from amyloid deposition . It has been noted in a small - size study that use of ursodeoxycholic acid could be of benefit in hepatic amyloidosis, with improvement in serum alkaline phosphatase and gamma - glutamyl transferase levels . The role of liver transplantation was studied extensively in familial variants of amyloidosis [19, 20, 21], to a lesser degree in primary (al) amyloidosis, but not in secondary reactive amyloidosis . However, the crux of treatment of secondary (aa) reactive amyloidosis lies at treating the underlying precipitating inflammatory condition . There has been a steady increase in survival in patients with reactive amyloidosis secondary to advances in treatment strategies for underlying inflammatory disorders . Excision of unicentric castleman's tumor often leads to regression of aa amyloidosis by suppressing cytokine production . However, our patient did not show an optimal clinical response even after excisional biopsy of the submandibular castleman's tumor . To our knowledge, this case is one amongst only few that presented systemic amyloidosis complicating castleman's disease . This case is unique in that extensive, diffuse amyloid deposits were observed in the liver with cholestatic jaundice . It is also the first reported case where secondary amyloidosis failed to regress even after removal of the primary trigger, the focal castleman's tumor . The authors do not have a direct financial relation with the commercial identities mentioned in the paper that might lead to a conflict of interest.
Molecular simulations of biomolecules are expressions of their underlying force fields, sampling the interactions of chemically bonded atoms through newtonian approximations of the quantum systems . The typical model, given in eq 1, is well - known . It approximates the energy of a molecular system as a thoroughly decomposable, easily differentiable sum of terms involving harmonic bonds, harmonic angles, lennard jones repulsion / dispersion terms, electrostatic interactions, and additional parameters guiding the dihedral preferences of the model . The additional parameters are both the least physically grounded and the most frequently edited part of most force fields; numerous forms of the expressions modifying the dihedral potential energy include screening factors applied between the electrostatic and lennard jones interactions of atoms connected by chains of three bonds, fourier series in the dihedral angles made by such atoms, and coupling terms between fourier series of consecutive dihedral atom chains . Changes in these parameters that have made considerable improvements in numerous force fields should be viewed in light of the fact that their adjusments to the system energy are small . The terms describe a regime between the high - frequency motions of bonded atoms and the low - frequency rearrangements of nonbonded chemical groups and appear to be a sort of keystone in numerous molecular models.1many molecular force fields can be classified into several lineages . While some force fields have been developed to reproduce bulk liquid properties, new models today tend to emerge as new levels of quantum mechanical theory become accessible and also as updates based on more extensive fitting data or inconsistencies with known biochemical data . Within each lineage, the models tend to evolve while retaining a significant portion of their parameters from previous work . Even comparatively minor changes can take years to gain acceptance, however, and any jump to parameter development based on a new level of quantum theory also requires significant effort to validate . The lineages of force fields are a natural consequence of the economics of force field development, particularly in the academic community where novelty and publications are essential . This environment has also driven the evolution of composite models where the various energy terms are shaped by different levels of quantum theory ., incorporated first into the amber ff94 force field, has remained in service for nearly 20 years . These charges were developed according to the kollmann resp method, using hf/6 - 31 g * quantum calculations to provide the target electrostatic potentials . Numerous other force fields have adopted the charge model, most of them distinguished by new dihedral fourier series, typically based on quantum calculations at the mp2 level with either a cc - pvtz or 6 - 31++g basis set . While the mismatch in quantum targets implies that the torsion terms are effectively modifying the short - ranged behavior of the charge set to fit a different potential energy surface, the force fields derived in this way have led to impressively stable protein behavior . In 2003, duan et al . Derived an alternative charge model, based on b3lyp / cc - pvtz quantum calculations in a polarizable continuum (pcm) solvent intended to mimic the interior of a protein . Backbone torsion fourier series were derived specifically for this new charge set at the mp2/cc - pvtz level of theory, also in the context of pcm solvent, to complete the amber ff03 force field . While the charges and single - point energies are derived with similar styles of qm theory, the standard torsion fitting procedure does not incorporate its own pcm solvent, and it is not trivial to extract the energy of the pcm - polarized wave functions in vacuum . Molecular mechanics gas - phase energies computed with charges derived in the context of pcm solvent have accordingly been shown to double - count polarization effects, as the statically polarized charges are likely to be further affected by direct interaction with the solvent . It is unclear how this imbalance may have affected the quality of simulations performed with ff03, but the force field has not become as widely used or further refined as ff94 and its derivatives . In recent work, we devised the implicitly polarized charge model (ipolq), based on a new quantum mechanical method that integrates the condensed - phase environment due to explicit water molecules into quantum calculations at the mp2 level of theory . In this method, the appropriate atomic partial charges of a nonpolarizable model are estimated to be halfway between the charges of the system in vacuum and those of a system fully polarized in the presence of a condensed - phase reaction field potential . The averaging accounts for the polarization energy and instantaneous rearrangements of dipoles in otherwise nonpolarizable models . The choice of tip4p - ew to represent the solution - phase environment while developing the protein force field supports the use of this water model for later simulations . Furthermore, the ipolq method provides a basis for understanding why essentially all fixed - charge water models carry a dipole moment of roughly 2.3 d: the dipole is halfway between the true condensed phase dipole of water, estimated to be 2.6 to 2.9 d, and the 1.85 d dipole of water in the gas phase . Charges for solutes can therefore be derived under physical approximations that also obtain very similar results to existing water models . While the ipolq approach offers numerous benefits for creating a self - consistent force field, the process of deriving charges is laborious, and our previous work did not assign torsion fourier terms to complete the model . In this article, we will extend the ipolq method to facilitate derivation of complete models, and we will also describe automation of the procedure, which greatly reduces the human effort needed to create new force fields for proteins and drug - like molecules . The amber ff14ipq force field is intended to be a direct alternative to other nonpolarizable amber force fields and contains no new functional forms . However, ff14ipq is also designed to consistently adhere to a specific level of quantum theory, mp2/cc - pvtz . Over the course of this study, we found two major obstacles to fitting a molecular mechanics (mm) expression to a quantum mechanical (qm) potential energy surface (pes) and devised our own solutions to each of them . The first concerns the means for adapting the charge set and torsion fourier series terms to work in concert . The second focuses on whether to make corrections for the inability of common mm models to reproduce certain high - energy features of the qm pes . The implicitly polarized charge method produces charges that, given certain assumptions detailed earlier, reflect the appropriate partial atomic charges of a nonpolarizable model intended for simulations in aqueous solution . The atomic partial charges of our ipolq amino acid charge set approximate electrostatic potentials around dipeptides; these potentials are halfway between the potential calculated for an unpolarized dipeptide (in numerous conformations) in vacuo and the potential calculated for the dipeptide (in the same conformations) after its electron density has been polarized by a solvent reaction field potential due to the time - averaged tip4p - ew water density sampled around each conformation . The electrostatic potentials needed for fitting charges in the condensed phase are laborious but straightforward to compute . It is less obvious how to extract the internal potential energy of the dipeptides in the condensed phase or make an equivalent averaging to derive an appropriate pes for fitting torsion parameters . Zgarbov and colleagues solved this problem by computing quantum mechanical energies of solutes at the hartree fock level of theory in the presence of the cosmo continuum solvent model, removing the solute solvent interaction energy and then comparing to molecular mechanics potential energies computed with a pb solvent model . In effect, they compared the internal energies of the qm and mm systems in the presence of equivalent continuum solvents . In our case, it is not as straightforward to decompose single - point energies at the mp2 level of theory, nor is it as certain whether a quantum - mechanical implicit solvent model could be substituted for tip4p - ew . We instead chose to extend the ipolq methodology and offer an alternative method for deriving torsion fourier series from gas - phase pess when the accompanying charge sets approximate polarization in a condensed - phase environment . It would be simple to derive torsion parameters for a set of charges derived strictly from the electrostatic potentials for solutes in vacuum: compute the single - point energies of many additional solute conformations at the same level of quantum theory, again in vacuum, and fit the mm energies of each conformation to match the qm results . Furthermore, the role of the torsion fourier series is to artificially correct errors in the nonbonded electrostatic and van der waals interactions between atoms on either end of a dihedral; the pes of a rotatable bond is mostly captured by the nonbonded parameters . We therefore sought a way to express the set of ipolq charges for a solute of interest, q, as a perturbation q of charges q derived for the solutes using only the vacuum quantum calculations:2it is simple to compute torsion parameters to match an mm pes q to a qm pes computed in vacuum . Changes in the pes of a molecule s rotatable bond due to immersing it in water could then be assumed to be adequately represented by changes in local dipoles as expressed by q . This appears to be a safe assumption, given that q q and that the van der waals parameters, which also strongly influence the potential energy changes due to rotation about a bond, are constant across both phases . However, the linear least - squares fit from which our ipolq charges q and our proposed vacuum charges q are derived is able to obtain many different solutions with similar levels of accuracy, particularly for buried atoms whose charges are not well determined . In our earlier work, we used large numbers of conformations of each solute in order to guard against this behavior . However, because residual indeterminacy could exist in either q or q, the difference q could still be amplified . We therefore extended the ipolq least - squares fitting procedure to fit both q and q simultaneously, with additional restraint equations to keep q small . The results are solutions for each of q and q related by a minimal, smooth perturbation q . The original matrix equation constructed in the ipolq fitting procedure may be expressed in shorthand:3here, a is the fitting matrix whose elements are derived from the molecular coordinates and the kernel of coulomb s law . Each row of a describes one linear equation through which the partial charges of a molecule in some conformation create an electrostatic potential at a nearby point in space . The electrostatic potential found by the target quantum method is stored in the vector u. each column of a solves for an independent charge variable; if multiple atoms of a molecule are constrained to have the same charges, then they contribute to the same column . Appended to a, the matrix v contains additional restraint equations that penalize the least - squares fit when particular charges stray from target values encoded in the vector vt appended to u. a more detailed statement of the linear least - squares problem is as follows:4here, the elements of the fitting matrix a(p, i)j pertain to the influence of all instances of the jth adjustable charge on the ith fitting point around the pth molecular conformation . The solution vector u on the right - hand side is filled with values of the electrostatic potential measured at many points rp, i . As was mentioned, there may be multiple instances of the same adjustable charge in each molecule . A general definition of the elements of a is then5where the summation runs over all atoms c bearing the fitted charge described by variable j. the various molecular conformations may be different poses of the same molecule or even a collection of poses of many different molecules . In the latter case (which best describes the original ipolq fit we performed), the matrix a becomes a sparse matrix due to the fact that not all systems contain all charge variables, but it is not often sparse enough to warrant a special storage format . This can require a large amount of memory, but simultaneous solution of all charge variables permits multiple systems to share the same charges . In our ipolq fit, for instance, we chose to follow the cornell charge set convention of giving similar charges to backbone n, h, c, and o atoms involved in the peptide bonds for neutral, positively charged, and negatively charged amino acids, compressing what could have been more than 80 charge variables into just 12, in the hope that this would expedite development of a common set of backbone torsion potentials . The restraint matrix v is appended to the fitting matrix a in order to keep the charges of certain atoms small and also to restrain the overall charges of certain groups of atoms: the fit is heavily penalized by vnc = 1.0 10 kcal / mole if the sum of atomic partial charges of neutral or ionic residues differs from the appropriate net charge (nc), qk for the kth system . In eq 5, p, j is 1 if the pth system contains the jth charge variable . To put all restraint constants vsa, j for specific atomic charges on the same scale, these constants were set proportional to the number of times nj the jth charge variable appeared in a6 in our ipolq fit, we set vsa0 to 1.0 10 kcal / mole . The extended least - squares fit for deriving ipolq charges as a perturbation of vacuum charges solves for twice as many variables based on the same molecular conformations . The extended problem to solve is simply7 in this system of equations, the original fitting matrix is replicated into several blocks of the extended system, and the solution vector contains electrostatic potentials computed from each molecular conformation in vacuum as well as the vacuum potential averaged with the electrostatic potential computed in the condensed phase u. the original restraint equations are still present, but they apply only to the vacuum charges q; additional restraint equations are added to strongly force the sum of all perturbation charges in each system to zero . An additional block matrix loosely restrains perturbation charges individually to zero using stiffness constants vgp scaled by the number of instances of each (perturbation) charge variable nj; numerical values of vgp are discussed below . In eq 7, i is the identity matrix; every perturbation charge is individually restrained . The extended system, which requires slightly more than four times the memory of the original problem and eight times the computation cost, yields q by applying eq 2 . The traditional least - squares approach for fitting torsion parameters is composed as shown in eq 8 . The fitting matrix is composed of the kernels t(p, i)j of the jth torsion term to be fitted for the ith conformation of the pth system, while other columns store energy adjustment constants cp applied to the pth chemical system present in the fitting data . The solution vector u in this equation contains the qm single - point energies of each conformation, less the average single - point energy of all systems with the same chemical composition, less the mm energy arising from other terms in the force field . The torsion terms are fitted so that the sum of their contributions compensates for inaccuracies in the molecular mechanics model to bring the relative energies estimated for different conformations of the same molecule into agreement with the qm pes.8 in principle, it would be feasible to leave the average single - point energy of each chemical system in u, but this would lead to a poorly conditioned matrix, as the energy adjustment constants cadj, here c1...ck, would need to take on very large values . It is also possible to compose u based on system conformational energies relative to the structure with the lowest quantum mechanical energy and to omit the constants cadj for each system . However, no quantum method is perfect, and omission of cadj would amplify the influence of a particular conformation over the fitted parameters . It is well - known to force field developers that the bond and angle terms that approximate high - frequency motions in molecular simulations are not consistent with the qm target models; aside from the breakdown of the harmonic approximation with increasing strain, the equilibrium bond length or angle is dependent on the chemical context . The inconsistency elevates the mm energies for structures optimized by qm methods and is therefore a sort of contaminant when fitting lower - frequency degrees of freedom, in particular torsion fourier series terms . Concern about introducing error by this method has led numerous groups to fit torsion fourier series by positing that a conformation of a molecule is defined strictly by its torsional degrees of freedom and making two slightly different variants of that conformation: relaxing all bond, angle, and nonbonded degrees of freedom by mm and qm approximations, respectively . The objective then becomes to fit torsion parameters such that the coordinates optimized by mm produce an energy most like the single - point energy found for the nuclear coordinates optimized according to qm . This method, hereafter the tandem optimization approach, relaxes much of the inconsistency arising from high - frequency degrees of freedom in the two pess, but it also introduces a new source of error in the nonbonded interactions between the different coordinate sets . While many investigators have accepted the trade - off, we performed an independent analysis, which led us to reject the approach in favor of a direct, one - to - one mapping between coordinates and energies . We analyzed the trade - off between error removed by relaxing high - frequency degrees of freedom and error introduced by changes in nonbonded interactions by computing mm energies for mm and qm optimized variants of 648 conformations in each of 15 amino acid dipeptides (over 9000 pairs of energies). The conformations sampled 1 and 2 at 20 intervals while the backbone was held in an -helical or -sheet conformation; 1, 2,, and dihedrals were held fixed during both optimizations . Mm optimizations and all mm energy evaluations were performed with a new variant of the amber ff99sb force field provided to us courtesy of james maier and professor carlos simmerling s research group . Qm optimizations, also performed for us by james maier, were performed at the mp2 level with the 6 - 311++g basis set . The mm and qm approximations are similar to those we have chosen for development of amber ff14ipq . First, we focused on the difference in the molecular mechanics energy computed for each variant of a given conformation; by construction, the mm optimized variant was always lower in energy, as scored by amber ff99sb . Table 1 gives the differences computed for 628 conformations of each dipeptide . By decomposing the mm energy, it is apparent that the mm and qm models do disagree about the optimal bond lengths and angles, and the energy differences in there terms are greater, sometimes much greater, than the energy differences arising from nonbonded terms . At face value, this result would appear to support the tandem optimization approach . Each difference is given as an average standard deviation, in kcal / mol . Includes lennard jones (lj) and electrostatic (elec) contributions . When fitting torsion fourier series to match a molecular mechanics pes to a qm pes, the objective is correct relative energies; the mm pes is otherwise very far removed from the qm pes, which includes factors such as electron nuclei interactions and nuclear repulsion . The mean energy of the qm pes is therefore subtracted, and an additional constant cadj is included in the fit to arbitrarily adjust the energy of a particular molecule, regardless of conformation, up or down, to bring the two pess into agreement . Because of this, the mean energy differences between the mm and qm optimized variants of each conformation will fall through the fit, absorbed into cadj . If the molecular mechanics and quantum models optimize bonds to different lengths, but the disagreement is consistent across all conformations of the molecule, then the mismatch in these high - frequency terms will have no effect on the fitted torsion parameters . Standard deviations of the difference, the variability of the disagreement between the qm and mm pes, is a much better indicator of possible contamination in a parameter fit . Table 1 shows that bond energy differences have a small deviation but angle energy differences carry a much higher deviation and therefore might contaminate the torsion parameter fit . Comparing the standard deviations in the energy differences arising from the sum of bond and angle terms to those arising from all nonbonded terms suggests that the tandem optimization approach offers a marginal benefit in most cases but is detrimental in cases such as ser and thr . The total nonbonded energy differences appear to be smaller than those of bonds, but the nonbonded energy is a sum of many interactions . The major amber molecular dynamics engines conveniently break nonbonded interactions into 14 contributions between atoms at either end of a dihedral group and all other contributions . These two parts of the lennard jones interactions are uncorrelated, but the sum of electrostatic 14 interactions tends to be strongly anticorrelated with the sum of all other electrostatic contributions, as shown in table 2 . As a consequence, the standard deviations of the differences in the electrostatic 14 nonbonded terms are as large or larger than the deviations in total nonbonded energy differences . Jones short - ranged interactions .) These 14 interactions form the base of the torsional pes around each rotatable bond and, of all nonbonded interactions, are the most strongly connected to the torsion potentials . This suggests that the contaminant introduced by the tandem optimization method, a mismatch in the nonbonded interactions, is actually worse than the contaminant being removed, the mismatch in energies due to high - frequency modes . Like table 1, this table compares mm energies computed for each of two optimized variants of a dipeptide conformation . We conclude that it is at least as sane to accept errors that might be introduced by a mismatch between molecular mechanics bond and angle geometries and those of the quantum target as to accept errors arising from a mismatch in electrostatic or steric interactions . For this reason, we chose to submit the mm optimized coordinates, which reflect the states that the molecular mechanics model will actually explore in simulations, to single - point quantum calculations and demand that our fitted molecular mechanics model reproduce the qm single - point energies calculated for precisely the same set of nuclear positions; details of our procedure for generating the actual fitting data for torsion fourier series can be found in the methods . Due to the one - to - one mapping of coordinates and energies, the fitting data suffers some contamination from angle terms in the mm approximation being inadequate to describe the qm pes . These terms are roughly 1 order of magnitude higher in energy than the torsion terms or nonbonded interactions in the mm model; as such, they could make erroneous contributions to the energy with the risk of torsion parameters becoming fitted against noise . However, the majority of angle strain is orthogonal to the torsional subspace of molecular motions . With adequate sampling of the torsional degrees of freedom in the fitting set, and with a consistent optimization of each set of coordinates relative to one model or the other we designed the amber ff14ipq force field to be similar to previous amber nonpolarizable force fields . Bond and angle parameters were taken from the existing ff99sb force field, along with most lennard jones parameters . Atomic partial charges were refitted according to the updated ipolq fitting procedure described in the theory section, using the same data produced in our previous study, but for future force field development, we automated the ipolq fitting cycle in the amber mdgx program . In our derivation of ipolq charges for amino acid side - chain analogues, we adjusted the lennard jones parameters of polar atoms in order to bring the computed hydration free energies into agreement with experiment . In this respect, the development philosophy of ff14ipq resembles that of the new gromos 54a8 force field . However, the interactions of these atoms with water were essentially the only changes that were of consequence during our hydration free energy calculations, and we found that larger radii fitted for most of the atom types intensified 14 nonbonded repulsion, making torsion parameters much more difficult to fit . To bring the lennard jones changes into ff14ipq, we made them applicable only between the polar atom types and the tip4p - ew oxygen type . The lennard jones lorentz berthelot mixing rule is broken for the interactions of these atom types with water, in the manner that charmm36 makes use of nbfix terms . With the bonded and nonbonded parameters established, the majority of the development in ff14 focused on torsion fourier series terms . A new module was added to the mdgx program to aid users in computing ipolq charges for arbitrary molecules . For a standard resp procedure, researchers must have a set of conformations of their molecule of interest; the conformations serve as inputs to tools such as the red server, which manages the necessary quantum calculations and performs the restrained charge fit . With the new ipolq module, researchers must have a set of conformations of their molecule immersed in the solvent of interest . The mdgx program will read the solvated conformations as restart files along with an appropriate topology and begin dynamics with the solute molecule held in a fixed position . The mdgx program automates the process of collecting the solvent charge density, computing the solvent reaction field potential (srfp), preparing inputs to a quantum program, and launching the calculations to ultimately produce grids of electrostatic potential computed for the molecule in vacuum and in the influence of the srfp . The mdgx ipolq module improves on the original protocol, always applying a shell of point charges around the average solvent charge density taken from the simulation . The charges in the shell are fitted to reproduce the srfp due to infinite electrostatics present in the simulation in the context of a quantum calculation on an isolated system . Furthermore, users can specify up to three concentric charge shells to increase the accuracy of the srfp, evaluate the srfp at additional sites throughout the solute volume, and even specify an interior shell of charges to reproduce the srfp in and around the solute without including point charges nearer than an arbitrary distance from solute atoms . The final feature could be useful for researchers who are concerned about qm basis functions adversely interacting with solvent charges, although we did not notice any effects on the ipolq results for some systems we tested with mp2/cc - pvtz calculations used in the original ipolq protocol (data not shown). In summary, mdgx manages a cycle of the ipolq procedure for a conformation of the molecule of interest; the output of independent runs on multiple solute conformations can be pooled and sent to the mdgx charge fitting module to derive a new charge set, which can then be used to update the solvated system s topology and start another round of calculations with the ipolq module . In this manner, mdgx manages nearly all aspects of the ipolq procedure until the solute charge model converges . The module supports both gaussian and orca quantum chemistry packages, performs dynamics with modest parallelism and efficient cpu execution, and launches quantum chemistry programs for parallel execution in accord with the molecular dynamics run to avoid wasting cpu cycles . Data for the fitting matrix a was derived from the same quantum data as the original ipolq charge set . The same protocol was followed for selecting fitting points from the electrostatic potentials evaluated around each conformation, but, due to memory constraints in the much larger matrix problem, only 3750 points per conformation were selected . (our earlier work showed that anywhere from 3000 to 5000 points per conformation yielded convergent results in the fitted charges .) This method implies two charge sets, one valid in vacuo and the other in solution . While the complete release version of ff14ipq takes the charges valid in solution, q + q in eq 7, we also considered a variant based on q in some studies of systems in vacuo, hereafter named v - ff14 and not distributed for general use . The fitting set for amber ff14ipq was created by molecular simulations and energy optimizations performed with the amber ff99sb force field and new variants under development in the simmerling group . All of these mm models are based on the cornell charge set and have been edited over more than a decade . The most significant feature of the force fields, to us, was the similarity in form and some parameters to the proposed amber ff14ipq force field . While it is impossible to completely sample the available conformational space in the fitting data, we chose the most recent amber force fields to simulate each molecule in the hope that the these models would sample the unrestrained degrees of freedom in a manner that reveals their biases so that those biases could be eliminated in ff14ipq . Nevertheless, critical dihedral angles near the center of each molecule were restrained to various values as we populated the fitting data set; the exact details of the simulations, such as cutoff, time step, and solvent model, are therefore of secondary importance to the choice of protein - like molecules, the array of restraints, and the number of snapshots collected for single - point energy calculations . The most common structures included in our fitting data set were blocked dipeptides, ace - xx - nme, where xx is an amino acid, although we also included ace - ala - ala - ala - nme and ace - gly - gly - gly - nme tetrapeptides to sample backbone and angles and ace - xx - xx - nme tripeptides to further reduce the influence of the blocking groups on these backbone dihedrals . In all, the torsion fitting data set consisted of nearly 28 000 structures whose single - point energies were computed in vacuum by mp2/cc - pvtz calculations . To sample side chain conformations, we obtained a set of some 17 000 dipeptide conformations for neutral asp, asn, deprotonated asp, cys, -protonated his, -protonated his, ionic his, ile, leu, phe, ser, thr, trp, tyr, and val from james maier and the simmerling group . Each amino acid was sampled in 628 conformations, exploring 1 and 2 angles at 20 intervals while holding the backbone in either an -helical or -sheet conformation . These amino acid conformations were among those used in our analysis of the effects of unrelaxed high - frequency degrees of freedom described in the theory section . To obtain a better sampling of backbone conformations and to reduce the risk of side chain dihedrals becoming coupled to particular backbone conformations, we generated another 180 conformations of each dipeptide by sampling and individually at 20 intervals with no restraints on any other degrees of freedom . To decorrelate the unrestrained degrees of freedom, we ran molecular dynamics simulations of each dipeptide while progressively advancing the and restraints . Dynamics were run in baths of tip4p - ew water at 300 k, with 100 ps intervals between each snapshot . Or were incremented with every snapshot, but each angle was completely rotated five times; as a result, separate snapshots at the same or coordinate were spaced by 1.8 ns of dynamics . To sample backbone conformations, reduce the dependence of the fitted parameters on ace and nme blocking groups, and increase side - chain sampling, we included a set of over 7000 tripeptides . We sampled all 20 amino acids with ala, gly, or ser adjacent in either direction in the peptide chain, restraining and individually at 15 intervals with no other restraints and running dynamics on each system at 450 k to generate multiple conformations at each value of or . Because there were so many of these systems, a generalized born solvent was used to generate the conformations . To further sample backbone conformations of two key amino acids, alanine and glycine tetrapeptides were sampled in 1296 conformations each, sampling the central residue s and space at 10 intervals on a two - dimensional grid . Aside from simultaneous restraints on and, no other restraints were used as dynamics were performed for 100 ps between snapshots to decorrelate other degrees of freedom . Snapshots of molecular dynamics trajectories are not suitable for direct incorporation into quantum calculations because of the potential for bond and angle high - frequency energy terms to contaminate the fitting data, as explained in the theory section . However, if the contributions from these terms are relaxed out with respect to either a mm or qm approximation, then the single - point qm energies calculated for each conformation then constitute an acceptable fitting set . We optimized each conformation with restraints on dihedrals involving four heavy atoms (but not hydrogens) in order to preserve the diversity of conformations created in our simulations while relaxing as much angle and bond strain as possible . We chose to optimize conformations with respect to the mm approximation because the calculations were cheap, allowing us to devote more time to evaluating single - point energies . Also, we felt that it was more instructive to evaluate the local minima of an mm model with respect to the mp2/cc - pvtz target, to examine states that an mm model might have a propensity to populate and to verify their energies with qm in the fitting data . All torsion parameters for the amber ff14ipq force field were fitted simultaneously . While the length of each fourier series and also the phase angles were taken from ff99sb, new atom types added by the simmerling group as well as new atom types required by the ipolq charge set for amino acids were included, and the glycine c and proline backbone nitrogen atoms were given their own unique atom types distinct from other amino acids . Including a new atom type implied replicating all bond, angle, and fourier series terms pertaining to the original type, thereby creating additional parameters to fit . All fourier series amplitudes were reoptimized by the standard linear least - squares approach in a single matrix equation to obtain the best overall fit for terms that appear in different contexts across multiple residues, as described in the theory section . Another new module of the mdgx program was created to perform the atom type branching, set up the matrix equation, and perform the linear least - squares fit . While it was an advantage to have the extensive fitting set described in the preceding section, all torsion fourier terms were still restrained loosely toward zero by a penalty of 2.0 10 kcal / mol (multiplied by the number of times each parameter appeared in the fitting matrix) to keep the amplitudes small and avoid overfitting . Torsion amplitudes were optimized to make molecular mechanics energies of the di-, tri-, and tetrapeptide systems computed in vacuo with charges from q agree with mp2/cc - pvtz single - point energies of the peptides also computed in vacuo . As explained in the theory section, we assume these torsion parameters to be transferable to describe the behavior of solvated peptides when paired with q. as a preliminary test of force field performance, we evaluated fidelity to the underlying mp2/cc - pvtz benchmark over the course of molecular mechanics energy minimization . The molecular conformations in the training set were already optimized with respect to a very similar mm model, but in the presence of one or more restraints on dihedral angles . Energy minimization of each conformation of all systems found in the fitting set was performed in vacuo with v - ff14, the variant of ff14ipq substituting q for the implicitly polarized charges found in the release version . New qm energy calculations were then performed on the resulting structures optimized by v - ff14 . These new conformations and energies were added to the training set, and new energy optimizations were performed in an iterative fashion until v - ff14 could score structures created by its own optimization consistently with those in its training set . Building on the fitting data of small peptides, we computed potentials of mean force (pmfs) for pairs of dihedral angles in blocked dipeptides at 298 k with a standard two - dimensional umbrella sampling technique . We collected data in 1296 windows spaced by 10 in and or in 1 and 2, depending on the amino acid and pmf . Each window was seeded from a continuous, incrementally restrained simulation similar to that used to generate conformations for the corresponding tetrapeptides prior to mp2/cc - pvtz calculations . After seeding, each window dihedral angles were initially restrained by an 8 kcal / molrad harmonic penalty function, but if some of the 10 bins were left undersampled, then more windows were added on a 5 grid with 16.0 kcal / molrad dihedral restraints to completely fill out the pmf . We began with simulations of penta - alanine (hereafter, ala(5)), the -helix k19, and -hairpins chignolin (starting structure pdb entry 1uao(32)) and the gb1 hairpin from the c - terminal fragment of protein g. furthermore, we performed microsecond length simulations of the globular proteins gb3 (starting structure pdb entry 1p7e, similar to protein g and containing a motif homologous to the gb1 hairpin) and lysozyme (4lzt) in solution . Each system was equilibrated with protein backbone atoms held under progressively decreasing restraints for up to 7 ns, depending on the size of the system . All simulations were performed with tip4p - ew water (the water model used to develop our solution phase charge set) in sufficient quantity to enclose the peptide and solvated protein systems by at least 10.0 within octahedral boxes after equilibration under constant pressure dynamics . Nonbonded interactions were calculated with a 10.0 cutoff on lennard jones interactions, a homogeneity approximation for long - ranged van der waals interactions, and smooth particle - mesh ewald electrostatics with direct and reciprocal space accuracies near 1 part in 200 000 (this corresponds to a direct sum tolerance of 5.0 10 and a direct space cutoff of 9.0 with the default mesh grid spacing of up to 1). A 2 fs time step was used in all simulations, along with the shake and settle algorithms to constrain the lengths of bonds to hydrogen . Most of the small systems were simulated at 277 k, by a langevin thermostat with collision frequency 3/ps, to replicated nmr conditions . The gb1 hairpin system was simulated at 298 k to investigate -sheet stability at room temperature . The updated ipolq procedure described in the theory section uses the same fitting data as the original method to derived q, but it also derives q, a set of charges appropriate for modeling electrostatics in vacuo . These two charge sets would be independent, and the updated q would be the same as the original, but for the fact that the restraint equations used to temper the fit of the original q instead temper values in q, and the perturbation q that relates q to q by eq 2 is subject to its own set of restraints . In our protocol, the size of q is controlled by setting the parameter vgp in eq 7 . Larger values of q could make it difficult to transfer torsion parameters developed for systems in vacuum to simulations in solution, but too small a q would imply q q, and neither set of charges would describe its intended environment well . We chose to set vgp to 0.005 kcal/(mole - instance), where e is the charge of a proton . This is half the stiffness of restraints holding charges of underdetermined sites to zero in this and earlier ipolq derivations . The fitting matrix is not well conditioned in either case: for main - chain amino acids, we found the (2-norm) condition number of the matrix a in eq 3 to be 1.8 10 and that of the extended matrix in eq 7 to be 2.2 10 . The possibility of a worst - case loss of precision compels us to use double precision arithmetic for what is already a very large matrix, but the results are nonetheless consistent . The earlier ipolq protocol, for instance, judged convergence of the charge set by the point at which successive generations changed the fitted charges by less than the inclusion of one of the fitting conformations . (these changes were on the order of 0.01e .) As before, we are more concerned with the effect that a different set of constraints might affect the outcome . The value of vgp we chose seems to have minor effects on the overall accuracy of q and q. averaged over all systems and conformations, the original q reproduces the target electrostatic potential with 1.82 kcal / mole root - mean - squared error (rmse), whereas q + q reproduces the same potentials with 1.89 kcal / mole rmse . The effects on selected systems are shown in the lower panel of figure 1 . Over the range we tested vgp, the effects on electrostatic potential rmse appear to begin to approach an asymptotic limit (half the difference between the electrostatic potentials computed in vacuo and in the condensed phase). If vgp is tuned even lower, then the rmse of q + q actually improves over the original q due to the fact that the perturbation charges are then much less restrained than the original charges . However, tuning vgp too low leads to undesirable effects such as ala c taking a charge of 0.21 at vgp = 0.0025, as opposed to 0.04 in the original q. accuracies and charge perturbations for all amino acids at vgp = 0.005 are shown in table 3 . Effects of the coupling constant vgp on q and the accuracy of electrostatic potentials for condensed - phase systems . Vgp determines the strength of the harmonic penalty restraining all charges q toward zero . Values of the root - mean - squared error (rmse) describe electrostatic potentials projected by each dipeptide s molecular mechanics charge set relative to the qm target . Vgp = 0 corresponds to partial charges by the original ipolq method before the extension, which allows us to express them as a perturbation to charges appropriate for simulations in vacuo . Errors and charge variations are expressed for a restraint of vgp = 0.005 kcal/(mole - instance) applied to all perturbation charges . Root - mean - squared error (rmse) of fitted mm charges in replicating the qm target electrostatic potential . The target is the average electrostatic potential of the solute s mp2/cc - pvtz wave function in vacuum and in the solvent reaction field potential due to tip4p - ew water . The target is the electrostatic potential of the solute s mp2/cc - pvtz wave function in vacuum . Maximum absolute deviation in partial charges unique to this residue; backbone atoms frequently showed q of 0.060.10, as shown in the supporting information . At vgp = 0.005, the majority of values in q are smaller than 0.05e, as shown by the top panel in figure 1; the larger values tend to be in buried methyl carbons, whose charges are small to begin with, and the atoms of polar head groups . Neither of these changes are likely to make torsion parameters developed with q less transferable because the electrostatic potential energy surface will change only if atoms with large values of q can rotate around nearby atoms possessing large of charges of their own . The perturbation does not, in fact, bring q + q to the polarity of the original q: the carbonyl carbon atom becomes less positively charged by as much as 0.05e, the oxygen becomes less negatively charged by 0.03e, and the polarity of the n however, the perturbation does make the backbone significantly more polar than q alone would describe it . As a consequence, the ipolq protocol continues to model protein backbones with more polarity than the cornell and duan charge sets, maintaining a principal finding of our earlier study . Torsion fourier series terms for all amino acids were fitted by a linear least - squares approach as described in the methods . The preliminary set of 28 000 structures and mp2/cc - pvtz single - point energies is, to our knowledge, the most extensive potential energy surface ever employed for this type of molecular mechanics parameter development . In addition to the atom types present in amber ff99sb and new atom types for c atoms introduced by the simmerling group, we included new types for each of the lennard jones modifications introduced to adjust hydration free energies of amino acid side - chain analogues in the initial ipolq derivation . We also included new types for the glycine c atom and proline backbone nitrogen because of the unique chemical and bonding structures around these atoms . When the electrostatics of each di-, tri-, and tetrapeptide are described by q, the fitted torsion fourier series terms complete a force field and describe the molecular mechanics energy of the system in vacuo . The rmse of these energy estimates relative to the mp2/cc - pvtz single - point energies for di- and tetrapeptides is given in table 4 . Also given in this table are the molecular mechanics (mm) energies obtained by reoptimizing a much smaller set of torsion fourier series terms, the parameters found in amber ff99sb, in conjunction with our newly derived q and the mm energy estimates that would have been obtained if no torsion terms were used . The overall contributions from the torsion terms are often small, only reducing the rmse of mm energy estimates by 1.3 to 1.5 kcal / mol relative to a model that has no such terms . The overall size of the torsion terms contributions may understate their importance, given the number of published force field improvements based on changes in these terms . In all cases, the charge set q fitted to reproduce the electrostatic potentials of blocked dipeptides in vacuo was used to estimate the molecular mechanics energy of each blocked dipeptide in vacuum . All energies are given in kcal / mol . The v - ff14 force field: q has been substituted for the implicitly polarized charge set in the release version . The ff99 force field, with q as derived for v - ff14 (identical to the force field in the first column, but with a smaller torsion parameter space). The new ff14ipq force field has many more torsion fourier series terms than the ff99sb force field: 427 to 67 . Most of the new parameters were added by including the simmerling group s unique c atom types . While the total number of torsion parameters increases nearly 7-fold from ff99sb to ff14ipq, table 4 shows that the number of parameters expressed in any particular system doubles at most . New atom types c8, 3c, and 2c for c have added new parameters for 1 and 2, which distinguish the side - chain rotamer energetics for protonated his, arg, and lys, the amino acids ile, thr, and val, and other amino acids . This partitioning is just one of many possible approaches, but it seems to have achieved a similar effect to an earlier extension of the ff99sb force field, which improved the rotamer propensities of residues ile, leu, asp, and asn by adding new atom types and fitting the newly minted torsion parameters to mp2/(aug)cc - pvtz energy profiles . The ff14ipq parameter space makes notable improvements over the ff99sb parameter space for ile, asp, and asn; it appears that providing only a single atom type for c forces the same set of torsion parameters to average between disparate potential energy surfaces and causes difficulty fitting the data . Tg atom type for glycine c likewise decouples two distinct potential energy surfaces: the dihedral angles between a backbone -hydrogen and the backbone polar hydrogen, or the backbone carbonyl oxygen, are sampled twice (roughly 120 apart) in glycine and only once in all other amino acids . These dihedrals contribute directly to the protein and propensities, and the new tg atom type improves the fit for glycine tetrapeptide by roughly 0.5 kcal / mol rmse and the fit for all other dipeptides by up to 0.1 kcal / mol rmse (data not shown). We also tried adding distinct atom types to the glycine -hydrogens, but this was not as effective as adding the tg atom type . In contrast, distinguishing the proline backbone nitrogen as tn has a negligible effect on the fit for any residues but the proline itself . Given the inherent difficulty in sampling dihedrals related to this atom type, we considered whether to include it at all, but it does decouple the proline profile from other amino acids and serves as a handle for future parameter development . With the expanded parameter set, the torsion potentials contribute more and more to the total molecular mechanics energy; their total contributions in ff14ipq are frequently double their contributions in the refitted ff99 parameter set . However, as shown by the final column of table 4, a model with no torsion potentials is only perhaps 1 to 2 kcal / mol less accurate . The larger torsion potential contributions in ff14ipq are being counterbalanced by larger energy adjustment constants cadj, which never appear in the force field . The number of adjustable terms and the size of cadj are only weakly correlated (pearson coefficient 0.40), perhaps because many copies of the torsion terms can appear in the total mm energy and also because the amplitudes of the terms themselves are so variable . Despite this, the sizes of cadj may be a useful indicator of whether a model is overfitted . The increases in cadj led us to track the sampling of each torsion parameter throughout the fitting set, as shown in the supporting information . With our exceptionally thorough data set, the torsion parameters related to rotatable bonds seem to be well - sampled, on the whole and in each system - dependent context in which they appear . Even with this degree of coverage, however, these fitted torsion parameters are not the final settings distributed as ff14ipq, whether in combination with the partial charge set q or q; they are, rather, a first draft . The following section presents results obtained when the draft model was allowed to guide geometry optimizations and thereby expose the ways in which its parameters could conspire to accumulate errors over the course of a simulation . Energy optimization served as a preliminary test of our fitting program and also of the robustness of our fitting data . Energy minimization of the fitting set s structures using the amber pmemd program and the preliminary v - ff14 force field confirmed that the mm energy computed for each initial structure matched that produced by the mdgx fitting module after solving eq 8 . After energy minimization in vacuo, some initial structures converged to the same final configuration, but a number of local minima were still produced for each di-, tri-, and tetrapeptide system . We computed mp2/cc - pvtz single - point energies for new structures in three tripeptide systems shown in figure 2 and compared them to the mm energy estimates to test whether the new model s 427 parameters were prone to overfitting . Energies of dipeptide and tripeptide conformations before and after energy minimization with the preliminary v - ff14 force field . All molecular mechanical energies are adjusted according to the adjustment constants found while fitting torsion parameters; quantum mechanical energies are normalized to a mean of zero . Hence, the energies of conformations found in the fitting data (black diamonds) lie directly on the trendlines, and the energies of system conformations optimized according to the preliminary v - ff14 (red, open squares) may not track it . The results in figure 2 show that v - ff14 is often able to guide small peptides into configurations that mp2/cc - pvtz calculations agree have lower potential energy . However, after energy minimization, v - ff14 tends to estimate the energy of each configuration as being more favorable than the mp2/cc - pvtz single - point energy . The degree to which v - ff14 departs from the 1:1 trendline with its benchmark may be small, as shown in table 5 . However, geometry optimizations in systems with his, arg, and lys led to mm energies that departed severely from the qm benchmark, despite the agreement maintained in the training set . By adding the structures freely optimized by v - ff14 back into the training set, the second generation v - ff14 nearly eliminated departures from the qm benchmark in arg and his and also showed minor improvements in its ability to optimize the structures of most other residues . V - ff14 is able to guide unrestrained energy minimizations of structures in its own training set and reduce the internal potential energy by up to tens of kcal / mol . (this may be realistic, as most training set structures were restrained in one or more torsional degrees of freedom .) However, when re - evaluated at the mp2/cc - pvtz level, the resulting structures were often not as optimal as molecular mechanics depicted . Negative numbers in the table indicate that v - ff14 strayed from its mp2 benchmark and estimated its optimizations to be too favorable . Gaps in the table indicate that a system was omitted from one generation, due to compute cluster downtime or sufficiently low error in the previous generation . Lysine presented more of a challenge: as shown in figure 3, the second generation v - ff14 apparently contained a new artificial minimum that was subsequently found in all optimizations of the dipeptide . We examined the molecular mechanics energies of lysine conformations in the original training set, the second - generation training set, and the final training set with respect to each generation of torsion parameters . Torsion parameters describing two of the rotatable bonds generate the severe departures from the benchmark seen in the first generation of the force field . First, a wildcard parameter with 3-fold periodicity describing rotation of the lysine side chain amino terminus takes on an amplitude of 4.99 . In the original training set, every conformation sampled the orientation of the terminus in a staggered conformation of the hydrogens at the crest of the cosine wave . The eclipsed conformation, favored by nearly 10 kcal / mol by this spurious parameter, was not sampled in the training set, and the result was six permutations of amino and aliphatic hydrogen interactions creating a nearly 60 kcal / mol fictitious energy release by adopting the unnatural, eclipsed conformation in every structure optimized by the first generation of the force field . A more serious problem occurred in the four - term fourier series describing interactions between the atom type c8, coined by the simmerling group for lysine and arginine c atoms, and the backbone carbonyl carbons . Because of its unique appearance in lysine and arginine, this fourier series strongly influences the backbone angles adopted by the residues . The original training set did not sample a 100 arc in for either residue, however, and the fourier series became fitted to produce an unnaturally low energy in the first generation, approximately 14 kcal / mol more favorable than any conformation of the angle should have allowed . Only some of the lysine structures fell into this trap when optimized by steepest descent energy minimization under the first generation of the force field, and the result was the two striations, which can be seen in figure 3 . In the second - generation training set, sampling in these torsion parameters improved considerably by including the structures trapped in the first generation s spurious minima, as shown in table 6 . Rotation of the lysine amino terminus needed to be sampled completely in order to get a transferable model, but this was accomplished by the third generation . Energies of lysine dipeptide conformations estimated by three generations of the v - ff14 force field . Each generation s fitting set contained all of the initial fitting set data plus conformations created by all previous generations . The torsion fourier series terms describing dihedral interactions of atom types c n cx c8 (backbone carbonyl carbon of any residue n - terminal to lysine or arginine, backbone nitrogen, c, and c of lysine or arginine) and x c8nl x (generic torsion affecting amino terminal hydrogens) evolve rapidly over three generations as sampling of the backbone angles and amino headgroup orientations becomes more complete . The set of torsion parameters optimized in the third generation of training was chosen for pairing with q and distributed as ff14ipq in ambertools14 (see http://ambermd.org). Blocked dipeptides, the simplest systems exhibiting protein - like backbone and side - chain dynamics, were studied extensively to characterize the effects of slight alterations in the charge set, q, which differentiate ff14ipq, our force field for simulations of proteins in water, from v - ff14, a force field used as a parameter fitting apparatus that is otherwise appropriate only for simulations of proteins in vacuum . Furthermore, while we found that torsion parameter refinement could eliminate catastrophic traps in the potential energy surfaces of some amino acids, the approach made modest improvements in the behavior of every other amino acid as well . We computed multiple two - dimensional potentials of mean force (pmfs) for alanine, glycine, and serine dipeptides to assess backbone propensities with either charge set and with alanine dipeptide to examine the impact of torsion refinement . As shown in figure 4, the pmfs of simple, nonpolar amino acids are nearly identical for each charge set, particularly in the populated regions of the ramachandran plot . By inspection, the polarization of charges slightly increases the alanine propensity toward (right - handed) -helices, decreases its propensity toward -sheets, and leaves the model s strongest tendency, toward poly proline ii backbone conformations, unchanged . Larger differences in all dipeptides appear near (,) = (0,0), as the backbone n h and c = o dipoles become favorably aligned to create a more favorable free energy under the ff14ipq model, which strengthens both dipoles considerably . However, the (,) = (0,0) arrangement remains strongly disfavored by steric clashes . The pmfs for alanine and glycine amino acids are also very close to those of the ff99sb force field . The lowest row of panels in figure 4 suggests that solvent effects that make the side - chain charges more polar also facilitate transitions between poly proline ii and left - handed -helicies, but the relative weights of each major conformation are unchanged . While it is not certain what differences in protein folding or dynamics a g of 0.25 kcal / mol in a low - energy region of (,) space could lead to, these plots suggest that q changes protein folding transition states more than equilibria . Potentials of mean force for blocked alanine, glycine, and serine dipeptides in water . The color scale for the preliminary versions of ff14ipq (leftmost panels) and v - ff14 (middle panels) measures g, the energy difference between any point in (,) space and the minimum free energy attainable in each model at 298 k. differences between models are shown on the rightmost panels in a separate color scheme . The importance of fitting torsion parameters in the context of an appropriate charge model is shown in figure 5 . In contrast to the changes introduced by fitting torsion parameters for v - ff14 to a gas - phase qm pes and then transferring them to work in ff14ipq, fitting torsion parameters in the context of ipolq charges, retroactively forcing this charge model to mimic a gas - phase qm pes, makes more substantial changes to the alanine pmf . The difference between this pmf and the ff14ipq pmf is much more frustrated than the difference between the ff14ipq and v - ff14 pmfs . The strong gradients evident in the difference map drive the -helical minimum approximately 15 to the south if the torsion parameters are fitted in the context of q and gas - phase quantum data . When working against an already polarized electrostatic model, the torsion parameters were also less effective at reproducing the gas - phase qm pes (data not shown). Difference plot of the alanine dipeptide pmf with torsion parameters derived for q rather than q. the color scheme is similar to difference plots in figure 4: here, solid red implies that a hypothetical (and incorrect) model fitting gas - phase quantum data in the context of charges appropriate to the solution - phase estimates a point in / space more than 1 kcal / mol more favorably than a properly tuned model; solid blue would imply that the incorrect model disfavors the conformation . Refining the torsion parameters has its own effects on the alanine dipeptide pmf, as shown in figure 6 . Even though the quantum mechanics target is unchanged and the initial set of torsion parameters had no serious problems optimizing the geometries of alanine (tetra)peptide, the third - generation model produces noticeable differences in the pmf . Notably, what was a saddle point between -helical and poly proline ii conformations in the first - generation ff14ipq becomes a local minimum in the third . Transitions between poly proline ii, -helical, and l--helical conformations also tranverse lower barriers according to the third generation of torsion parameters, and in some of the high - energy regions of the pmf, the differences between the first and final generations are as great as those shown in figure 5 . While the magnitudes of the differences are comparable to those in figure 5, however, the locations of all the major backbone conformational minima remain in the same places, and the most significant differences reside in high - energy regions of the pmf, which stands in contrast to the consequences of fitting torsion parameters with the wrong charge set . As before, the influence of these changes on protein folding is not discernible from the pmf alone, but the differences again appear mostly in highly strained configurations, suggesting that the refined torsion parameters depict more frequent transitions between major backbone conformations . Potential of mean force for blocked alanine dipeptide in the release version of ff14ipq and comparison to the initial model . Both plots refer to combinations of condensed - phase charges with torsion parameters fitted to reproduce gas - phase quantum data . Peformance of the release version of ff14ipq, specifically the third generation of torsion parameters paired with the condensed - phase appropriate q, was evaluated on ala(5), -hairpins chignolin and protein g c - terminal fragment, the -helical miniprotein k19, two variants of the miniprotein trp cage, and globular proteins gb3 and lysozyme . Sources of each protein structure, as well as sequences of the miniproteins, are given in table 7 . Each of the miniproteins simulated in this study was selected to evaluate ff14ipq s performance on a particular secondary structure element . Simulations combined third - generation torsion parameters with q. protein sequence; blocking groups are indicated by ace- and -nme . The ala(5) system has recently become a standard diagnostic of a force field s ability to balance three major backbone configurations and reproduce nmr j - coupling results . Best and colleagues made a comprehensive assessment of modern force fields with respect to nmr data from graf and co - workers, calculating mean values for each model s reproduction of 11 order parameters . The karplus relations used to calculate order parameters from the md simulations are sensitive to their own coefficients, but best and colleagues took three different sets of karplus coefficients and posited that a value of 2.25 or less under all three karplus relations indicated a high - quality force field . The results for ff14ipq using the same karplus coefficients and quadruplicate 375 ns simulations of the unblocked peptide with protonated c - terminus are shown in table 8 . The details of the simulated system were intended to match the acidic conditions of the nmr experiments; new charges were derived specifically for the protonated c - terminal alanine and are given in the supporting information . The mean values obtained with the original karplus coefficients used by graf and two sets of dft - based karplus coefficients from case and colleagues were 1.3 0.0, 2.6 0.1, and 1.5 0.0, respectively . (the error bars are standard deviations of the from each of the four 375 ns simulations .) The major drivers of the scores are disagreement with the j(c, c) coupling for the second alanine residue and the j(hn, c) coupling for the third residue . While ff14ipq does not meet best s standard by one of the dft results, it does score very well by the other two, and the j(hn, c) coupling is known to be difficult for the karplus relation itself . By best s definitions of each secondary structure element, ff14ipq models the central residue of ala(5) in poly proline ii, -helical, and -sheet conformations for 56, 18, and 14% of the pooled trajectories, respectively . Dft - based karplus coefficients from case . Ff14ipq also stabilizes larger -sheet structures, as indicated by the plots in figure 7 . The gb1 hairpin from protein g appears to be challenging for other fixed - charge force fields to stabilize (emilio gallicchio, personal communication) but maintains its secondary structure throughout the 250 ns simulations whether simulated with blocking groups or without . While it is reassuring that ff14ipq stabilizes a -sheet structure, the hairpin should be only approximately 30% folded at 298 k. one element that appears to stabilize the system considerably is the ionic interaction between the termini: it remains intact for virtually the entire simulation with a mean and modal distance between the termini of 3.5 0.7 . We investigated the stability of the blocked peptide with quadruplicate 600 ns runs and found no significant unfolding of the antiparallel -sheet arrangement (data not shown; the 250 ns simulation reported in figure 7 is representative). Given that the melting curves for this peptide and other -hairpins are well - established, it may be possible to chip away at the stability of these structures in ff14ipq, and this is probably easier than to try and obtain the right structure from a model that cannot stabilize the peptide at all . Backbone stability of the -hairpin from protein g over 250 ns . Blocked and unblocked forms of the peptide were simulated, and backbone rmsd is plotted for both variants . The dssp chart below the rmsd plots refers to the blocked peptide system and indicates that the antiparallel -sheet is maintained throughout the simulation . The chignolin system could be simulated economically on equally long time scales and appears to fluctuate between two major backbone states . Neither of them is very far from the structure obtained after equilibration; figure 8 shows backbone rmsd relative to the first structure in the nmr ensemble, but if it is calculated relative to the first frame of the simulation after restrained equilibration, then the rmsd would fluctuate between 0.7 and 1.4 . In addition to calculating the standard, overall backbone positional root - mean - squared deviation (rmsd) relative the first nmr model, we computed the way in which individual residues deviate from the nmr ensemble as a whole . This was accomplished by making optimal alignments of the simulated backbones to each of the 18 chignolin nmr models found in the pdb and then computing the rmsd of each particular residue without further position alignment . The minimum residue rmsd to any of the models in the nmr ensemble was recorded for each frame of the trajectory to indicate how far from any of the plausible structures our simulations might have departed . The lower panel of figure 8 shows histograms of these per - residue backbone position rmsds for each residue over the course of the simulation . This confirms that the backbone adopts two major conformations, with larger fluctuations in the zwitterionic, unblocked terminal residues . The arrangement of the termini follows the gb1 hairpin system: the zwitterionic termini begin the simulation in contact with one another . In chignolin, the termini fluctuate in concert most of the time, although for 7% of the simulation the two ionic groups separate by more than 4.5 . The -hairpin fold is maintained throughout the simulation, but the state adopted in the middle of the simulation appears to be slightly outside the nmr structure ensemble . Further simulations may be able to fold the peptide, test the pathway against the known mechanism, and suggest whether changes to the backbone backbone interactions would tighten up the equilibrium structural results . Backbone rmsd in the top plot is calculated relative to the first nmr structure; per - residue backbone rmsd reflects the deviation of each residue s backbone from the closest possible match out of the entire nmr ensemble . Over the course of 1 s, residues 112 exhibit consistent helicity, while the c - terminus transiently explores alternative coil conformations and sometimes even forms a packed double - helical structure, as shown in figures 9 and 10 . The experimental data suggest that the protein s first 18 residues should consistently maintain an -helix, but the ff14ipq results are otherwise in qualitative agreement with experiment and previous simulations of this system . Of particular interest was the degree to which highly solvent - exposed lysine residues in the c - terminal region might make hydrogen bonds with the backbone . Jones radii in both the lysine amino headgroup and the backbone oxygen were altered when interacting with water for agreement with hydration free energies but allowed to maintain their original interaction with one another in the interest of keeping repulsion between nearby atoms from disrupting the internal potential energy surface . Interactions between lysine and the backbone oxygen may not be well tuned: much has been done to reduce the backbone oxygen s affinity for water, but its affinity for amino groups is only balanced, if at all, by the fact that amino groups lennard jones radii were reduced to provide greater affinity for water . We calculated the minimum distance between each lysine headgroup on k19 with any backbone oxygen atom throughout the simulation . As shown in figure 11, lysines 4 and 9 rarely make contact with backbone carbonyl groups, but lysines 14 and 19 are much more likely to form hydrogen bonds to the peptide backbone . Defining an amino group to be hydrogen - bonded to a carbonyl if the nitrogen and oxygen atoms come within 3.2 of each other, lysines 4, 9, 14, and 19 are bonded to the backbone in 4, 7, 17, and 27% of the snapshots, respectively . While the interaction between some groups may be stronger than is realistic, their interactions remain transient . The nitrogen atom types in the lysine headgroup and the charged amino teminus of our -hairpin systems are the same, and the oxygen atom types in carbonyl and carboxylate groups were altered in a similar manner with respect to their precursors in ff99 . The artificial behaviors seen in each system are likely to have a common origin . The time axis applies to both the rmsd plot in the top panel and the dssp plot in the lower panel . The k19 peptide is predominantly -helical, with some instability at the c - terminus . Residues 1619 begin to adopt a metastable 310 helical conformation near the middle of the simulation . All conformations have been aligned relative to the stable backbone of residues 214 . Radial distributions of lysine head groups and backbone oxygen atoms in the k19 system .. as shown in figure 12, both trp cage simulations showed very stable backbone configurations over the 500 ns simulations . The baseline rmsd of approximately 1.2 may indicate different preferences of the ipolq charge set compared to the parameters used in nmr refinement, but larger departures from the backbone configuration depicted in the nmr models were merely transient . Figure 12 shows histograms of per - residue backbone position rmsds as were calculated for chignolin . Most residues display low rmsds under this test, and most importantly the pro - pro - pro sequence near the c - terminus remains stable . Some residues, in particular the asp - gly - gly - pro sequence in simulations of neidigh s trp cage (pdb code 1l2y(45)), show a weakly bimodal distribution, suggesting that the simulations explore an alternate conformation not seen in the nmr ensemble . When simulating a hyperstable trp cage mutant (pdb code 1rij(46)), the asp - gly - gly - pro sequence again departs from the nmr ensemble more significantly than other regions of the protein . The conformations of these residues are good candidates for future analysis and refinement of ff14ipq . Backbone positional root - mean - squared deviations (rmsds) for trp cage miniprotein simulations . Simulations of each of two trp cage proteins are indicated by their respective pdb codes . The top panel shows overall backbone rmsd to the first published nmr model for each system . Lower panels show histograms of per - residue backbone rmsd to the closest possible match out of all published nmr models, darkened to indicate increasing occupancy at a particular deviation . Even though the ipolq charge model is directed toward solvated molecules, ff14ipq is expected to have application to globular proteins . Most residues on proteins of even a few hundred residues have some degree of solvent exposure, and the majority of buried residues will be nonpolar and therefore little different when represented by ipolq as opposed to other charge models . Simulations of both gb3 and lysozyme showed that, overall, ff14ipq stabilizes the crystallographic backbone configurations of both proteins (figure 13). Gb3 and lysozyme (56 and 129 residues, respectively) were simulated with ff14ipq in baths of tip4p - ew water for 1 s . Multiple simulations of gb3 are shown, each performed with a different generation of the ff14ipq torsion parameters . See figure 3 for the accuracy of each generation in predicting energetics of lysine dipeptide . Because we began simulations of gb3 before realizing the importance of iterative torsion parameter refinement, the system provides an indication of the symptoms of underfitted parameters when simulating a complex biomolecule . The first generation of torsion parameters caused one of the lysine - containing loops (residues 916) to take on alternative conformations that drove the overal rmsd significantly higher . Application of the second - generation torsion parameters, which fixed an artificial minimum in the lysine backbone angle, greatly diminished these excursions, as shown in figure 14 . However, another very short loop of the protein, residues 3941 connecting -sheet to -helical structures, makes excursions from the x - ray backbone structure in any generation . It may be significant that this loop contains an aspartate residue: in the torsion fitting results (table 4), aspartate presented one of the most difficult potential energy surfaces for our torsion parameters to capture . While ff14ipq seems to provide a better fit than would be possible with ff99, the greatly expanded parameter space that is almost certainly the basis for this improvement may have allowed some overfitting that we have not yet been able to eliminate with our iterative scheme . Alternatively, aspartate polarization in solution may not be well captured by our approximations . (a third possibility remains, whereby in solution this loop really does take on conformations not seen in the crystal lattice .) We intend that future releases of ff14ipq will build on all of the existing quantum data with further refinements to improve the description of larger protein systems . Per - residue backbone rmsd for gb3, with three generations of the ff14ipq model . Per - residue rmsd was calculated in the same manner as was done for trp cage, but the reference ensemble comprised only the one x - ray structure . Loops that make significant departures from the x - ray structure in solution - phase simulations are emphasized on the x axis . The simulations performed to date with ff14ipq suggest that the force field is ready to guide much longer time scale experiments with proteins and solvated peptides . While some results indicate possible weaknesses in ff14ipq, it is straightforward to develop hypotheses for their origins in the parameter development . It is encouraging to get this level of performance from a new force field and to see the model improve over generations of new fitting data . Before completing our molecular mechanics model based on the ipolq charge set, we needed a set of charges compatible with the gas - phase quantum calculations available to guide our torsion parameter optimization . This, in turn, required us to revisit the way in which we derived charges, decomposing the ipolq charges into foundational values describing the electrostatics of solutes in vacuo and perturbations describing the manner in which atoms polarize in aqueous solution . Because of the iterative nature of the ipolq procedure, we must consider whether this latest change will require additional iterations of molecular simulations with fixed solutes followed by charge fitting . First, although the polarities of certain bonds have changed somewhat, the individual charges of polar side - chain atoms and overall dipoles of the molecules did not change very much . Restraints on q seem to have the strongest effects on buried atoms or groups: increasing vgp reveals indeterminacy in the model fitting but is not likely to affect hydration free energies . Indeed, we chose this approach to exploit the fact that there exist many partial charge models that reproduce a molecule s electrostatic potential with nearly the same accuracy: increasing vgp chooses two models that are most similar in terms of the mean - squared value of q . Second, the objective of the ipolq procedure is a solvent reaction field potential (srfp) that is consistent with the way a solute polarizes; the result is a set of charges that is consistent with the srfp . Restraints on q can make minor changes to the result, but they should not change the objective . It would therefore be appropriate to apply the original ipolq procedure until convergence and then to make other stipulations about how the model s partial charges reproduce the fitting data . One surprising result of this analysis is that the electrostatic potentials of solutes in vacuo are harder to fit than their potentials in the condensed phase when the wave function has been computed in the presence of a polarizing charge density . This occurs in spite of the fact that the charges for polar groups, including the backbone, become significantly larger in the condensed phase to represent stronger electrostatic fields . While an analysis of why this occurs is beyond the scope of this work, it is a worthy subject for future studies and may inform the design of polarizable charge models . In another study, extension of the standard resp protocol, which is similar in spirit to our extended ipolq scheme if one takes the vacuum charges to be dresp s baseline charge model . Dresp employs variable restraint stiffnesses on polar and nonpolar atoms to accommodate different polarizabilities, something that our arbitrary constant vgp does not support . We had thought that, with a complete charge parameter scheme and no obvious reasons to make further changes to bonded parameters or lennard jones terms, producing viable torsion parameters would be the simplest step in developing ff14ipq . Beyond the aforementioned issues with double - counting solvent contributions, there were many hurdles in deriving a transferable torsion parameter set, and this aspect of the model development required as much effort as the charge derivation . We hope that our experience and the programs we designed to meet each challenge will benefit future ab initio force field development . We found inclusion of new atom types and the new fourier series terms they generated to be very helpful in fitting the vacuum - phase potential energy data . The new types introduced to accommodate the ipolq charge set did not introduce many new parameters, as the atom types they replaced were already found in unique bonded arrangements . However, introducing atom types specific to individual residues introduced many new terms that could be fitted to the unique potential energy surface of the residue and relieve the burden that would have been placed on generic terms . In this manner, including a new atom type for c made improvements in the rmse for nearly all dipeptide systems, as the and torsions of the uniquely achiral center of glycine could then be decoupled from those of all other, chiral amino acids . While we had wanted to include separate atom types for the c atoms of positively and negatively charged residues to match the unique charges derived for all backbone atoms in these residues, we were not confident in our ability to generate enough fitting data to sample the and torsion space for these individual residues . In particular, sampling and for consecutive arginine or lysine residues would require mp2/cc - pvtz calculations on large tripeptide systems at very high computational cost . Expansion of the data set in this manner will likely be part of the next version of ff14ipq . Our study also highlights the perils of adding new parameters without adequate sampling . While we believe it is correct to assign unique parameters to unique chemical environments, if the degrees of freedom governed by each parameter might take on values in simulations that are not present in the training set, then catastrophic excursions into artificial minima can occur . Data collected with the first - generation ff14ipq showed unnatural backbone angles adopted by lysine residues in the gb3 protein, which disappeared in subsequent generations trained with complete sampling of this rotatable bond . While such cases might be apparent to an especially careful investigator, we programmed the mdgx fitting routines to produce a great deal of information on the sampling of each parameter, and we did not connect higher backbone fluctuations in the gb3 protein loops to undersampling in particular parameters until significant improvements were seen in the second generation of the force field and a complete breakdown of the molecular mechanics energy was determined for each of the hundreds of lysine structures in each training set . Perhaps ore important is our finding that the descriptions of residues that do not exhibit such catastrophic artificial minima can still be improved by the simple approach of reintroducing the products of molecular simulations back into the model s training data . Whatever artificial minima were removed from the other residues by the third generation of ff14ipq must have been shallow and probably involved combinations of several parameters, implying a very large conformational space to sample in order to obtain transferable parameters on the first attempt . If allowed to drive simulations, then any molecular model should be expected to overpopulate regions of the conformational space in which it scores too favorably . However, because all of the torsion parameters discussed in this work are based on cosines, an unrefined model s propensity to overstate the favorable nature of some conformations may conversely create a tendency to overestimate barrier heights . If this is true, then it may not be a coincidence that the third generation of ff14ipq models transition states in alanine dipeptide with lower barriers than the first generation . In our charge parameter development, we were forced to alter the radii of certain polar atoms, mostly by making them larger, to adjust hydration free energies of amino acid side - chain analogues into agreement with experiment . In this work, we were forced to break the combining rule when integrating these atoms into a complete force field because the larger radii of nearby atoms would clash and make the internal potential energy surfaces of polar amino acids very hard to model . We have covered the possible implications of this decision in the k19 system, where lysine head groups appear to interact too strongly with the backbone . The special combining rules for these polar atoms would also affect the stability of backbone backbone contacts, particularly -sheets and -helicies, and are likely the source of the overstabilization reported for the gb1 -hairpins . Goetz and colleagues reported strong performance for the ipolq charge set when studying aggregation of ionic amino acids, but in that system the correct result was for the amino acids to aggregate . While a prototype version of ff14ipq was able to form these aggregates with the correct radial distributions, their study did not test whether the contacts formed might, in fact, be too strong . More recently, debiec, gronenborn, and chong reported that the ipolq charge set produced the correct association constants for charged amino acid side chains when the full amino acids were considered: positively charged side chains showed affinity for both negatively charged side chains and the backbone carbonyl group, and the competition drove the equilibrium of side chains contacting one another in the right direction . However, they did not rule out the possibility that the side chain to backbone contacts were themselves overstated, as our results on k19 lead us to believe . Debiec and co - workers also did not have available to them the special combining rules we adopted for ff14ipq . When these rules are applied, they found that ff14ipq overstabilized salt bridge formation between charged amino acid side - chain analogues by about 1 kcal / mol (personal communication). We are collaborating with debiec and others to find a pair - specific lennard jones matrix that will not create excessive 1:4 strain but will properly balance the strength of polar interactions . We also intend to add new residues to the palette, including nucleic acids and phospholipids . The folding and aggregation of these residues is dominated by electrostatic interactions, which will demand the proper balance between charged groups interacting with one another and also with water . For proteins jones interactions will still contain special combining rules but that the departures from a standard rule will be smaller than they are in ff14ipq . Some of the lennard jones interactions in ipolq itself can be traced to limitations of the nuclear - centered charge model . For instance, the nuclear - centered charges cannot portray the hydroxyl group with as strong a polarity as it should have without making larger errors elsewhere in the electrostatic field . The hydroxyl oxygen radius was reduced to correct alcohol hydration free energies, but this probably would not have been necessary with a virtual site scheme that could model the correct polarity . For lipids and nucleic acids, any new departures from a standard lennard jones combining rule may hold clues to the inherent limitations of a nonpolarizable, nuclear - centered charge model.
This is especially true about microorganisms, which reside and thrive in almost all environments on earth, including some considered extremely harsh . Common environmental factors that affect the activities of microorganisms include temperature, ph, water availability, nutrient limitation, presence of various chemicals, osmolarity, pressure, and radiation . Consequently, for every microorganism the ability to adapt rapidly to changes in environments is essential for its survival and prosperity . Many single stress - induced regulatory circuits have been identified, which enable cells to cope with specific stresses . However, given that microbial cells live in a dynamic environment where multiple factors fluctuate constantly at the same time, stress responses are generally carried out by a regulatory network composed of a series of individual circuits which are highly connected . Most of our understanding of microbial stress response mechanisms has come from the study of model microorganisms, particularly escherichia coli and bacillus subtilis . Extensive physiological and genetic analyses of the stress response systems in these two bacteria have helped us to elucidate the complexity of the process, function of critical proteins, and regulation . While model organisms will continue to provide insights into the fundamental properties of the stress response systems, efforts should be extended to other microorganisms, especially those that are of scientific, environmental, and economic importance . As one of representatives, the family of shewanellaceae (order alteromonadales, class -proteobacteria) is emerging in recent years . The genus shewanella consists of rod - shaped, gram - negative, aerobic or facultatively anaerobic, polarly flagellated, readily cultivated -proteobacteria [58]. Shewanellae are renowned for its ability to use a diverse range of electron acceptors for anaerobic respiration, including fumarate, nitrate, nitrite, thiosulfate, elemental sulfur, trimethylamine n - oxide (tmao), dimethyl sulfoxide (dmso), fe(iii), mn(iii) and (iv), cr(vi), u(vi), as(v), v(v), and others [10, 11]. As a result of this property, shewanellae have drawn much attention in the fields of bioremediation, biogeochemical circulation of minerals, and bioelectricity [12, 13]. In addition, shewanellae have now served as the model for ecological and evolutionary studies at the whole genome level because of its diverse habitats and the availability of up to 26 genome sequences [14, 15]. However, stress responses have focused nearly exclusively on shewanella oneidensis, the first genome of the shewanellae to be sequenced . The availability of the genome sequence allowed development of high - throughput technologies such as microarrays and proteomics tools, with which an array of assays has been carried out to decipher the ability of s. oneidensis to respond to and survive external stresses . While impacts of most of common environmental factors have been examined, oxidative stress imposed by h2o2 is surprisingly untouched . In this paper, we consider all insights into the stress response mechanisms revealed thus far in s. oneidensis and broaden our discussion to other sequenced species if necessary . Stress response to sudden fluctuation in growth temperature, has become a model system for studying the impact of environmental stresses on biological systems . The hallmark of this adaptive cellular response is the induction of a limited set of proteins, called heat shock proteins (hsps) or cold shock proteins (csps). In general, hsps play important roles in protein folding, degradation, assembly of protein complexes, and transport of proteins across membranes whereas csps function as rna chaperons to regulate ribosomal translation, rate of mrna degradation and termination of transcription [1719]. Using whole - genome dna microarrays, temporal gene expression profiles of s. oneidensis mr-1 in response to temperature variations have been investigated [20, 21]. Both heat and cold shock responses appear to share a couple of common features, including that approximately 15% of the total genes are significantly affected (p <0.05) over a 25-min period, that the global changes in mrnas are rapid and transient, and that a similar set of proteins are induced to manage energy production and protein damage . For instance, most of genes encoding enzymes in the entner - doudoroff pathway and the pentose cycle are highly induced upon a temperature alteration . In the case of heat shock response, two lines of evidence suggest that s. oneidensis copes with the situation with mechanism similar to that employed by e. coli . First, the majority of the genes that showed homology to known hsps in e. coli such as dnak, dnaj, groel, groes, grpe, htpg, and lon / la proteases were highly induced . Second, the identified consensus sequences (cttgaaa-13/15bp - ccccat) of both bacteria for heat shock gene promoters are virtually the same (figure 1), indicating that the induction of most hsps owns to a rapid and transient increase in the intracellular concentration of an alternative factor, encoded by rpoh . After numerous attempts, we failed to remove rpoh from the genome, implicating that is essential in s. oneidensis (unpublished result). Additionally, some hypothetical proteins (i.e., so2017) are under the control of, suggesting that s. oneidensis recruits new proteins to overcome increased temperature (table 1). Unlike e. coli, possesses three (of which two (so1648 and so2787) are cold inducible) whereas e. coli has nine (of which four are cold inducible). Both so1648 and so2787 are important in growth at low temperatures evidenced in the mutational analysis . The s. oneidensis genome carries two more genes encoding csd(cold shock domain)-containing proteins (so0733, 203 aa; so1732, 224 aa) whose c - terminal lacks sequence similarity to any known proteins . Intriguingly, such a structure has been found only in eukaryotes, with the exception of mycobacterium . Neither so0733 nor so1732 is found to be induced upon a decrease in temperature or influences growth at low temperature, indicating that these csd - containing proteins may not be involved in cold stress response . S. piezotolerans wp3 is another shewanella that has been studied in respect of response to low temperatures . Strikingly, none of its csps are cold inducible, suggesting that these proteins may not play an indispensible role in the process . These include increased production of epa (eicosapentaenoic acid) and bcfa (branched - chain fatty acid), induced expression of rna helicase dead which may facilitate transcription, morphological changes in cell membrane, and elevated assembly of lateral flagella (the organism possesses both polar and lateral flagella . ). In addition, a novel filamentous phage (sw1) is found to be significantly induced at low temperature but the significance of this event in the cold adaptation of s. piezotolerans wp3 is unknown . Microorganisms live in a volatile environment where extracellular ph changes frequently . To minimize the acid- or alkaline - induced damage, various adaptive strategies have evolved [28, 29]. Studies on e. coli have revealed that bacterial cells activate outward h pumps such as k / proton antiporters in response to acute cytoplasmic acidification and sodium proton antiporters, which bring in 2 h for each na extruded, to adapt to alkaline ph in the presence of na . To survive upon prolonged acid stress exposure, cells rely on the arginine and glutamate decarboxylase / antiporter systems, which are thought to counteract external acidification through the consumption of intracellular protons and the generation of alkaline amines . Additional acid tolerance responses include regulation of proton permeability by induction of membrane proteins and lipid modification enzyme . In the case of alkaline stress, amino acid metabolic enzymes such as tryptophan deaminase (tnaa) and o - acetylserine sulfhydrylase a (cysk) are induced to reverse alkalinization by metabolizing amino acids to produce acidic products . The response of s. oneidensis to acid and alkaline stresses intersects with other stresses evidenced by elevated expression of rpos, a central regulator of stationary - phase gene expression . It is reasonable to speculate that s. oneidensis cells upon altered ph mimic those at the stationary phase . In respect of response to acidic ph, the most important and effective player of e. coli in mediating acid resistance is the glutamate - dependent (gad) system, which is missing in all sequenced shewanellae . Additionally, none of genes encoding h ex - pumps are found to be induced . Instead, proteins showing substantial induction are rather diverse, including those functioning in cell envelope structure (e.g., csg genes), glycogen biosynthesis (glg operon), fatty acid metabolism (fadba), glutamate synthesis (gltbd), phosphate transport (so1724 and pstb-1), and regulation (e.g., rpos and phou). This observation indicates that the molecular effects of acute acidic ph are profound and multifarious . Upon alkaline ph, as in e. coli na / h antiporter systems (nhaa) are particularly important in maintaining a ph - homeostatic mechanism, thus enabling s. oneidensis to survive and adapt to external alkaline conditions . The most important one is regulation of aquaporins in the outer membrane for water intake by the stationary - phase sigma factor, rpos . It is common that upon the stress condition k uptake is activated and k ions are maintained at high levels . Additionally, cells accumulate neutral, polar, small molecules, such as glycine betaine (gb), proline, trehalose, or ectoine . These compatible solutes serve as osmoprotectants and are synthesized and/or imported into the cell . Many shewanella species are marine microorganisms and therefore are naturally tolerant to relatively high levels of salt . Although some like s. oneidensis, are obtained from freshwater environments, they are able to grow in the presence of up to 0.6 m nacl . Genes encoding k uptake proteins, na efflux system components, and glutamate synthesis are found to be highly induced . Nonetheless, some novel mechanisms are observed . Genes encoding proteins involved in accumulation of compatible osmolytes are either missing in the genome or transcriptionally unaffected when encountered stress . Interestingly, genes encoding tca cycle are particularly active, probably producing much needed atp for ion transport . This may also explain that s. oneidensis shows reduced motility and chemotaxis responding capability under the stress given that the assembly of flagella is extremely energy consuming . Although dna is the major chromophore in general, effects of radiation are in fact pleiotropic [35, 36]. S. oneidensis, one of the most radiation - sensitive organisms known so far, is approximately 1 order of magnitude more susceptible to all wavelengths of solar uv, uv, and ionizing radiation than e. coli [35, 3740]. This is strikingly because the organism similar to e. coli possesses the complete set of genes for photo - reactivation, and nucleotide excision repair, and sos response, primary mechanisms that protect cells from dna damages and radiation - induced oxidative stress [16, 41, 42]. All of these s. oneidensis genes appear to be functional and crucial in the cellular response to radiation, supported by significant upregulation in transcriptional analyses . It is interesting to note that shewanella strains vary significantly in their susceptibility to radiation although compared to e. coli they are still much less resistant . The general trend is that the more radiation exposure is in the habitat where the organisms are isolated the less sensitive they are . For instance, s. oneidensis mr-1 from lake sediment and s. putrefaciens 200 from a crude oil pipeline are more sensitive to radiation than s. algae from the surface of a red alga and s. oneidensis mr-4 from the surface of the black sea . It has been suggested that the hypersensitivity to radiation may be in part due to the activation of prophage [3840]. Radiation has been used as a standard approach to induce prophage in a variety of bacteria [43, 44]. In s. oneidensis, upon radiation the majority of lambdaso, muso1, and muso2 genes are induced and phage particles have been found in the cultures, indicating that a great number of cells are lysed by lytic phages . It has also been implicated that a large number of iron - containing proteins may be partially accountable for the susceptibility . Compared to e. coli which hosts only five to seven cytochrome c proteins, s. oneidensis contains 41 such proteins, some of which are electron transport proteins and essential in respiration [45, 46]. Damages on these proteins by reactive oxygen species (ros) generated in cells upon radiation would likely cause two detrimental results . First, damaged proteins per se may be dysfunctional, directly reducing ability to survive or thrive . Second, damaged proteins release irons into cultures, which further induce ros production . This second wave of ros may be more fatal because it comes at the onset of recovery of seriously damaged cells . Furthermore, the finding that the intracellular mn / fe concentration ratios correlate well with resistance to radiation may explain the hypersensitivity of s. oneidensis, which has the lowest ratio among bacteria tested so far [35, 49]. Many of metal elements are required for microbial growth mostly as cofactors in metabolic pathways . Shewanellae have attracted much attention because of their ability to reduce metal ions including chromium, cobalt, iron, manganese, technetium, uranium, and vanadium, some of which are not needed and highly toxic for most organisms [10, 51, 52]. At the low level these metal ions are taken as electron acceptors by cells and mildly induced some stress - associated genes . However, at the high concentration some of them elicited a distinctively different pattern [5460]. The cellular resistance mechanisms displayed by microorganisms are diverse and include biosorption, diminished intracellular accumulation through either direct obstruction of the ion uptake system or active chromate efflux, precipitation, and reduction of metals to less toxic form . Multiple regulatory circuits are found to work together to cope with the stress response of s. oneidensis to heavy metal compounds . The major ones include those modulating oxidative stress protection, detoxification, protein stress protection, iron acquisition, and dna repair . The molecular response of s. oneidensis to heavy metal shock elicits a distinctively different transcriptional profile compared with metal reduction [5360]. This observation is consistent with that metal reduction and toxicity resistance mechanisms are to be unlinked cellular processes . Responses of s. oneidensis to acute stresses imposed by a variety of heavy metals share a common strategy: survive first and then exert both general and specific stress responses . As a result, s. oneidensis up - regulates its resistance - nodulation - cell division (rnd) protein family genes that facilitate cation export and thus confer heavy metal resistance . Once the first line of defense is initiated, cells employ both general and specific stress responses that are inseparable from each other to recover from the crisis . Alternative sigma factors including rpos, rpoh, rpoe, along with stress - response - related genes are induced, leading to induction of a variety of detoxification, resistance, and transport functions . Such coordinated expression of stress response and detoxification mechanisms in s. oneidensis may offer an advantage to thrive in anoxic metal - reducing conditions in aquatic sediment and submerged soil systems where substantial amounts of heavy metals can be generated . The first is that genes / proteins involved in iron transport are transcriptionally active and implicated to play an important role in the process . Although induction of siderophore biosynthetic and iron transport genes may not be a direct consequence of intracellular iron limitation, several lines of evidence suggest that it is more likely to be indirect by interfering with the fur (ferric uptake regulator) protein, which eventually results in derepression of the iron regulon . Several reports have demonstrated that co, mn, or other divalent cations interact with the fur - binding sites [62, 63]. Moreover, iron - chelating siderophores from other microorganisms have been shown to be able to bind other metals, such as thorium, uranium, vanadium, and plutonium [64, 65]. By increasing siderophore production the other is that sulfur transport and assimilation is promoted . While the underlying mechanism is currently unknown, an explanation is offered . In s. oneidensis, reactive oxygen species (ros) produced in cells by heavy metal stresses can damage iron - containing proteins . As cysteine residues in these proteins are essential to their functions, an extra mount of cysteine is needed for protection . To this end, cells elevate transportation of inorganic sulfate which is reduced and incorporated into bioorganic compounds via assimilatory sulfate reduction, which is the major route of cysteine biosynthesis in most microorganisms . As a potential strategy for the reductive immobilization or detoxification of environmental contaminants, in situ bioremediation has received much interest and attention in last 20 years and are becoming more prevalent today . As its intrinsic feature, the application puts its work force, mostly bacteria, in situ facing the unpredictability of individual microbial processes and constant fluctuations in environments . Thanks to the availability of the s. oneidensis genome sequence, stress responses of the microorganism have been extensively investigated, generating a handful of insights into mechanisms adopted to cope with detrimental conditions . Nonetheless, adaptive mechanisms of shewanella to environmental stresses are still a large playing field for three reasons . First, a number of common stressful agents, especially reactive oxygen species, are not visited . Second, the complex components and regulation in the bacterial stress responses discussed in this paper are mostly based on transcriptional profiling and thus experimental validation is urgently warranted . Last, but definitely not the least, the genus is composed of members which are not only isolated from extremely diverse habitats but also lack unifying phenotypic features, prompting exploration to be extended to other ecological groups of the shewanellae.
Vascular leiomyoma (vl) is an uncommon benign tumor composed of smooth muscle cell and vascular endothelium.1 intracranial involvement is quite rare: in fact, only two reports in the literature described an angioleiomyoma involving the cerebellopontine angle (cpa) and internal auditory canal (iac).23 we present the case of a patient with vl localized within the geniculate ganglion and the initial labyrinthine segment of the facial nerve . To our knowledge, no other vl has been previously described in these sites . In august 2012, a 45-year - old man came to our department complaining of a grade iii facial paralysis (house - brackmann scale) that had arisen 5 months earlier . Otoscopy, as well as pure - tone audiometry, was normal with no tinnitus and no vestibular symptoms . Magnetic resonance imaging (mri), coronal t1-weighted images revealed a hyperintense, 8-to-10-mm enhancing lesion with contrast englobing the geniculate ganglion without any alteration of the other cranial nerves . Preoperative magnetic resonance imaging, coronal t1-weighted images; the arrowhead indicates 8 to 10 mm hyperintense enhancing lesion with contrast, englobing the geniculate ganglion . We planned an intact canal wall tympanomastoidectomy (icwt) with eventual facial nerve grafting . We were able to remove the entire lesion, identifying a cleavage plane with the geniculate ganglion and, thus, preserving the anatomical integrity of the nerve (fig . Postoperatively, the patient had a mild conductive hearing loss (pta 25). Intact canal wall tympanomastoidectomy, tumor arising from the geniculate ganglion and the labyrinthine segment of the facial nerve (arrowheads). Intact canal wall tympanomastoidectomy, complete removal of the lesion preserving the integrity of the nerve; the arrowhead indicates the imprint of the tumor on the geniculate ganglion . Postoperative histology showed cells of medium size with an elongated nucleus and irregular eosinophilic cytoplasm, organized in small bundles . In the context of this lesion, numerous vascular spaces were evident with flat endothelium that were compressed by the lesion . Immunohistochemical studies showed abundant positivity of tumor cells for smooth muscle actin and negativity for s100 . 4). Geniculate ganglion tumor; the arrowheads indicate the presence of cells with elongated nucleus, eosinophilic cytoplasm organized in small bundles; positivity for smooth muscle actin (immunohistochemical, 10). At 3-month follow - up the patient was tumor - free with a recovery of the facial palsy to grade ii . An mri performed at this time confirmed the absence of any expansive lesions involving the geniculate ganglion . Vl is a benign tumor, with vascular and smooth muscle components, first reported by stout et al (11 cases) in 1937.1 vl is rare in the head and neck, wang et al4 in a study published in 2004 analyzed 160 patients with vl affecting these sites; only 21 had a tumor localization in this area . Usually, vl develops in the oral or sinonasal cavities, the parotid, submandibular region, carotid sheath, and retropharynx . Karagama et al2 and pepper et al3 were the first to describe two cases of vl of the iac and the cpa . To our knowledge, our patient is the first case of vl arising within the geniculate ganglion involving the mastoid and tympanic segment of the facial nerve . Morimoto et al5 proposed a classification system that divides vl into three histological subtypes: capillary or solid, cavernous, and venous . Some studies have suggested that the solid subtype is the most frequent in the head and neck region.4 microscopically, vl is generally characterized by a circumscribed nodule with well - organized smooth muscle bundles that extend in fingerlike projections from the smooth muscle in the vessel walls . The cells show eosinophilic cytoplasm and elongated basophilic nuclei with tapered endings (cigar - shaped nuclei).145 vascular spaces, which are lined by a single layer of endothelial cells, are a constant feature in vl and have an intimate association with neoplastic cells . Immunohistochemical studies were positive for muscle - specific actin, desmin, and vimentin and negative for s100 protein, cd34, and cd31.3 histological differential diagnoses include peripheral nerve sheath tumor (neurofibroma and schwannoma) and solitary fibrous tumor.13 computed tomography (ct) or mri have no specific findings for preoperative diagnosis of vl,6 mass enhancement subsequent to contrast administration, owing to the vascular nature of the lesion, might be characteristic . Therefore, only surgical removal and histological examination make it possible to establish a diagnosis with certainty . The best treatment strategy for lesions involving the facial nerve has not yet been fully established . A conservative strategy is often adopted in the presence of normal facial nerve function or minimal preoperative dysfunction.7 in contrast, others proposed not delaying surgery at all, pointing out the increased risk for hearing loss and the better results of facial reconstruction in those patients having no facial palsy.8 when surgical management is selected, the decision regarding the surgical approach to use depends on tumor size and site . When the tumor extends from the geniculate ganglion to the tympanic segment of the nerve, as in our case, the transmastoid extralabyrinthine approach allows access to both these portions and to the beginning of the labyrinthine portion.9 unlike our case, facial nerve integrity may be spared on rare occasions; more frequently, nerve reconstruction is required . Falcioni et al,7 in a retrospective case review analyzing 28 patients undergoing surgery for primary facial nerve tumors, preserved the anatomic integrity of the nerve in 4 cases . All the others required a nerve interruption followed by immediate reconstruction using a sural nerve graft . Also, yamaki et al,10 in three cases of geniculate neurinoma after removal of the lesion, achieved facial nerve reconstruction with intratemporal anastomosis of the great auricular nerve . They preferred to use the greater auricular nerve because it can be readily obtained from the neighboring anatomical field . To conclude our study showed that every effort should be made to preserve the anatomic and functional integrity of the facial nerve.
Since the beginning of mankind nature has been contributing considerably to drug discovery for human beings by providing remedial treatments . One of nature's treasures is the marine biotope, which occupies almost three quarters of the earth's surface (fenical, 1993; whitehead, 1999). Marine natural products play an increasingly important role in biomedical research and drug development, either directly as drugs or as lead structures for bioinspired chemical drug synthesis (molinski et al ., many marine natural products, especially those isolated from macroorganisms, have already undergone clinical trials (newman and cragg, 2004). During the last decades, however, repeated isolation of known metabolites and a reduced hitrate of novel compounds from marine macroorganisms were observed . Hence, natural product chemists are turning their interest to so far less investigated drug sources, such as marine fungi and bacteria, which turned out to be a vast untapped reservoir of metabolic diversity . Thus, research on chemistry of natural products derived from marine microorganisms has increased tremendously in recent years due to the demand for compounds having potential pharmaceutical applications or economical value as cosmetics, drugs, fine chemicals and functional personalcare products (andersen and williams, 2000). In contrast to macroorganisms, microorganisms represent promising natural product sources having the advantage of feasible and sustainable production of large quantities of secondary metabolites with reasonable cost, by largescale cultivation and fermentation of the source organisms (waites et al ., 2001). Furthermore, to adapt and survive in the marine ecosystem, characterized by very special conditions that differ from those found in other habitats, marine microorganisms sometimes accumulate structurally unique bioactive secondary metabolites not found in terrestrial organisms (bhakuni and rawat, 2005). Natural products research still has an enormous unexploited potential, and the significant advantages and disadvantages of natural productderived molecules as drug candidates for development have been highlighted in numerous articles (rogers, 2004). The importance of natural product discovery from microorganisms started only after the largescale production of penicillin (1) during world war ii (numbers 1101 in bold refer to chemical structures 1101 at the end of this article). After the end of the war, pharmaceutical companies refocused on the search for new bioactive biomolecules . In the 1970s for example, cholesterol biosynthesis inhibitors, compactin (2) (larsen et al ., 2007) and mevinolin (3) (araki and konoike, 1997) were discovered . The discovery of compactin and mevinolin enabled the development of the highly successful statin therapeutics (4) (endo, 1992), which even today are considered as block buster in pharmaceutical sales . The discovery of streptomycin, gentamicin, omegamycin (5) and other antibiotics pushed the pharmaceutical industry to implement large research and developing programs based on natural product discovery, with a recent emphasis on marine microbial fermentation based technologies . The water column of the oceans contains approximately 10 bacterial cells per millilitre (hagstrm et al ., 2002). Marine bacteria and fungi are of great interest as novel and rich sources of biologically active products . They live in close association with softbodied marine organisms, which lack obvious structural defence mechanisms, and thus rely on chemical defence by production of bioactive secondary metabolites, either by themselves or by associated microflora, to survive in their extreme habitat (jensen and fenical, 1994). In the last decades, the number of reported secondary metabolites from marine bacteria and fungi has steadily increased (fenical, 1993; kobayashi and ishibashi, 1993; bernan et al ., 1997; faulkner, 1997; 1998; 1999; 2000; 2001; blunt et al ., 2003; 2004; 2005; 2006; 2007; 2008; 2009; hill, 2003), thus reflecting the growing attention by groups from academia and industry . In the year 2007 alone 961 new compounds were described from marine microorganisms reflecting an increase of 24% compared with the number of compounds reported for 2006 (blunt et al ., 2009). In the following, selected examples of new secondary metabolites from bacteria and fungi, published in the period 200709, that live in association with marine macroorganisms, such as sponges, algae and mangrove plants, are presented, with special emphasis on bioactive products and their modes of their action, as well as source organisms and place of origin . Chemical structures are only shown for new compounds, or for previously reported compounds with newly reported biological activities . Since the discovery of penicillin in 1928 (fleming, 1929), intensive studies, mainly on soilderived bacteria and fungi, demonstrated that microorganisms are a rich source of structurally unique bioactive substances (fenical, 1993). The increasing need for new antimicrobial agents able to control emerging diseases or resistant strains of microorganisms inspired a growing number of research groups to explore the oceans for new bioactive compounds . Throughout the years, extensive screening programs were developed worldwide and great efforts have been devoted aiming of the isolation of new metabolites from marine microorganisms . Cultures of the marine bacterial isolate brevibacillus laterosporus png276 obtained from papua new guinea yielded a new lipopeptide named tauramamide (6), together with its methyl (7) and ethyl esters (8). Structures were elucidated by analysis of nmr and ms data and by chemical degradation as well as by total synthesis of tauramamide (6) and tauramamide ethyl ester (8). Compounds 6 and 8 showed potent [minimum inhibitory concentration (mic) values of 0.11 m] and relatively selective activity against the important grampositive human pathogen enterococcus sp . The ethyl ester (8) showed weaker activity against multidrugresistant staphylococcus aureus, but neither compound was appreciably active against the yeast c. albicans . Tauramamide (6) is a new lipopeptide antibiotic that contains two d amino acids and is acylated at the nterminus . Both structural features are hallmarks of nonribosomal peptide synthase biosynthetic origin (desjardine et al ., 2007). A member of the new bacterial genus marinispora (strain nps008920) was isolated from a sediment sample collected in cocos lagoon, guam . Chemical investigation of this strain afforded a series of novel 2alkylidene5alkyl4oxazolidinones, lipoxazolidinone a (9), b (10) and c (11). Compounds 911 showed broad spectrum antimicrobial activities similar to those of the commercial antibiotic linezolid (zyvox) (barbachyn and ford, 2003). Lipoxazolidinones a (9), b (10) and c (11) and the hydrolysis product 12 were screened against a panel of various grampositive and gramnegative bacteria . Compound 9 showed broad spectrum activity, with mic values ranging from 1.56 to 15.57 m against grampositive bacteria and 37.38 m against two strains of haemophilus influenzae . Compounds 10 and 11 showed also broad spectrum antibacterial activity, albeit with lesser overall potency than 9 . In contrast, the hydrolysis product of 12 showed only weak activity against mssa (methicillinsensitive s. aureus), indicating the importance of an intact oxazolidinone ring system . While the oxazolidinone heterocycle is a common structural motif shared by the lipoxazolidinones and linezolid, the compounds are clearly distinguished as 4 and 2oxazolidinones, respectively, and each class is uniquely substituted . Thus, the 4oxazolidinones offer a unique scaffold of compounds with antibiotic therapeutic potential (macherla et al ., 2007). The new marine actinomycete, nps12745, was recently isolated and described from a marine sediment collected off the coast of san diego, california . The analysis of the fulllength 16s rrna sequence indicated that nps12745 is a novel strain of the recently described marine actinomycete genus marinispora . Chemical investigation of this novel strain yielded a series of new chlorinated bisindole pyrroles, lynamicins ae (1317). The bisindole pyrrole derivatives are small molecules, and include chromopyrrolic acid which was previously isolated from chromobacterium violaceum (hoshino et al ., 1993) as well as lycogarubins ac known from the myxomycete lycogala epidendrum (frde et al ., 1994; hashimoto et al ., the antibacterial activity of the compounds against a panel of grampositive and gramnegative bacteria was studied and substances 14 and 15 were found to be active against s. aureus (mssa, mrsa: methicillin resistant), staphylococcus epidermidis and enterococcus faecalis, suggesting potential for treatment of nosocomial infections (mcarthur et al ., 2008). Screening of 100 bacteria, which were isolated from intestinal tract of fish that landed on the baluchistan coast that borders the gulf of karachi, pakistan, afforded an isolate of pseudomonas stutzeri (cmg 1030) that showed pronounced inhibitory activity against several pathogenic bacteria, including mrsa strains . Chemical investigation of the ethyl acetate extract yielded a new antibacterial metabolite named zafrin (4methyl5,6,7,8tetrahydro1 (4h)phenanthrenone) (18). The mic of zafrin (235.85589.62 m) compared favourably with other novel antimicrobials such as 2,4diacetylphloroglucinol (2.384.76 mm) (isnansetyo et al ., 2003). Interestingly, the killing rate of zafrin against bacillus subtilis was faster than for ampicillin, vancomycin or tetracycline . Zafrin does not target the bacterial cell wall and its pattern of lysis resembles that of compounds such as nisin (14.91 m) and triton x100, which disrupt the cell membrane . It was suggested that the mode of action of zafrin is via the disruption of the cytoplasmic membranes, since the molecule is amphiphilic (uzair et al ., 2008). Alaa 2000, which was isolated from the marine red alga laurenica spectabilis collected from rasgharib coast of the red sea, egypt, was found to be active against pathogenic microorganisms with mic values ranging from 0.1 to 10 g ml . Chemical and biological screening of the crude extract of this strain afforded the new bioactive compound ayamycin [1,1dichloro4ethyl5(4nitrophenyl)hexan2one] (19), which is structurally unique since it contains both chlorine and rarely observed nitro groups, beside being structurally related to compounds such as chrysophanol 8methyl ether (20), asphodelin (21) and justicidin b (22). The isolated compounds were tested for their antimicrobial activities against grampositive and gramnegative bacteria as well as against pathogenic fungi such as candida albicans, aspergillus niger and botrytis fabae . The most active compound was the new ayamycin (19) with mic values ranging from 0.31 to 1.57 m (elgendy et al ., 2008). From a marine sediment sample (collected near la jolla, ca) at a depth of 51 m, the actinomycete strain cnq418 was isolated . This strain shared 89.1% 16s rnra gene sequence similarity with its nearest neighbour streptomyces sannurensis . Two prominent products named marinopyrroles a (23) and b (24), were isolated and identified as new secondary metabolites . Xray analysis of marinopyrrole b showed that the natural product exists as an atropoenantiomer with the mconfiguration . The newly isolated substances 23 and 24 displayed noteworthy activity against methicillinresistant s. aureus, with an mic90 of less than 2 m . For each of the compounds, cytotoxicity against a human cancer cell line (hct116: colon carcinoma) was less pronounced (8.8 and 9.0 m for compounds 23 and 24 respectively) (hughes et al ., 2008). The marinederived fungus nigrospora sp . Was isolated from a sea fan that was collected near similan island, thailand . When grown in 500 ml erlenmeyer flasks containing potato dextrose broth for 4 weeks, this strain produced four new metabolites named nigrospoxydons a c (2527) and nigrosporapyrone (28), together with nine known compounds . The crude ethyl acetate extract obtained from the culture broth showed antibacterial activity against standard s. aureus atcc 25923 (sa) and methicillinresistant s. aureus (mrsa), with mic values of 64 and 128 g ml respectively . Only compound 25 and the known compound (+) epoxydon (29) showed activity against both strains, while the remaining compounds were inactive . Compound 25 was more active than 29 against sa (mic 152.38 m), but was less active against mrsa (mic> 304.76 m). Compound 29 gave an mic value of 820.51 m against both strains (trisuwan et al . A marine aspergillus species (family trichocomaceae) was isolated from the surface of the marine brown alga sargassum horneri collected at gadeok island, busan, korea . The fungal broth yielded a new polyoxygenated decalin derivative, dehydroxychlorofusarielin b (30), which was found to exhibit mild antibacterial activity against s. aureus, methicillinresistant s. aureus, and multidrugresistant s. aureus with mic values of 142.36 m for all strains (nguyen et al ., 2007). Fungi of the genus penicillium are known to produce a large variety of compounds with a wide range of biological and pharmacological activities . The fungal broth yielded two new metabolites, penicipyrone (31) and penicilactone (32), together with three known macrolides (+) brefeldin a (33) (+) brefeldin c (34) and 7oxobrefeldin a (35). Compounds 32, 33 and 35 were tested for antimicrobial activity against methicillinresistant s. aureus sk1 and microsporum gypseum shmu4 . Compound 33 showed the strongest antifungal activity against m. gypseum shmu4 with mic value of 228.57 m, whereas the remaining compounds were inactive (mic> 700 m). When tested against methicillinresistant s. aureus sk1 all compounds gave mic values of> 700 m (trisuwan et al ., 2009). The culture broth of a marine fungal strain belonging to the genus exophiala (family herpotrichiellaceae) afforded the new aspyrone derivatives chlorohydroaspyrones a (36) and b (37). The fungal strain was isolated from the surface of the marine sponge halichondria panicea collected on bogil island, jeonnam province, korea . Compounds 36 and 37 displayed moderate to weak antibacterial activity when tested against s. aureus, methicillinresistant s. aureus or multidrugresistant s. aureus, showing mic values of 284.09, 568.18 and 568.18 m, respectively, for compound 36, as well as 284.09, 284.09 and 568.18 m, respectively, for compound 37 two new compounds, named xanalteric acids i (38) and ii (39), were isolated from the fungus alternaria sp ., isolated from fresh healthy leaves of the mangrove plant sonneratia alba (sonneratiaceae), collected in dong zhai gang mangrove garden on hainan island, china . Compounds 38 and 39 exhibited weak antibiotic activity against multidrugresistant s. aureus with mic values of 686.81343.40 m (kjer et al ., 2009). The marinederived fungus, ascochyta sp . Ngb4, was isolated from a floating scrap of festering rope that had been collected at a fishing port in nagasaki prefecture, japan . Chemical investigation of this strain yielded a new spirodioxynaphthalene metabolite, named ascochytatin (40). The relative stereochemistry of the compound was determined by xray crystallographic analysis, and the absolute stereochemistry was identified by the modified mosher's method . A sensitive screening method for antibacterial agents that inhibit essential encoding genes (yycg / yycf) for the bacterial twocomponent regulatory system (tcs), a fundamental system of bacterial response to environmental stress, the difference in sensitivity of b. subtilis 168 and cnm2000 towards 40 suggested that 40 inhibited the function of tcs (yycg / yycf) in b. subtilis . Compound 40 exhibited relatively strong and specific activity against grampositive bacteria and against the yeast c. albicans . Compound 40 exhibited cytotoxicity to both a549 (human lung carcinoma cell line) and jurkat cells (human leukaemia cell line) with ec50 values of 4.8 and 6.3 m respectively (kanoh et al ., 2008). F14 was isolated from seawater collected from the mangrove stand at kei ling ha lo wai, sai kung, hong kong . Chemical investigation of its fermentation broth yielded nine compounds, which were tested for antifouling activity towards macro and microfouling organisms . Antilarval activity was assessed in settlement inhibition assays against the barnacle balanus amphitrite and the bryozoan bugula neritina . Cinnamic acid (41) and bis (2ethylhexyl) phthalate (42) were found to be potential natural antifouling agents inhibiting larval settlement of b. neritina (ec50 = 77.77 14.19 m and lc50> 1351.35 m) and b. amphitrite (ec50 = 23.54 4.49, treatment with high concentration of bis (2ethylhexyl) phthalate (42) (> 256.4 m) was found to cause aggregation of barnacle cyprids due to possible interactions of this compound with the hydrophobic sites on cell membranes . Notably, bis(2ethylhexyl)phthalate is a common plasticizer, which was proven in this study to be produced by the fungus itself and not as a result of labware contamination . The compounds were further tested for antibacterial activity using standard disc diffusion assay against six bacterial species, obtained from natural marine biofilms in hong kong water . The bacterial strains included four larval settlementinducing bacteria: loktanella hongkongensis (ust950701009), micrococcus luteus (ust950701006), rhodovulum sp . (ust010723008) and two marine pathogenic bacteria: pseudoalteromonas piscida (ust010620005) and vibrio harveyi (ust020129010). Cinnamic acid (41), cyclo(phepro) (43) and cyclo(valpro) (44) showed antibacterial activity against l. hongkongensis with mic values of 1351.35, 819.67 and 813.0 m respectively . Cyclo(phepro) (43) showed further antibacterial activity against m. luteus and ruegeria sp . With mics of 409.83 and 819.67 m (strain g 100/2), which was isolated in a screening programme for new marine secondary metabolites from endophytes of several types of algae, together with three known substances . The antifungal activity of chaetocyclinone a (45) was tested against selected phytopathogenic fungi . The compound exhibited an inhibitory activity at a dose of 89.1 m against phytophthora infestans . The other new compounds showed neither antibacterial nor antifungal activity in a standard agar plate diffusion assay . No cytotoxic properties against the tumour cell lines hm02 (stomach), hepg2 (liver) and mcf7 (breast) could be observed up to a concentration of 28.7 m . The results suggested the compound to be derived from the polyketide pathway (lsgen et al ., 2007). A new difuranxanthone, asperxanthone (48), and a new biphenyl, asperbiphenyl (49), were obtained from a fungal strain, identified as aspergillus sp . (mf93), which was isolated from sea water collected in quanzhou gulf, fujian province, china . Compounds 48 and 49 showed moderate inhibitory activity against tobacco mosaic virus, a typical plant virus of the tobamovirus group, with inhibitory rates of 62.9% and 35.5%, at concentrations of 0.62 and 0.48 mm, respectively (wu et al ., 2009). Marine microorganisms are often taxonomically unique, which makes them interesting as potential sources of new drug leads . One of the major areas of research on marine natural products is devoted to the discovery of new anticancer drugs . In 1997, a novel depsipeptide named thiocoraline was isolated from the mycelial extract of the bacterium micromonospora marina associated with a marine soft coral in the indian ocean . Thiocoraline inhibited dna polymerase and is currently in preclinical phase by the pharmaceutical company pharmamar (romero et al . 768), which was isolated from the red alga acanthophora spicifera, was active towards a panel of human tumour cell lines . Chemical investigation of this fungus yielded the novel macrolide apralactone a (50), a 14membered phenyl acetic acid macrolactone, as well as six further curvularin macrolides . The isolated compounds were tested against 36 human tumour cell lines, comprising 14 different solid tumour types . The novel macrolide apralactone a (50) showed moderate concentrationdependent cytotoxicity with a mean ec50 value of 9.87 m . The most active metabolite (+) (10e,15r)10,11dehydrocurvularin (51), displayed concentrationdependent cytotoxicity with a mean ec50 value of 1.25 m, combined with significant in vitro tumour cell selectivity towards nine of the 36 tested tumour cell lines, which indicated 25% of selectivity (using an individual ec50 value <1/2 of the mean ec50 value as threshold for above average sensitivity). These nine above average sensitive cell lines included bxf 1218l (bladder cancer, ec50 = 0.43 m), bxf t24 (bladder cancer, ec50 = 0.5 m), cnxf sf268 (glioblastoma, ec50 = 0.36 m), lxfa 289l (lung adenocarcinoma, ec50 = 0.28 m), maxf 401nl (mammary cancer, ec50 = 0.4 m), mexf 462nl (melanoma, ec50 = 0.38 m), mexf 514l (melanoma, ec50 = 0.5 m), ovxf 899l (ovarian cancer, ec50 = 0.58 m) and prxf pc3 m (prostate cancer, ec50 = 0.4 m) (greve et al ., 2008). Two new natural products (z)6benzylidene3hydroxymethyl1,4dimethyl3methylsulfanylpiperazine2,5dione (52) and (3s,3r)3(3hydroxybutyl)7methoxyphthalide (53), together with three known compounds were isolated from the culture broth of an unidentified marinederived fungus of the order pleosporales (strain crif2). Compound 54, which was previously known only synthetically, was isolated for the first time as a natural product . Compounds 52 and 54 exhibited weak cytotoxic activity when tested against a panel of cancer cell lines (prachyawarakorn et al ., 2008). Investigation of natural products produced by the marinederived fungus spicellum roseum, isolated from the sponge ectyplasia perox collected from the waters around the caribbean island of dominica, afforded two new cyclohexadepsipeptides, named spicellamide a (55) and spicellamide b (56). The absolute configuration of the compounds was deduced after hydrolysis using marfey's method, chiral chromatography, as well as noesy and modelling data . The isolated peptides were tested for their cytotoxic activity using the celltiterblue cell viability assay, against rat neuroblastoma b104 cell line . Compound 56 exhibited an ec50 value of 10.03 m while compound 55 was less active with an ec50 of 49.83 m (kralj et al . (strain cnt016), which was isolated from a marine mud sample collected in palau island, yielded two new secondary metabolites named spiromassaritone (57) and massariphenone (58). The isolated compounds were subjected to cytotoxicity assays against the human colon carcinoma cell line (hct116) and also screened for antimicrobial activity . The crude extract as well as however, none of the compounds isolated showed significant activity against hct116, c. albicans or s. aureus (abdelwahab et al ., 2007). The marinederived fungus aspergillus carbonarius was isolated from a marine sediment collected at weizhou island of china . Chemical investigation of this strain when grown in liquid medium afforded two new secondary metabolites, carbonarones a (59) and b (60). Inhibition of the human leukaemia cell lines k562 was measured by the sulforhodamine b (srb) assay . The cytotoxic effects of 59 and 60 were preliminarily evaluated against k562 (human leukaemia), p388 (murine leukaemia), a549 (human lung carcinoma), bel7402 (human hepatoma) and hl60 (human promyelocytic leukaemia) cell lines . Both compounds 59 and 60 exhibited moderate antiproliferative activity against k562 cell lines with ec50 values of 244.54 and 121.39 m, respectively, while they were inactive against the other cell lines tested (ec50> 436.68 m) (zhang et al ., 2007). The fungus aspergillus ustus was isolated from the marine sponge suberites domuncula, which had been collected from the adriatic sea . From cultures of this strain grown on both biomalt agar and barleyspelt solid media seven new drimane sesquiterpenoids (6167), including the threoisomers 66 and 67, the crude etoac extract of a. ustus displayed cytotoxic activity against the murine lymphoma cell line l5178y at a concentration of 10 g ml . Compounds 64, 65 and the known compound res11492 (68) exhibited ec50 values of 13.11, 1.77 and 4.75 m, respectively, with 65 being the most active congener discovered in this study . All other compounds were inactive at the range of concentrations analysed (0.110 g ml). All cytotoxic compounds featured an olefinic ester side chain comprising two (63 and 64) or three conjugated olefinic double bonds (68) with a terminal carboxylic, aldehyde or methyl substituent (liu et al ., 2009). The fungus petriella sp . Isolated from the sponge s. domuncula that had been cultured in aquaria for 4 weeks yielded three new infectopyrone derivatives (6971) together with the cyclic tetrapeptide wf3161 (72). The cyclic tetrapeptide wf3161 was primarily responsible for this activity; the ec50 value was <0.18 m (proksch et al ., 2008). The marinederived fungus aspergillus aculeatus cri32304a, obtained from the marine sponge xestospongia testudinaria collected from ton sai bay, phi phi islands, krabi province, thailand, afforded a new tyrosinederived metabolite, aspergillusol a (73), in large quantities . Aspergillusol a selectively inhibited glucosidase from the yeast saccharomyces cerevisiae (ec50 = 465 2 m), but it was inactive towards glucosidase from the bacterium bacillus stearothermophilus (ec50 = 1060 20 m). The compound also displayed weak cytotoxic activity towards molt3 (acute lymphoblastic leukaemia), hucca1 (human lung cholangiocarcinoma), and a549 cell lines with ec50 values of 19, 50 and 74 m respectively (ingavat et al . Aspergiolide a (74), a novel anthraquinone derivative with naphtho[1,2,3de]chromene2,7dione skeleton, was isolated from cultures of the marinederived fungus aspergillus glaucus . The fungal strain was obtained from a marine sediment collected from mangrove roots in fujian province, china . The compound selectively inhibited the proliferation of a549, hl60, bel7402 and p388 cancer cell lines (du et al ., 2007). Recently, animal tests with mice indicated that aspergiolide a also inhibited tumour growth in vivo (sun et al ., 2009). A marinederived isolate of the common terrestrial fungus, aspergillus versicolor (mstmf495), was recovered from a beach sand sample collected at low tide from cottesloe, western australia . The fungal strain yielded a new alkaloid, cottoquinazoline a (75), and two new cyclopentapeptides, cotteslosins a (76) and b (77). Cotteslosin a (76) exhibited weak cytotoxic activity against human melanoma (mm418c5, ec50 = 103.93 m), prostate (du145, ec50 = 141.73 m), and breast (t47d, ec50 = 148.03 m) cancer cell lines (fremlin et al ., 2009). Fungi of the genus pestalotiopsis are characterized by their extensive distribution and wide genetic and biological diversity ., obtained from fresh healthy leaf material of rhizophora mucronata (rhizophoraceae) collected in dong zhai gangmangrove garden on hainan island, china, yielded five new cytosporones j n (7882), new coumarins, pestalasins a e (8387), a new alkaloid named pestalotiopsoid a (88) (xu et al . F (8994) (xu et al ., 2009b). Among the isolated compounds only pestalotiopsone f (94) exhibited moderate cytotoxicity, with an ec50 value of 26.89 m, when tested against the murine cancer cell line l5178y (xu et al . Three new oxaspiro[4.4]lactam containing diketopiperazine alkaloids, 6methoxyspirotryprostatin b (95), 18oxotryprostatin a (96) and 14hydroxyterezine d (97), as well as 14norpseurotin a (98) and the 29nordammarane triterpenoid 6,16diacetoxy25hydroxy3,7dioxy29nordammara1,17(20)dien21oic acid (99) were obtained from marinederived aspergillus sydowi pfw113 . The fungal strain had been isolated from a driftwood sample (pfw1) collected from the beach of baishamen, hainan, china . Compounds 9597 displayed weak cytotoxic activity against a549 cells, with ec50 values of 8.29, 1.28 and 7.31 m respectively . Compound 95 also exhibited slight cytotoxicity against hl60 cells (ec50 = 9.71 m). Furthermore, compounds 98 and 99 showed significant antimicrobial activities against escherichia coli, b. subtilis and micrococcus lysoleikticus with mic values of 3.74, 14.97 and 7.49 m for compound 98, and 10.65, 5.33 and 10.65 m for compound 99, respectively (zhang et al ., 2008b). (eg5), collected from waters around egypt (suez canal, egypt). Chemical investigation of the fungal culture resulted in the isolation of the new chromone derivative, chromanone a (100), which was evaluated for its cancer chemopreventive activity, especially for prevention of the initiation stage of carcinogenesis by modulation of carcinogen metabolizing enzymes . Carcinogens are activated by cytochrome p450 1a (cyp1a) and detoxified by glutathione stransferases (gst), quinine reductase (qr), and epoxide hydrolase (meh). Chromanone a (100), in 18.18 m concentration, inhibited cyp1a activity up to 60% of the stimulatedcyp1a in murine hepatoma cells (hepa1c1c7), and significantly induced gst but not total thiols at low concentrations . The compound did not affect qr activity, but it significantly enhanced meh activity in hepa1c1c7 cells (p <0.050.01) in a dosedependant manner . Also, chromanone a (100) showed potent radical scavenging activity against hydroxyl radicals starting from a dose of 45.45 m (p <0.05), which may be responsible for its inhibitory effect on induced dna damage in cells (gamaleldeen et al ., diabetes mellitus is a debilitating and often lifethreatening disorder with increasing incidence throughout the world (who, 1985). Literature surveys show that more than 400 plant species were reported to have antidiabetic activity, and most of the antidiabetic natural products were, so far, isolated from plants (mukherjee, 1981; rai, 1995). In contrast, marine bacteria and fungi are poorly investigated for antidiabetic activity, but may be of great promise in the search for new antidiabetic drugs for the future . Sf5060, isolated from an intertidal sediment collected at gejae island, korea, yielded the known compound aquastatin a (101). The compound exhibited potent inhibitory activity against protein tyrosine phosphatase 1b (ptp1b) with an ec50 value of 0.19 m . Protein tyrosine phosphatases (ptps) constitute a large family of enzymes, which are responsible for modulation of tyrosine phosphorylationdependent cellular events . Studies demonstrated that ptp1b, an intracellular nonreceptor type ptp, negatively regulates insulin and leptinreceptor mediated signalling pathways . Thus, its inhibition may represent an outstanding, novel therapy for type 2 diabetes and obesity . Aquastatin a (101) was found to inhibit ptp1b activity in a competitive and selective manner as demonstrated by kinetic analyses and testing over a small panel of other ptps respectively . In addition, hydrolyzing studies of the compound suggested that the dihydroxypentadecyl benzoic acid moiety present in the molecule was responsible for the inhibitory activity (seo et al ., 2009). Table 1 contains a selection of the most active secondary metabolites isolated from marine bacteria and fungi during the period between 2007 and 2009 . Marinederived fungi and bacteria constitute a promising source of unique metabolites with considerable pharmaceutical and therapeutical potential . Common biological assays usually focus on antimicrobial and cytotoxic activities as demonstrated throughout the literature . Whereas more effective and safe drugs in the field on infectious diseases and cancer are certainly needed, many other pharmacologically active compounds may be overlooked . Thus, it is suggested to broaden biological screens for the discovery of exceptional and rarely investigated biological activities, which may be important for the therapy of chronic diseases . Examples mentioned in this review include the potent ptp1b inhibiting activity of aquastatin a (101) which may be a promising therapeutic agent for treatment of type 2 diabetes and obesity, as well as the carcinogen metabolizing enzymes modulatory activity of chromanone a (100), which could be helpful in preventing the initiation stage of carcinogenesis . Such activities trigger the continued interest in marine microbial natural products and reflect the need for more intensive investigation of their chemical and pharmacological properties . Literature surveys showed that fungi of the genus aspergillus are among the most heavily studied fungal strains as shown in our review (6 investigated aspergillus species out of a total of 20 fungal species). This genus is characterized by its worldwide and frequent distribution, and its species are causative agents of stored products decay, thus they are also important in view of health hazards . Nevertheless, members of this genus are renowned for their ability to produce a huge number of structurally unprecedented bioactive metabolites . Studies showed that the tendency of biosynthetic genes to form adjacent clusters proves helpful in allocating secondary metabolite genes present in fungal genomes (keller et al ., 2005, fox and howlett, 2008), yet such an analysis cannot predict gene expression and, thus, product formation . Recent advancements in molecular biology of fungal secondary metabolism may offer a better insight into how biogenetic gene clusters are regulated and whether their expression is affected by environmental changes and culture conditions (delany et al ., 2000; keller et al ., 2005, fox and howlett, 2008). A better understanding of such regulation and the influencing factors may help to induce and optimize secondary metabolite production under laboratory conditions to yield bioactive natural products with significant pharmaceutical potential.
If one accepts the behavioral model of the factors related to the development of insomnia and its transition from to chronic or insomnia proposed by spielman and colleagues,12 a question logically arises about the optimal means of treating short - term insomnia . Possible strategies might range from watchful waiting, as it is likely that a substantial number of persons with short - term insomnia may have complaints that improve with time . An alternative might be to intervene aggressively in those with short - term insomnia in order to prevent the development of chronic insomnia . If clinicians were able to determine when and how to intervene in the transition from acute to chronic insomnia, a substantial number of cases of chronic insomnia might be prevented from developing . Unfortunately, little information has accrued on the best ways to identify at - risk individuals and intervene in the process . One study suggested that it may be possible to identify vulnerable individuals with a questionnaire that assesses the effect of stress on their sleep.13 this form of secondary prevention might establish the utility of heightened surveillance of persons at risk for developing chronic insomnia and justify more aggressive interventions in primary care . Cognitive behavioral treatment of insomnia has demonstrated efficacy,1416 but its use is limited by the availability of qualified practitioners . Many clinicians have few options for employing behavioral interventions for insomnia and may have difficulty identifying qualified persons to whom interested patients can be referred . Limited reimbursements for behavioral interventions for sleep disorders may also limit the willingness of practitioners to provide these services.17 although behavioral treatments thus are effective for insomnia, the limited number of qualified practitioners limits the availability of this treatment in many situations . For many clinicians, the most readily available treatment for short - term insomnia may be pharmacotherapy . One of the most common interventions for short - term insomnia is thus pharmacotherapy with a hypnotic agent . Currently available therapies include benzodiazepines, non - benzodiazepine gabaergic medications, the melatonin receptor agonist ramelteon, and sedating antidepressants.18 many patients report use of over - the - counter medications most of which contain antihistamines but evidence suggests that while the sedating effect of antihistamines may be helpful for a few days it becomes indistinguishable after several days.19 probably the most commonly used sedating antidepressant is trazodone, but evidence for its efficacy is limited and it may have significant adverse effects.20 benzodiazepines are often used to treat short - term insomnia but clinicians may be concerned about the possible development of tolerance in some patients . Little specific information is available to guide the choice of one agent over another in short - term insomnia, although clinician preferences and concerns about the development of dependence may be important factors in medication selection . Clinicians are increasingly likely to prescribe one of the nonbenzodiazepine gabaergic medications such as zolpidem, eszopiclone, or zaleplon in preference to benzodiazepines . Although effective and safe pharmacologic treatments are available for insomnia, the prescribing information for most medications such as zolpidem include the recommendation that it be taken only when the user will be able to devote 8 hours or more to sleep . In addition, most current pharmacotherapeutic agents do not have demonstrated efficacy in promoting sleep maintenance, although formulations of zolpidem and eszopiclone may be useful in promoting sleep throughout the night.21 as noted, motn insomnia arises in individuals who may have little or no difficulty in initiating sleep but who awaken after several hours and then have difficulty returning to sleep . As noted above, a significant number of persons may complain of nocturnal awakenings followed by protracted wakefulness . A challenge in treating this prevalent form of insomnia arises from the length of action of medications typically used for insomnia, including benzodiazepines such as temazepam or nonbenzodiazepine gabaergic agents such as zolpidem . These medications have half - lives that range from 1.4 to 4.5 hours for zolpidem to more than 20 hours for several of the benzodiazepines . Even those medications with short half lives may have significant residual daytime sedating effects when used for motn insomnia . Sleep medications may have significant residual daytime effects that affect cognitive skills such as reaction time, motor coordination and memory.22 these skills may be particularly relevant to driving an automobile, a skill that may often be used by insomnia patients in the morning after having taken a medication . Several studies have shown that medications such as zolpidem may have significant negative impacts on real - world driving ability after having been taken at night.23,24 it is thus unsurprising that several studies have used driving as a criterion for evaluating the morning after effects of insomnia medication use . In one study, both the long half - life drug flurazepam and the shorter half life drug temazepam had significant effects on driving 12 hours after they had been taken . Betts and birtle25 evaluated the effect of single doses of either flurazepam 15 mg or temazepam 20 mg administered in counter - balanced order on driving performance on a closed course . The driving performance of 12 participants, all of whom were women, was evaluated in the morning between 9.00 and 11.00 am, 12 hours after they had taken the drug . Participants in each condition displayed substantially more driving errors in driving over a course with barriers that required weaving or the equivalent of changing lanes . These results thus show that residual daytime effects on driving performance may be present even with the relatively shorter half - life medication temazepam . Some evidence has accumulated for similar residual effects of the non - benzodiazepine drugs . In a controlled environment, partinen et al26 gave zolpidem 10 mg, temazepam 20 mg, or placebo to women with insomnia at 2.00 am and studied their performance in a driving simulator at 7.30 am, 5.5 hours after taking the medication . A battery of computer - administered neuropsychological measures was completed in the morning and polysomnographies (psgs) were done each night . Participants were 23 women with primary insomnia as defined as having a) complaint of difficulty in initiating or maintaining sleep, or of nonrestorative sleep, for 1 month; b) the sleep disturbance or related daytime fatigue caused clinically significant impairment, and c) the sleep disturbance did not occur in the context of other disorders . All were between 35 and 60 years of age (mean age 49.5 years), had driver s licenses for at least 5 years, and reported driving at least 5000 km each year . The primary endpoint for the study was the mean time to a collision in the driving simulator, a variable that reflected sustained attention and reaction time in the driving simulator . No group differences were found across conditions (placebo, zolpidem, temazepam) in mean time to a collision . In secondary endpoints, neither reaction time nor memory score showed significant group differences, however, a significant effect was found for zolpidem in a measure of how well the person was able to maintain lane control over 100 km of simulated driving . Although these group results suggest little effect of the drugs on driving and memory performances, the authors of this study note that they observed substantial interindividual differences in performance that were possibly clinically significant . The total number of accidents in the driving simulator, for example, was 6 for participants in the drug conditions but only 1 in the baseline and placebo conditions . Several participants thus showed much worse performance in the simulator after drug administration, suggesting that some individuals make be substantially impaired even 5.5 hours after ingestion of either temazepam or zolpidem . Clinicians should therefore be alert to the possibility that some individuals may be highly susceptible to residual effects of sleep medications and exercise appropriate caution in prescribing them for patients who may have to engage in complex mental activities the morning after taking them . Another study27 compared the residual daytime sedating effects of either zaleplon 10 mg or zolpidem 10 mg after motn awakening . Thirty - seven adults with insomnia (mean age 44 years) received either a medication or placebo four hours after bedtime, and residual sedation was evaluated by sleep latency tests at hourly intervals after they awakened 4 hours later and for up to 7 hours after taking the medication . Residual effects of the medications were assessed through repeated sleep latency tests as well with self - reports of concentration ability and the digit symbol substitution test (dsst). The dsst is a paper and pencil test that requires individuals to rapidly substitute numbers in a series of boxes according to a key while their performance is timed . It is often considered a measure of psychomotor speed and attention that is sensitive to the effects of a sedating medication . Persons receiving the 10 mg dose of zolpidem showed significant effects on the sleep latency test, self - report of concentration, and dsst compared to zaleplon 10 mg and placebo for up to 7 hours after having received the drug . These results thus suggest that a standard dose of zolpidem (10 mg) may be associated with significant cognitive effects as long as seven hours after taking it . Currently - available treatments for insomnia thus may pose significant risks for individuals alertness, psychomotor speed, and driving performance the morning after they are taken . These effects may be present in susceptible individuals for as long as 7 hours after taking a drug such as zolpidem, and it is possible that residual effects may be present for even longer periods if longer half life medications are used . It is thus clear that although currently available medications are clearly efficacious in promoting sleep onset in individuals with insomnia and may be useful in improving sleep maintenance, they may have lingering effects the morning after they are taken that have important implications for patients function and safety . The need for new agents that improve sleep hoch et al for example, showed that sleep restriction could improve sleep continuity in older persons, decreasing motn awakenings . Other studies have been consistent in suggesting that the behavioral technique of sleep restriction can improve sleep continuity . In this strategy, the patient is asked to keep a sleep diary for several weeks in order to determine the amount of time spent in bed and the amount of time actually spent in sleep . The patient is then asked to spend slightly less time in bed than he or she is usually asleep . This treatment strategy has been shown to decrease motn awakenings in studies of chronic insomnia14 and might be expected to be effective in other contexts as well . As noted above, however, the limited availability of qualified persons to deliver behavioral sleep medicine services is an important drawback so that pharmacotherapy will continue to be an important option for motn insomnia as well . Given the problems with residual daytime sedation and the possible effects of the time required for oral doses of sleep medications to take effect, the usefulness of several novel formulations of sleep medications have been investigated.28 given the possibility that sublingual administration might be associated with more rapid onset of action of zolpidem, several studies have evaluated this route of administration for this medication . Staner et al29 investigated the utility of a sublingual formulation of zolpidem using it to assist in sleep initiation after participants had napped during the day . In this model of insomnia, having participants nap in the afternoon before later trying to reinitiate sleep served to reduce sleep drive so that the they would have difficulty in falling asleep . In a group of 21 healthy volunteers with a mean age of 26.7 years, 2 doses of sublingual zolpidem (5 and 10 mg) were compared to a standard oral formulation of zolpidem (10 mg). Study results showed that the sublingual 10 mg dose produced shorter latencies to persistent sleep compared to the standard oral dose (12.8 vs 18.4 minutes) with similar effects on sleep onset latency and latency to stage one sleep . No significant group differences were observed for sleep maintenance variables or on subjective measures of sleep quality and all side effects were moderate or mild and resolved . These authors conclude that a sublingual formulation of zolpidem may be clinically useful given the reduction in time to sleep onset observed in this study . Roth and colleagues30 evaluated the pharmacokinetic and pharmacodynamic characteristics of low - dose sublingual zolpidem . In this study, 24 participants (mean age 34.7 years, 13 men and 11 women) who were healthy and without sleep complaints completed a double - blind, placebo - controlled crossover trial of the daytime sedating effects of 3 doses of sublingual zolpidem (1.0, 1.75, and 3.5 mg). The purpose of this study was thus to evaluate the sedating effects of several doses of zolpidem during the day, provide data on the time course of objective and subjective effects of the medication, and allow an investigation of the pharmacokinetic characteristics of these doses and their relation of the effects of the medication to its blood levels . Daytime sedation was measured objectively by the digit symbol substitution test (dsst) and subjectively via self - report ratings of sedation on a visual analog scale (vas). Memory for words and reaction time were also measured to provide additional objective evaluations of the cognitive effects of the medication . Blood samples for pharmacokinetic evaluations were collected prior to the initial dose and for periods up to 12 hours after each dose . Results of this study showed that both the 1.75 mg and 3.5 mg dose, but not the 1.0 mg, of sublingual zolpidem showed significant sedating effects on the dsst at 20 minutes after drug administration . The effect of the 3.5 mg dose was substantially greater than that of the 1.75 mg dose on this measure . Significant effects of the medication compared to placebo lasted up to 90 minutes, although participants dsst scores did not return to levels essentially identical to those in the placebo group until 180 minutes after drug administration . Subjective reports of sedation paralleled results of objective testing, although participants reports of sedation did not return to baseline levels until 300 minutes after drug administration . The 3.5 mg dose showed maximum effects on reaction time at 20 minutes, while the 1.75 mg and 1.0 mg doses showed maximum change at 1 hour . For the two lower doses, the observed change in reaction time was not statistically significantly different from placebo . On the word recall measure, the time of maximal change from placebo was 20 minutes for the 3.5 mg dose but 1 hour for the 1.75 and 1.0 mg doses . Here again, the changes from baseline for the lower two doses were not significantly different from placebo levels . Pharmacokinetic analyses showed that for these participants the maximal drug concentration and areas under the curve were proportional to the dose . The half - life of zolpidem was slightly longer than 2.3 to 2.5 hours, with a time to maximal concentration of 36 to 38 minutes . A pharmacokinetic study with elderly persons was presented by krystal et al (reported in lankford31). In that study, the pharmacokinetics of sublingual zolpidem were studied in healthy elderly volunteers . In the elderly, at the 3.5 mg dose both the maximum concentration and the area under the curve were elevated relative to younger adults . The 1.75 mg dose in the elderly resulted in maximum concentrations and areas under the curve that were somewhat lower than those found in younger adults . The elimination half - life, however, was similar for older as well as younger persons . Lankford suggests that this finding supports the recommendation that the lower dose of sublingual zolpidem may be most appropriate as a starting point for older persons.31 in another study, roth and colleagues32 report a randomized, double - blind, placebo - controlled, three - way crossover trial of sublingual zolpidem tartrate in 82 adults (mean age = 45.9 years; 24 men and 58 women) with a dsm - iv diagnosis of primary insomnia and a history of motn insomnia with an average of 2.2 awakenings per night . Individuals were eligible to participate if, in a preliminary evaluation, they showed psg evidence of prolonged time in returning to sleep after a scheduled motn awakening . Individuals participated in three treatment episodes consisting of two consecutive nights of dosing with placebo, sublingual zolpidem 1.75 mg, or sublingual zolpidem 3.5 mg . Dose of placebo or medication was administered after awakening the participants four hours after initial lights out (without regard to the participant s sleep stage at time of awakening). Treatment episodes were separated by a 5- to 12-day washout period and delivered in a randomization sequence in which all participants received each treatment condition . The primary efficacy variable was the participant s average latency to persistent sleep after the motn awakening . Study results showed significant effects of both doses of sublingual zolpidem on key sleep parameters . Both doses of zolpidem were associated with a significant decrease in psg - measured latency to persistent sleep (from a mean of 28.1 minutes for placebo to 16.9 and 9.7 minutes for the 1.75 mg and 3.5 mg doses of zolpidem, respectively) as well as in patient - reported sleep onset latency . Both doses of zolpidem were associated with significant increases in total sleep time both as measured by psg and patient report . The 3.5 mg dose of zolpidem was associated with significantly better ratings of sleep quality in comparison to placebo, as well as in ratings of level of refreshed sleep and ability to function the next day . This study included evaluation of the objective and subjective effects of zolpidem treatment on morning functioning . Participants completed the dsst and a visual analog scale (vas) asking for self - report of level of sedation on each morning of the treatment periods . Results of this evaluation showed that neither dsst performance nor the va s ratings differed significantly across conditions five hours after administration of zolpidem or placebo . It may be noted, however, that subjects reported some subjective sedation up to 5 hours after medication administration . It is thus not clear precisely how long patients must sleep to avoid any subjective morning sedation . No serious side effects were noted in this study, although 14 of the 82 participants reported at least one adverse event during the study . All adverse events were mild and of short duration, allowing the authors to suggest that sublingual zolpidem may be a safe and effective treatment for motn insomnia . Studies of sublingual zolpidem thus show that this form of the drug is likely to be effective in short - term insomnia . This dosing form s rapid onset of action may confer benefits for patients taking the medication at typical bedtimes, as shown in the study by staner et al.29 standard doses may be associated with clinically significant residual daytime sedation so that standard doses may not be safely used in patients with motn insomnia . At lower doses, however, the residual daytime effects of zolpidem may be reduced and it may thus be useful in motn insomnia . It should be noted, however, that at least one study suggested that the subjective sedating effects of zolpidem persisted as long as five hours after dosing, a factor that may be important depending on time of administration and time of arising . Still, although standard 5 and 10 mg doses may be associated with morning sedation if taken after midnight,27 lower doses of sublingual zolpidem may be less likely to cause morning sedation and may thus be useful for treating short - term insomnia characterized by difficulty in initiating sleep and in motn insomnia.
Jamaica, like many other caribbean countries, has experienced an increase in the prevalence of diabetes over the last 50 years . In cross - sectional studies of selected adult populations, the prevalence of diabetes has increased from 1% in 1960 to between 13%17.9% in the mid-1990s [14]. Data from the 2008 island - wide jamaican health and lifestyle survey estimated the prevalence of diabetes among 1574-year - old jamaicans to be 7.9% after adjusting for sampling methodology . The role of the well - established risk factors for diabetes in jamaica such as obesity, hypertension and family history of the disease has been investigated . In the spanish town study, few studies in the caribbean and other regions undergoing the epidemiological transition have examined the contribution of novel risk factors on diabetes risk using population based data . Sleep is one such risk factor for diabetes that has not been previously explored . In western societies, a decreasing trend in sleep hours in the society coincides with the increasing diabetes prevalence . Sleep duration extended beyond or curtailed before the normal sleep period of 7 - 8 hours has been associated with metabolic and endocrine changes [9, 10]. Curtailed sleep has been shown to increase insulin resistance, a precursor of type 2 diabetes . Shortened sleep duration has also been demonstrated to produce an adverse hormonal profile (increase in cortisol, ghrelin, and a reduction in leptin) and increased inflammatory markers [1012]. Sleep deprivation would therefore be expected to affect both diabetes prevalence and glycemic control . While several cross sectional and cohort studies have investigated the relationship between sleep duration, diabetes prevalence and glucose control, none of these studies have been conducted in black populations undergoing the epidemiological transition [1316]. Additionally, the relationship between sleep and diabetes has not been consistently demonstrated in black populations . We therefore examined the relationship between sleep duration and diabetes prevalence and glycaemic control (assessed by glycosylated haemoglobin) in jamaican adults . We also evaluated whether any relationship between sleep and diabetes prevalence was independent of traditional risk factors for the disease such as age, obesity and family history of diabetes . The jamaican health and lifestyle survey ii was a cross - sectional survey conducted by the epidemiology research unit of the tropical medical research institute (tmri) and the ministry of health, between 2007 and 2008 . The primary sampling units were enumeration districts randomly selected from each of the fourteen parishes in the island using probability proportionate to size . Within each enumeration district, systematic random sampling was used to select households beginning at a random starting site . Within each household, a single respondent between the ages of 1574 was selected using the kish methodology . Once an eligible individual was selected to participate in the study, interviewers were required to revisit households where the participant was not home at the time of first contact three times before the household participant was deemed a refusal . Approximately 30 participants per enumeration district were selected resulting in a final sample of 2848 persons . The survey included an interviewer administered questionnaire where information on demographics, education, socioeconomic status, personal and family medical history and emotional health was collected . Trained interviewers measured height in centimetres using a portable stadiometer and weight in kilograms using an electronic digital scale . The body mass index (bmi) was calculated using the weight (kilograms) divided by height (metres) squared . Participants were seated for 5 minutes before measurement and 3 measurements (each one minute apart) were performed . Participants were visited on a subsequent day in order to obtain fasting finger - stick blood samples for glucose using an accutrend glucometer (roche diagnostics gmbh, germany). Glycosylated haemoglobin was also measured from the capillary blood sample using a nycocard hba1c reader (axis shield) in those with diabetes or an elevated fasting glucose at evaluation . The study was approved by the university of the west indies, faculty of medical sciences, ethics committee and written informed consent was obtained from each participant before enrolment . Sleep quality was assessed by asking the question do you wake up several times during your sleep? Participants were classified as having diabetes if they had a fasting capillary glucose 6.1 mmol / l when tested or if they responded yes to the question, have you been prescribed medication for your diabetes? Participants with a glycosylated haemoglobin of 7% or less were classified as having controlled diabetes, while those with a value above 7% were classified as having uncontrolled diabetes . Participants were classified as hypertensive if their mean systolic blood pressure (sbp) 140 mmhg and or mean diastolic blood pressure (dbp) 90 mmhg or if they answered yes to the question have you been prescribed medication for your high blood pressure? Participants were classified as depressed if they had seriously considered suicide in the last year or if they answered yes to either of the following questions during the past month have you been bothered a lot by little interest or pleasure in doing things? Or during the past month have you been bothered a lot by feeling down, depressed or hopeless? . They were also classified as depressed if they answered positively to at least three of the following questions: during the past month have you been bothered a lot by: (i) feeling sad or lonely, (ii) feeling guilty or worthless, (iii) change in appetite or (iv) change in sleeping patterns? . Respondents were categorised into three groups: high activity, medium activity or low activity / inactive - based on their response to a study questionnaire which included questions on work - related and leisure time physical activity . Participants were categorised nondrinkers if they answered no to the question do you ever drink alcohol? Or if they reported abstaining from alcoholic beverages for more than a year . Participants were classified as smokers if they reported smoking 100 or more cigarettes in their life . Data were analysed using stata version 9.1 (stata corporation, college station, tex). Participants were divided into six groups based on the total hours spent sleeping: <6 hrs 6 - 7 hrs, 7 - 8 hrs, 8 - 9 hrs, 9 - 10 hrs and> 10 hrs . Analysis was done separately for men and women as there was evidence of interaction between sleep duration and sex on preliminary analysis . Trends across sleep duration categories were assessed using a non - parametric test for trend . Those sleeping 8 - 9 hours were used as the referent group based on a smoothed plot of diabetes prevalence and hours of sleep which did not show any significant difference in the odds of diabetes in men or women sleeping between 6 and 9 hours . Adjusted odds ratios were obtained from a base model that included age, obesity and family history of diabetes . Additional confounders were added to this model and only those variables that significantly improved the model were retained . To assess the effect of sleep on the control of diabetes, analysis was limited to those participants who were identified as having diabetes for more than one year . Multivariable logistic regression adjusting for age, sex and insulin use, using good versus poor control as the outcome, was used to assess the effect of sleep on diabetes control . Analysis for this study was restricted to respondents who had complete data for diabetes diagnosis, sleep duration, and potential confounders . Of the 2,848 individuals who participated the final sample had a higher proportion of urban dwellers than the total sample (63% versus 57%; p = 0.02) but there were no other differences in the demographic factors assessed . The population was predominantly black (94.3%), female (69%), with a mean (se) age of 42 16 years, bmi of 27.6 6.6 kg / m, and reported sleep duration of 8.2 1.8 hours . Approximately 12% (men 11%, women 13%) had diabetes, with 29% diagnosed with diabetes at the time of the survey . The median duration of diabetes was 7.0 years, with 18% reporting treatment with insulin . Women were more likely to have depression / depressive symptoms, live in crowded conditions, be classified as physically inactive and report a family history of diabetes . Women had a higher mean bmi and were more likely to report receiving advanced education than men . Sex specific characteristics of the sample by sleep duration category are presented in tables 2 and 3 . In both sexes, there was an inverse relationship between bmi and reported hours of sleep . In men, the proportion of alcohol drinkers was inversely associated with the number of hours of sleep . In women, age, post - secondary education and high physical activity were inversely associated with the number of hours of sleep . In univariate analysis, using participants sleeping between 8 - 9 hours per night as the referent, men who reported sleeping more than 10 hours per night had an increased likelihood of diabetes (or [95% ci] = 3.97 [1.5710.1]). When adjusted for age, bmi and family history of diabetes the odds of diabetes increased for each of the sleep categories and also became significant in those who reported sleeping less than 6 hours with an or [95% ci] = 2.65 [1.096.48] (see table 4). Adjustment for depression, alcohol use, smoking, physical activity and education did not significantly affect these findings . In univariate analysis in women, there was no difference in the likelihood of diabetes in any sleep category compared to the referent group . After adjusting for age, bmi, and family history of diabetes the odds of diabetes were significantly lower in those who reported sleeping less than 6 hours (or [95% ci] = 0.43 [0.230.78]) (see table 4). The results were unchanged after adjusting this model for depression, physical activity, alcohol use, smoking, crowding and educational status . The relationship between reported hours of sleep and the prevalence of diabetes did not change when analysis was restricted to those participants with established diabetes or those diagnosed as having diabetes at the time of the survey, though many of the associations were no longer statistically significant due to the reduced sample size . Obesity did not modify the association between sleep and diabetes prevalence in this study . Reported duration of sleep and diabetes control was examined in 228 participants (26% men) who had diabetes for more than 1 year . Approximately two - thirds (69%) had an hba 1c less than 7.0% . Participants who had controlled diabetes were older (median age (interquartile range) 59 [4868] versus 54 (4463) years) but there were no differences in the reported hours of sleep between the groups . Using those sleeping 8 - 9 hours as the referent, there was no significant association between good diabetes control and sleep duration on univariate analysis . This remained unchanged after adjusting for insulin use and age (data not shown). We found a significant relationship between sleep duration and diabetes prevalence among jamaican adults, although the relationship differed between the sexes . In men, both shorter and longer hours of sleep were associated with an increased likelihood of diabetes, and this relationship remained significant after adjustment for potential confounders . Conversely in women, shorter hours of sleep were associated with a reduced likelihood of diabetes, even after adjustment for the same confounders . Our findings in men are consistent with those of other studies which have examined the relationship between sleep duration and diabetes . In a cross sectional study of 323 men and 417 women aged 2164 years, chaput found an increased likelihood of diabetes for those sleeping 5 - 6 hours and 9 - 10 hours in both sexes . Other studies, such as the lifestyle survey of korean men have also found an association between either short or long sleep duration and diabetes prevalence in men . Contrary to what was expected, women who slept less than 6 hours had a reduced likelihood of diabetes . While some studies have found no association between sleep deprivation and diabetes or sex differences in the association between short sleep duration and diabetes [13, 24], none of these studies have suggested that there might be a protective effect from lack of sleep . While these women had a higher bmi, they were more physically active and better educated . The association between sleep and diabetes was still significant after adjustment for these potential confounders . In a study of korean men, sleep deprivation was associated with diabetes prevalence in the nonobese participants; however, we found no difference in the association according to body mass index in the jamaican women . Sex differences in the relationship between sleep and diabetes prevalence could be attributed to biological differences in sexual hormones that affect sleep or differences in stress and the stress response . Short and long sleep duration has been shown to affect metabolic and endocrine control [7, 9]. Sleep deprivation affects serum leptin (an appetite suppressive hormone) and ghrelin (an appetite stimulatory hormone). There are few data examining the effect of sleep on these hormone levels but hours of sleep have been negatively correlated with ghrelin levels and sleep deprivation has been shown to result in lower levels of leptin . Sleep deprivation may also reduce the amount of rapid eye movement sleep, interfering with brain glucose utilization and resulting in increased serum glucose levels . Sleep disorders are associated with increased sympathetic activity, which leads to an increase in gluconeogenesis and glycogen breakdown, thus inducing insulin resistance . The association between sleep and diabetes prevalence may be a result of confounding from obesity . Lack of sleep as well as longer hours of sleep has also been associated with increased obesity . Obesity, a strong diabetes risk factor, can also result in poor sleep quality and interrupted sleep . In our analysis, the effect of the association was still present even when adjusting for differences in bmi . Sleep apnea, which can result in insulin resistance, can also be considered another risk factor for diabetes and may result in excessive sleepiness and longer reported hours of sleep . . Some complications, such as peripheral neuropathy, which are worse at night, can interfere with the ability to sleep and thereby result in an association between sleep and diabetes control . Additionally, insulin, typically reserved for patients with poor glucose control, may be associated with a higher risk of hypoglycaemia, especially at nights and this may also interfere with sleeping patterns in patients concerned about this risk . The relationship between diabetes and sleep, however, was still present even when restricted only to those diagnosed with diabetes at the time of the survey suggesting that the association may not be a result of having diabetes or its treatment . We did not demonstrate an association between sleep and diabetes control in our analysis even with adjustment for age or insulin use this may be a result of insufficient power to detect this effect . Overall glucose control was good in this sample with over two - thirds having an hba1c of 7% or less . There was no evidence that sex modified the relationship between glucose control and hours of sleep . While there was no statistically significant difference in the prevalence of depression or poor quality sleep with reported hours of sleep in the study, these conditions appeared to be more common in those who reported sleeping the recommended number of hours . Adjusting for depression did not make any difference in the association between diabetes prevalence and hours of sleep in the analysis . It was not found to be a significant predictor of the presence of diabetes, and adjusting for sleep quality did not affect the association between diabetes and hours of sleep in multivariable analysis . The female predominance has been seen in our previous national survey and may reflect the household structure in jamaica; as for participants to be eligible for selection, they needed to spend at least 3 nights at home . In other analyses, we have corrected this by applying survey sampling weights; however, as we were interested in the relationship between sleep and diabetes, we did not make the adjustment for this paper . The cross - sectional design restricts the ability to infer causality as the temporal relationship between sleep duration and diabetes cannot be established; however, previously conducted cohorts have shown that sleep duration is a likely risk factor for diabetes mellitus . Another limitation of the study was the inability to account for sleep apnoea and diabetic complications including chronic pain which were not measured and could potentially produce a relationship between sleep and diabetes prevalence by interfering with sleep or through changes in physical activity . The definition of diabetes was based on capillary glucose and not plasma; however, this technique still provides a useful estimate of diabetes prevalence from a large nationally representative sample and had been considered as an acceptable means for diagnosing diabetes in previous who criteria . Glycosylated haemoglobin was measured using a point of care testing machine (nycocard) which may tend to underestimate the true value but allows subjects to be appropriately classified for clinical purposes and provides a better estimate of overall glucose control than a single finger stick glucose measurement . Sleep duration and quality were self - reported which may be subjective, but this is considered equally effective as actigraphy in measuring sleep patterns of individuals . As the assessment took place at one point in time, we may have failed to identify changes in the pattern of sleep that may be of importance in this association . Our findings support the growing evidence that sleep may play a role in the prevention of diabetes, particularly in black men living in developing countries . We are unaware of any other studies conducted in predominantly black developing countries to examine this issue . Adequate sleep may be an important public health initiative to stem the diabetes epidemic in the region . Further study of the reasons for the sex differences in the association between sleep and diabetes and to determine the physiological and biological mechanisms underlying the associations between sleep duration and diabetes needs to be conducted.
Several clinically significant antibiotics as well as widely used drugs against common diseases have been derived from this unique genus affiliated with the order actinomycetales . Diverse members of this order including streptomyces have been isolated from previously unexplored natural habitats to nourish the current microbial antibiotic searching programs [2, 3]. Jaj06 is a gram - positive, moderately halophilic streptomyces strain, which has previously been isolated from hypersaline coastal solar saltern at tuticorin, india . This strain produces an antimicrobial polyketide compound which has been reported to have potent antimicrobial activity against a set of bacteria and yeast with significant minimal inhibitory concentrations . Production of antibiotic compound in jaj06 has been reported to have seawater dependence . Like many other complex medium components, seawater has nondefined composition which might affect the reproducibility of the production profile of microorganisms . To maintain a reproducible production profile, media components and their optimum levels are critical to the secondary metabolites produced by microorganisms . In the field of antibiotics, much effort was directed toward optimizing production rates and directing the product spectrum . Production of microbial secondary metabolites in a microbial system can be improved by optimization of physical parameters and nutritional constituents of production medium . The optimization experiments are usually performed using nonstatistical one - factor - at - a - time [9, 10] and statistical experimental design approaches [11, 12]. However, the former one is highly laborious and time consuming than the statistical methods . Consequently, statistical experimental design techniques especially plackett - burman design (pbd) and response surface methodology (rsm) are widely used to select the significant variables and obtain the optimal levels, respectively [7, 1118]. The application of these statistical experimental design techniques in media optimization can result in improved product yields, reduced process variability, reduced time, and overall costs, compared with conventional practice of single factor optimization [7, 13]. Plackett - burman design has been applied by several researchers to select influencing factors among the constituents of complex medium [1517]. Optimization of selected, highly influencing factors can be done using response surface methodology with either central composite design (ccd) or box - behnken design experiments . In present work, efforts taken to expel the seawater from the production medium of jaj06, with incorporation of chemically defined salt formulations . Further, in order to enhance the antibiotic production by jaj06, production medium was optimized using a successive optimization strategy, selection of media components that significantly influence the antibiotic production using plackett - burman design and optimization of these media components using rsm with box - behnken design . Jaj06 was previously isolated from a coastal solar saltern soil and extensively studied for their antibacterial secondary metabolite . The strain was maintained over the surface of isp4 agar slants supplemented with 3% of sea salt (w / v). A nutrient medium contained 10 g of starch, 4 g of yeast extract, 2 g of peptone, 1 g of mgso4, 2 g of caco3, 0.04 g of feso47h2o, and 0.1 g of kbr in 1 l of sterile seawater was taken as basal production medium for further modification and statistical optimization . In order to replace the chemically nondefined seawater, two defined salt formulations were screened and compared with seawater to support antibiotic compound in jaj06 . According to previous reports, two salt formulations were prepared with some modifications: salt formulation i contained 12 g of nacl, 0.35 g of kcl, 0.22 g of cacl22h2o, 10.7 mg of h3bo3, 7.3 mg of srcl2, 1.3 mg of naf, and 26 g of cocl26h2o in 1 liter of deionized water; salt formulation ii contained 15 g of kcl, 0.22 g of cacl22h2o, 10.7 mg of h3bo3, 7.5 mg of srcl2, 1.3 mg of naf, and 26 g of cocl26h2o in 1 liter of deionized water . Spore suspension of jaj06 was prepared in distilled water from cultures grown on modified isp-4 medium supplemented with 3% of sea salt (w / v) at 30c for 7 days . The suspension was then added to isp-2 broth in 250 ml erlenmeyer flask at a rate of 10 spores in 50 ml liquid medium . Cultures were incubated on a shaker at 120 rpm at 30c for 3 days and used as seed stocks . For antibiotic production, strain jaj06 was inoculated into production medium and incubated on a shaker at 120 rpm for 8 days at 30c . Cell - free supernatant of fermentation broth was recovered by centrifuging it at 10000 rpm for 10 min . Ethyl acetate was added to the supernatant in 1: 1 proportion and the mixture was agitated for 10 min . The solvent layer containing antibiotic substance was separated from broth and it was further centrifuged at 5000 rpm for 15 min to remove traces of fermentation broth . The antimicrobial crude extract was concentrated tenfold using a rotational vacuum concentrator and used for antibacterial assay . The extracted crude substance was assayed in triplicate for their antimicrobial activity against bacillus subtilis mtcc 441 by agar diffusion plate assay [7, 20]. The crude extract was loaded on 6 mm sterile discs, dried, and placed on nutrient agar plate inoculated with b. subtilis suspension adjusted to a mcfarland standard of 0.5, which is equivalent to 1.5 10 cfu / ml . The plates were incubated at 37c for 24 h and the inhibition zone formed around the disc was measured with transparent ruler in millimeter . . Confirmed that the size of the zones of inhibition can be considered as measure of antibiotic titre . Therefore, the antibiotic activity was expressed as units of activity per millilitre the crude substance of culture, where 1 u was defined as a 1.0 mm annular clearing around the antibiotic disk . Plackett - burman design (pbd) was employed for screening the most significant medium components for growth and antimicrobial compound production by streptomyces sp ., pa, usa) was used for the experimental designs and subsequent analysis of the experimental data . In the experimental design, 7 medium components (independent variables) were screened by representing them at two levels, low () and high (+) in 12 trials . Table 1 shows media components, symbol code, and actual low and high level of the variables . Table 2 shows the detail of the design, each row represents a trial, and each column represents an independent variable . The experiment was carried out in triplicate and the average antimicrobial activity against b. subtitles was taken as the response . The variables with confidence levels above 90% were considered to have significant effect on antimicrobial compound production and thus used for further optimization . Response surface methodology (rsm) was used with box - behnken design to optimize the selected media constituents: starch, kbr, and caco3 for enhanced antibiotic production in jaj06 . The three medium components (independent variables) were studied at three different levels: (), (0), and (+) for low, intermediate, and high concentrations, respectively (table 3). The experiment was carried out in 17 trials (table 4) with five replicates at the centre point and the values of responses were the mean of two replications . For statistical calculations, coding of the factors was done according to the following equation: (1)xi = xix0xi, i=0,1,2,3,,n, where xi was the coded value of an independent factor, xi was the actual value of an independent factor, x0 was the actual value of an independent factor at the center point, and xi was the step change value . For predicting the optimal point, a second - order model was fitted to correlate the relationship between independent variables and response . The behaviour of the system was explained by the following quadratic equation: (2)y=0+ixi+ijxixj+iixi2, where y is the predicted response, 0 is the intercept term, i is the linear coefficient, ij is the quadratic coefficient, ii is the interaction coefficient, and xixj represent the independent variables . The design expert trial package (version 7.0) the statistical significance of the model was verified by applying the analysis of variance (anova). Overall model significance was determined using fisher's f - test and its associated probability p(f). Lack of fit values lower than 0.05 indicates that there might be a contribution to the variables - response relationship that the model does not take into account . The quality of the polynomial model equation was judged statistically by coefficient of determination (r) and adjusted r. the fitted polynomial equation was then expressed in the form of three - dimensional response surface plots, to illustrate the relationship between the responses and the experimental levels of each independent variable . The design expert's numerical optimization method was employed to optimize the level of each variable for maximum response . The combination of different optimized variables, which yielded the maximum response, was experimentally validated by culturing jaj06 in optimized and unoptimized production medium . The cell - free culture broths were collected and extracted with equal volume of ethyl acetate and the top organic layer was dried for further analysis . The dried ethyl acetate extracts were resuspended in methanol and assayed as above for antibiotic activity . The maximal antibiotic activity of jaj06 in three different media prepared separately with nondefined sterile seawater and two chemically defined salt formulations was examined in shake flask cultures . Growth and antibiotic activity exerted by streptomyces sp . However growth rate in both seawater and sodium chloride based salt formulation seemed to be the same, while the growth rate and antibiotic activity are slightly lower in salt formulation ii than those of the other two . The main effect of each variable on antibiotic activity was calculated to determine the medium components which influence the antimicrobial compound production by streptomyces sp . Table 6 represents the effect, standard error, t - value, p value, and confidence level of each component from the result of antibiotic assay given in table 2 . The medium components were screened at the confidence level of 92.5% on the basis of their effects . It was indicated that starch (x1), kbr (x5), and caco3 (x7) had apparent influences in antibiotic production by jaj06 . Confidence levels of these three variables were higher than 90% which indicates their significant contributions than those of other media components . The same was confirmed from the pareto graph (figure 1) in which higher effects were presented in the upper portion and then progress down to the lower effects . It directly shows that the most important factors influencing antimicrobial compound production were starch, kbr, and caco3 . Based on the results of the plackett - burman design, starch, kbr, and caco3 were chosen as most influencing media components and further optimised using response surface methodology . In this approach, the batch runs were conducted in box - behnken design experiments and results to determine the effect of independent factors on the response along with the predicted values are shown in table 4 . The regression equation coefficients were calculated and the data was fitted to a second - order polynomial equation . The response of antibiotic activity (y) can be expressed in terms of the following regression equation: (3)y (antibiotic activity) = 165.34 + 13.54a+7.29b 5.83c0.43ab+2.50ac+0.85bc 14.13a221.63b218.71c2, where y represented the response of antibiotic activity, and a, b, and c were the coded values of starch, kbr, and caco3, respectively . The anova was performed to inspect the second - order response surface model and results are given in table 7 . The anova showed that the model was highly significant, as it was evident from the low p value (<0.0001) of the fisher's f - test . Significance of model was also supported by statistically insignificant lack of fit, as was evident from the lower calculated f value (4.01). The model r of 0.9831 implied that model equation could explain 98.31% of the total variation which suggested a good agreement between predicated values and experimental data . Diagnostic plots were drawn to judge the model adequacy and clarify the signs of any problems in the experimental data . Plot of observed response (antibiotic activity) versus predicted reponse is shown in figure 2(a). In this case, predicted values were in agreement with observed ones in the range of the operating variables . The normal probability plot of the studentized residuals was used to check for normality of residuals (figure 2(b)). A linear pattern observed in this plot suggests that there was no sign of any problem in the experimental data . Figure 2(c) represents a plot of studentized residuals versus predicted values to check for constant error . Residuals displayed randomness in scattering and suggested that the variance of the original observation was constant . The three - dimensional (3d) response surface plots were drawn to illustrate the individual and interactive effects of starch, kbr, and caco3 on antibiotic production by jaj06 (figure 3). Each 3d plot presented the effects of two variables while the rest one was held at middle level . There was insignificant mutual interaction between starch and kbr as well as starch and caco3 (figures 3(a) and 3(b)), respectively . With the increase of the concentration of starch from 2 to 17.5 g / l (coded value, 1.0 to 0.75), the antibiotic activity significantly increased at moderate concentration of kbr (coded value, 0.5); however, decreased antibiotic activity was observed with any further increase of kbr even at high concentration of starch (figure 3(a)). When the concentration of caco3 was just beyond the middle level (coded value, 0.25), the antibiotic activity increased with increasing concentration of starch from 2 to 15 g / l (coded value, 0.1 to 0.5) and further increase of starch to its high level resulted mild decrease in antibiotic production (figure 3(b)). With the increase of the concentration of kbr from 0.02 to 1.1 g / l (coded value, 1 to 0.15), the antibiotic activity significantly increased to certain level at a low concentration of caco3 (coded value, 1) and further slightly increased at a moderate level of caco3 (coded value, 0.25). Lower concentration of caco3 was found to be supportive for antibiotic activity; however the activity was suppressed when the concentration of caco3 was higher in production medium . On the basis of numerical optimization, the quadratic model predicted that the maximum antibiotic activity was 169.07 u / ml, when the optimal values of test factors in the coded units were starch = 0.37, kbr = 0.24, and caco3 = 0.20 (figure 4), which were 7.4 g / l starch, 0.048 g / l kbr, and 0.8 g / l caco3, respectively . The validation of the statistical results using the optimized medium was accomplished by carrying out shake - flask experiments in triplicate . The maximum antibiotic activity unit obtained experimentally was found to be 173.3 u / ml, (table 8) which is in close agreement with the prediction value (169.07 u / ml). Therefore, the developed model was considered to be accurate and reliable for predicting the production of antibiotic by streptomyces sp . The final optimized medium contained 7.4 g of starch, 4 g of yeast extract, 2 g of peptone, 1 g of mgso4, 0.8 g of caco3, 0.04 g of feso47h2o, and 0.048 g of kbr in 1 l of sodium chloride based salt formulation i. their antibiotic producing capability is not a static property and it can be significantly affected by constituents of production medium [24, 25]. . Undefined nature of seawater can affect the reproducibility of the production profile of actinomycetes . In this study, the seawater has been expelled from the production medium with incorporation of chemically defined salt formulations . Among the two defined salt formulation, this type of chemically defined salt formulation has already been reported for consistent production of bioactive compounds from seawater dependent salinispora tropica strain nps21184 . Small manipulations in the culture medium composition can exert significant effect on secondary metabolites biosynthesis in microorganisms [7, 26]. Several researchers working on antibiotics discovery programs have applied pbd and rsm as statistical tools to recognize, manipulate and optimize influencing medium constituents and recorded the increased antibiotic production . For instance, wang et al ., applied rsm approach for medium optimization for antibiotic production by xenorhabdus bovienii and recorded 37.8% increase in antibiotic activity . Recently, chen et al . Reported 2.7-fold increase in antibiotic production by bacillus sp . Zjuibe-076 using rsm approach . In the present study, pbd and rsm were applied for medium optimization for antibiotic production by streptomyces sp . Jaj06 and recorded 26.8% increase in antibiotic activity . The results of pbd revealed that the crucial media components related to the antibiotic production by jaj06 were starch, kbr, and caco3 . Raytapadar and paul reported starch as a significant media component for production of antibiotic from streptomyces aburaviensis 1da-28 . Similarly, caco3 has been identified as a crucial ingredients related to the production of cyclic hexapeptide antibiotic by streptomyces alboflavus . Rsm was found to be very effective in optimizing the selected medium components evident from positive diagnostic plots (figure 2) and r value 0.9831 which was comparable with the earlier reports [7, 27]. Jaj06 as a function of various salt compositions and levels of ingredients in production medium . Pbd and rsm were found to be very effective in selecting and optimizing the medium components in manageable number of experimental trials with overall 26.8% increase in antibiotic activity . Moreover, the optimum culture medium obtained in this experiment will be useful for further study with large scale fermentation in a fermenter for the efficient production of antibiotic from this streptomyces sp.
Body image is a multidimensional issue comprising cognitive, perceptual, and behavioral aspects, which begins early in life . There are different theories for the explanation of body image disturbances . Based on the objectification theory, children, like adults, internalize the social values and schema of attractiveness, success, and power . These schemas are advertised by the western media, such as action figures, and toys include which thin women and masculine men . The mass media influences children's beliefs, attitudes, and behaviors . Cognitive behavioral theories explain how feelings and behaviors can affect body image disturbances . However, it seems that the bio - psychosocial model can provide a better explanation . Based on this model, body mass index (bmi) is considered as an important predictive factor in body satisfaction and related behaviors such as dieting . One of the most important psychological determinants for body image dissatisfaction is self - esteem and negative affect about body . A number of social contributing factors, such as role models and pressure from external sources, can influence body image disturbance . Researches argue that these models can explain the body image concerns and body dissatisfaction (bd) experienced by children as well as adults . Body image concerns and a desire for an ideal body (thinness for women and masculinity for men) began as a predominately western phenomenon . Thinness is perceived as the ideal and thin individuals are considered to be smart, successful, and attractive . Research shows that children aged between 7 and 11 years believe that obese children are lazier and unhappier than thin children . Also, they believe that obese children are less liked by others including their parents . In another study, approximately 40% of children aged between 7 and 10 years believe being thin is important for both genders . The preference for thinness is an important variable in the development of self - esteem and body satisfaction . Different studies focusing on children reveal that weight gain is associated with higher negative body image . Body image dissatisfaction and concerns about having the ideal body is recognized as a health problem . Body image dissatisfaction can be harmful to people of all age groups; however, it can be especially harmful during childhood . Bd can result in eating disorders, low self - steam, depression and anxiety, poor interpersonal skills during childhood, unhealthy body change activities, and susceptibility to suicide . Bd has been reported as a serious risk factor for unsafe weight reduction methods, severe exercise, and use of drugs for weight reduction or muscle enhancement . The side effects of these methods may be more harmful during childhood than other ages as they may impact growth . Several studies argue that the foundation of one's body image and the ideal body is developed during childhood . This body concern, at this age, could be due to increasing awareness about body, weight status, and desire for attractiveness . These factors, in addition to external pressure from family, peer groups, or the media could induce bd . Found that thin boys desire to be larger, but only 44% of thin girls desire to be larger . Other studies also emphasize that more boys than girls harbor a desire to be larger . Therefore, in order to achieve the ideal body, weight management strategies differ between boys and girls . Girls follow strict diets and use weight reduction pills, while boys prefer to use muscle enhancing products such, as steroids and creatine, increase their food intake, and engage in heavy exercise . The majority of research about asian children focuses on immigrant children such as asian - americans . In iran, limited research exists about body image and associated issues, especially during childhood and adolescence . This study has been designed on account of the serious health consequences of bd, and the fact that this phenomenon begins in early childhood . This study evaluates antifat attitudes and body weight concerns and their relationship with gender, age, academic grades, parental education, and bmi in elementary (preadolescent) school children . This cross sectional study was undertaken in kerman, one of the largest cities in iran . Five - hundred elementary school students, including 242 girls and 248 boys, participated in this study . Assessment of body image (shape) was done using children's collins body figure rating scale (bfrs, 1999). Children were asked to select one of the pictures that they think more suitable for these statements: (1) which one is like you? (current body image) (2) which one do you look like? (desired or ideal body image). The subjects were classified into three groups based on absolute number: no bd (bd = 0), mild dissatisfaction (bd = 1), and moderate (notable) dissatisfaction (bd> 1). . Cut off 1 was selected to ensure adequate sample size in mild and moderate bd groups . In this study bd bmi was divided to four categories underweight (<5 percentiles), normal weight (5 and 85 percentiles), overweight (85 and 95 percentiles) and obese (> 95 percentiles) for girls and boys . One national study had showed that these cutoff points are appropriate to be used for iranian children and adolescents . Two additional questions about desire for friends were asked (using children's bfrs): (a) which one do you think has more friends? (b) which one do you like to be a friend with? Other demographic variables were included gender, age, educational grade, and parental education . Statistical analysis was done using spss 16 (statistical package for social sciences), and significance considered 0.05 level . For descriptive analysis, we used central tendency and dispersion (mean standards deviation). Chi - square tests, one - way analysis of variance, t - test, pearson and spearman correlations ad nonparametric tests were used for additional analysis . Assessment of body image (shape) was done using children's collins body figure rating scale (bfrs, 1999). Children were asked to select one of the pictures that they think more suitable for these statements: (1) which one is like you? (current body image) (2) which one do you look like? (desired or ideal body image). The subjects were classified into three groups based on absolute number: no bd (bd = 0), mild dissatisfaction (bd = 1), and moderate (notable) dissatisfaction (bd> 1). . Cut off 1 was selected to ensure adequate sample size in mild and moderate bd groups . In this study bd bmi was divided to four categories underweight (<5 percentiles), normal weight (5 and 85 percentiles), overweight (85 and 95 percentiles) and obese (> 95 percentiles) for girls and boys . One national study had showed that these cutoff points are appropriate to be used for iranian children and adolescents . Two additional questions about desire for friends were asked (using children's bfrs): (a) which one do you think has more friends? (b) which one do you like to be a friend with? Other demographic variables were included gender, age, educational grade, and parental education . Statistical analysis was done using spss 16 (statistical package for social sciences), and significance considered 0.05 level . For descriptive analysis, we used central tendency and dispersion (mean standards deviation). Chi - square tests, one - way analysis of variance, t - test, pearson and spearman correlations ad nonparametric tests were used for additional analysis . The mean weight of participants was 32.40 (12.01) in girls and 31.78 (9.41) in boys; there was no significant gender difference . In addition, there was no significant gender difference in terms of height (girls = 135 cm 18.44, boys = 134 cm 15.72 [p> 0.05]). Selected characteristics of respondents there was a significant difference in bmi (categorized) between boys and girls (p <0.05). Although there was a significant relationship between bmi and age (p <0.05) and academic grades (p <0.05) for both genders, there was a significant relationship between bmi and maternal education (p <0.05) for girls . There was a significant difference in the current body image score for girls (3.79 1.45) and boys (4.31 1.31) (p <0.05). In addition, there was a significant gender difference related to ideal body images (3.45 1.45 for girls and 4.32 1.36 for boys [p <0.05]). Further, there was a significant difference in bd (subtracting the ideal body from the current body) between boys and girls (p <0.05). There was no relationship between bd and education of parents, age, or academic grades . However, there was a significant relationship between bd and bmi (df = 2, p <0.05). Categorization of bd based on gender antifat attitudes were assessed by two additional questions relating to selecting friends: who do you think has more friends? (s3) and who would you like to be friends with? Tables 3 and 4 summarize the images that were selected by both boys and girls . Results demonstrate a significant difference in the manner in which boys and girls answered these questions (p <0.05). Responses to the question which one do you think has more friend? The selection of figures (antifat attitudes) had no relationship with boys bmi, however, thinner girls (lower bmi) selected images of thinner children . Correlation between some of variables was shown in table 5 . According to the results, boys with higher bd assessed obese children as being more friendly (r = 0.141, p <0.01); however, they preferred to be friends with thinner children (r = 0.21, p <0.01). Although girls with higher bd preferred to be friends with thinner children (r = 0.208, p <0.01), there was no correlation between girls bd and their selection of friends (r = 0.125, p> 0.05). In some of the studies, 60% of girls expressed a desire to be thin versus 38% of boys . Also, studies focusing on asian children highlight the same gender difference . One national study in iran, highlights children's desire to be thin, especially girls . In a survey conducted as part of the aforementioned study, approximately 25% of children and adolescents believed they were of normal weight, while 50% of children and adolescents believed they were obese . Socio - cultural messages about manhood are associated with a strong and muscular body especially, in asian cultures . In iranian culture, men are expected to be brave and protect women; therefore, it is not surprising that boys desire to be larger . Some studies argue that gender differences in perception of body size based on the figure rating scale may be due to boy's desire for masculinity . As these figures are designed to represent silhouettes ranging from thin to obese (small versus large), masculinity is ignored in this scale; consequently, boys select silhouettes with larger bodies . Body mass index is introduced as a risk factor of bd in adolescents and adults, especially for women . Research undertaken by davison et al . Demonstrates a correlation between bmi and bd in preschool girls . In this study, nearly 27% of children were underweight, and 13% of children were obese . In a previous study, approximately 17.3% of iranian children and adolescents were underweight, and 17.7% were overweight or obese . This difference may be due to the younger age group and smaller sample size of this study . Given the high rates of childhood obesity and its impact on bd and eating disorders, serious preventive planning is required which encompass school - based programs . Messages about the ideal body affect individuals, especially children, via the media, their family, and peer groups . The desire to be accepted by family and friends can cause body concerns and a preoccupation with achieving an ideal body . Parental reactions and attitudes toward the appearance of their children can lead to bd among children . The opinions of parents have a greater impact on elementary school children than younger children . Researches argue that children have negative opinions regarding their overweight peers (antifat attitudes). Understanding the reasons children harbor antifat attitudes is important to ensuring the success of interventional programs . Children that are exposed to negative messages about weight are prone to developing negative attitudes toward obese individuals, regardless of their bmi . In a study conducted by cramer and steinwert, children aged between 3 and 5 years viewed thin or average pictures of children as being while cramer and steinwert's research did not highlight any gender differences in this regard, holub observed more negative attitudes toward obese people in preschool girls than boys . This study found a relationship between bmi and girls perception of children with more friends . This finding emphasizes the body image disturbances issues experienced by girls . According to the relationship between bmi and bd, social and familial messages about thinness have a greater impact on girls than boys; this may explain by boys desire to be larger . It appears that body image concerns and its consequences, such as eating disorders, are increasing in asian cultures, including in iran . One iranian study has shown that the number of girls who are overweight or obese is greater among those with highly educated mothers . Therefore, it is necessary to study the predictive factors of obesity and body image disturbances and associated behaviors (such as body management strategies) among iranian children and families . Healthcare planners, researchers, educators, and parents are concerned about body image issues and its consequences in childhood . It is recommended that asian countries work to identify the related risk factors particularly, during childhood . Identifying the risk factors of body image and related issues will be helpful in terms of developing preventive and therapeutic protocols . Given the limitations of this study, it is recommended that future studies further explore body image concerns and its associated behaviors or consequences among iranian children.
Orexins - a and -b, which are also called hypocretins, have recently been identified as novel hypothalamic neuropeptides (1). They strongly activate the appetite of animals through action in the lateral hypothalamus, and have been implicated in the central regulation of metabolic rate and glucose homeostasis . On the other hand, it inhibits the orexinergic pathway of acceleration of food intake in the lateral hypothalamic area . Both orexin - a and -b are derived from a common precursor preproorexin, which consists of 131 amino acid residues, by proteolytic processing (2). Orexin - a and -b consist of 33 and 28 amino acids, and their molecular weight are 3561 and 2899, respectively . There are two types of orexin receptors, both of which are coupled with g - protein . The affinity of orexin 1 receptor (ox1r) is 50 times higher to orexin - a than to orexin - b, while orexin 2 receptor (ox2r) shows equal affinity to both types of ligands . Orexinergic neurons are known to project to multiple neuronal systems (3), and the physiological role of orexin in the central nervous system (cns) has been elucidated at least partly . The gene expression of orexin is regulated by blood glucose and feeding (4, 5). Moreover, orexin regulates the sleep - wake cycle and other behaviors such as grooming through a dopaminergic pathway in cns . Depletion of orexin in cns plays a central role in the pathogenesis of narcolepsy in both animals and man (6, 7). The orexin gene is expressed in the testis (8), ovary, intestine (9), pancreas (10), and adrenals (11). Although orexin - a occurs in blood in a substantial amount, little is known about the action mechanism of orexin - a in peripheral tissues . Orexin - a, but not orexin - b, permeates the blood brain barrier (12). Orexin - a, when administered intra - peritoneally to the rat, stimulates insulin secretion by direct action on the pancreatic islets (10). We have already demonstrated that orexin - a stimulates catecholamine synthesis in bovine adrenal medullary cells via the ox1r route (13). To our knowledge the present study was designed to determine the plasma level of orexin and its relationship with other metabolic and anthropometric markers in obese children . Forty - seven obese japanese children, consisting of 31 boys and 16 girls, who visited the clinic for obese children at either the university of yamanashi or the university of occupational and environmental health, were consecutively enrolled in the study (table 1table 1 anthropometric data in control and obese children). According to the criteria for obesity in childhood adopted by the ministry of health, labor and welfare in japan, a child was considered to be obese when the body weight exceeded 120% of the standard body weight, which is defined as the mean body weight corresponding to the height for that age obtained from the national statistics for japanese school children in 1990 . The ages of the subjects were 10.4 0.5 (mean s.e.m .) Yr, and their percentage overweight was 42.9 1.9% . Blood was drawn after an overnight fast and they were subjected to anthropometric measurements including height, body weight, waist circumference, hip circumference, and triceps and subscapular skinfold thicknesses . The age - matched control group for measuring orexin and leptin (mean age, 10.4 0.3 yr) consisted of 26 nonobese children, 13 boys and 13 girls (table 1). Blood was drawn after an overnight fast . Since there were no significant sex - related differences among the anthropometric and clinical laboratory data in either the obese or control children, the boys and girls the human study committee of the university of yamanashi and the university of occupational and environmental health approved this study . Informed consent was obtained either from each subject or from his or her parents as appropriate . Anthropometric measurements were performed, as described previously (14), by the medical staff at both clinics . In brief, height was measured to the nearest 0.1 cm and body weight to the nearest 0.1 kg using a stadiometer . The waist circumference was measured at the level of the umbilicus, and hip circumference at the level of maximum extension of the buttocks, to the nearest 0.1 cm . Skinfold thickness was measured to the nearest 0.1 cm using the skinfold calipers at triceps (halfway between the acromion and the olecranon) and subscapular (1 cm below the inferior angle of the scapula). The percentage overweight was calculated using a small programmed calculator (pocket growth checker gen-185, sumitomo pharmaceuticals co., osaka, japan). The percentage body fat, based on the sum of triceps and subscapular skinfold thicknesses, was obtained using brozek s equation (15), after body density was calculated according to nagamine s formula (16). Orexin - a in human plasma was assayed by a radioimmunoassay kit (peninsula laboartory inc ., leptin in human plasma was assayed by an enzyme - linked immunosorbent assay kit (ibl co., ltd ., fujioka, gunma, japan). Clinical blood biochemical data were obtained from the clinical laboratories of both the university hospital of yamanashi and the university hospital of occupational and environmental health . Low - density lipoprotein - cholesterol (ldl - c) was calculated using the friedewald equation [ldl - c = tc hdl - c the statistical analyses were performed using spss version 11.0.1j (spss japan inc ., tokyo, japan). Forty - seven obese japanese children, consisting of 31 boys and 16 girls, who visited the clinic for obese children at either the university of yamanashi or the university of occupational and environmental health, were consecutively enrolled in the study (table 1table 1 anthropometric data in control and obese children). According to the criteria for obesity in childhood adopted by the ministry of health, labor and welfare in japan, a child was considered to be obese when the body weight exceeded 120% of the standard body weight, which is defined as the mean body weight corresponding to the height for that age obtained from the national statistics for japanese school children in 1990 . The ages of the subjects were 10.4 0.5 (mean s.e.m .) Yr, and their percentage overweight was 42.9 1.9% . Blood was drawn after an overnight fast and they were subjected to anthropometric measurements including height, body weight, waist circumference, hip circumference, and triceps and subscapular skinfold thicknesses . The age - matched control group for measuring orexin and leptin (mean age, 10.4 0.3 yr) consisted of 26 nonobese children, 13 boys and 13 girls (table 1). Blood was drawn after an overnight fast . Since there were no significant sex - related differences among the anthropometric and clinical laboratory data in either the obese or control children, the boys and girls the human study committee of the university of yamanashi and the university of occupational and environmental health approved this study . Informed consent was obtained either from each subject or from his or her parents as appropriate . Anthropometric measurements were performed, as described previously (14), by the medical staff at both clinics . In brief, height was measured to the nearest 0.1 cm and body weight to the nearest 0.1 kg using a stadiometer . The waist circumference was measured at the level of the umbilicus, and hip circumference at the level of maximum extension of the buttocks, to the nearest 0.1 cm . Skinfold thickness was measured to the nearest 0.1 cm using the skinfold calipers at triceps (halfway between the acromion and the olecranon) and subscapular (1 cm below the inferior angle of the scapula). The percentage overweight was calculated using a small programmed calculator (pocket growth checker gen-185, sumitomo pharmaceuticals co., osaka, japan). The percentage body fat, based on the sum of triceps and subscapular skinfold thicknesses, was obtained using brozek s equation (15), after body density was calculated according to nagamine s formula (16). Orexin - a in human plasma was assayed by a radioimmunoassay kit (peninsula laboartory inc ., belmont, ca, usa). Leptin in human plasma was assayed by an enzyme - linked immunosorbent assay kit (ibl co., ltd ., fujioka, gunma, japan). Clinical blood biochemical data were obtained from the clinical laboratories of both the university hospital of yamanashi and the university hospital of occupational and environmental health . Low - density lipoprotein - cholesterol (ldl - c) was calculated using the friedewald equation [ldl - c = tc hdl - c the statistical analyses were performed using spss version 11.0.1j (spss japan inc ., tokyo, japan). Table 2table 2 data on clinical blood biochemistry for obese children (n=47) summarizes the data on clinical blood biochemistry for obese children . Plasma orexin - a concentration was higher in obese children (17.0 0.4 pg / ml; p<0.001) than in the control children (13.5 1.1 pg / ml) (fig . There is no significant sex - related difference in the plasma concentration of either orexin or leptin . Obese vs. control: p<0.001 for both indices . ). Similarly, plasma leptin concentration was higher in obese children (12.0 1.0 ng / ml; p<0.001) than in the control children (5.2 0.4 ng / ml) (fig . There is no significant sex - related difference in the plasma concentration of either orexin or leptin . Table 3table 3 correlation between anthropometric indices and plasma level of orexin - a or leptin in obese children (n=47) summarizes relationships between the anthropometric indices and the plasma level of orexin - a or leptin in obese children . The plasma orexin - a level was correlated significantly with the waist - to - hip ratio only, while the leptin level was correlated with percentage overweight, waist circumference and percentage body fat . The relationship between plasma levels of orexin - a and leptin . 2 correlation between plasma levels of orexin - a and leptin in obese children (n=47).. there was a highly significant positive correlation between the two parameters . None of the data on clinical blood biochemistry shown in table 2 was correlated with the plasma level of orexin - a or leptin . Correlation between plasma levels of orexin - a and leptin in obese children (n=47). Plasma levels of both orexin - a and leptin were higher in the obese than the control children, and orexin - a was correlated positively with leptin . However, both parameters were correlated with only limited numbers of anthropometric indices, and with no other data of clinical blood biochemistry . (18) reported that plasma immunoreactive orexin - a level was 6.91 0.85 pg / ml in healthy adults after extraction of orexin - a by sep pak chromatography from acidified plasma . (19) reported that the basal level of orexin - a was 29.9 1.6 pg / ml without extraction, using the same commercial kit as ours . They also reported that the plasma orexin - a level increased to 38.947.9 pg / ml during fasting for 710 days in the treatment of psychosomatic diseases in nonobese women (19). The reported level by arihara et al . (19) were higher than ours . In our study, orexin - a reported (20) in subjects, whose ages ranged from 23 to 79 yr, that plasma orexin - a level was higher in the elderly adults than in the younger ones . However, whether the difference in plasma concentrations between our study and other studies is dependent on the ages of the subjects needs to be studied further . The high level of orexin, which promotes appetite, in obese children, observed here, implies that their appetite is not suppressed regardless of their obesity . The high plasma level of orexin - a appeared to parallel the level of leptin in the present study . If the orexinergic effect parallels that of leptin in the hypothalamic regulation of appetite, this association can, at least partly, explain the apparent leptin resistance in the present obese children . However, this is unlikely because leptin is known to inhibit the orexinergic pathway of acceleration of food intake in the lateral hypothalamic area . Adam et al . Reported (21) that the plasma orexin level was inversely correlated with body mass index in the range of 2059 kg / m in adults . (22) reported that plasma orexin - a level was positively correlated with either body mass index or fat mass in lean and normal weight adults with chronic obstructive pulmonary diseases . Patients with narcolepsy show a low concentration of orexin - a in cerebrospinal fluid (23, 24), however, their plasma levels of orexin were reported to be either low (23) or normal (24). Around half of the orexinergic neurons in the hypothalamus are under the inhibitory regulation of leptin . Thus, orexin is secreted by fasting in order to conserve energy in the central nervous system . Whether the peripheral mechanism related to orexin is also regulated by leptin or this dual action of orexin in central and peripheral mechanisms needs to be studied further . In the present study, plasma orexin - a level was correlated significantly with the waist - to - hip ratio but not with waist circumference . On the other hand, the opposite was the case with leptin . Lissner et al . (25) reported in swedish women that hip circumference showed a health - promoting effect, and that the effect was independent of the adverse health effect of waist circumference . They suggested that waist - to - hip ratio and waist circumference had mutually different implications in health and longevity of men . Thus, the correlation profile of the two indices may be different from each other.
A 31-year - old multiparous turkish woman was scheduled for cesarean section under spinal anesthesia at 37 weeks and five days gestation because of hemorrhage due to secondary placenta previa . Invasive blood pressure, central venous pressure, and heart rate were stable during the surgery . Rates of maternal hypertension, pre - eclampsia, anemia, and infection in the pregnant chronic dialysis patient are high . However, our findings suggest that with careful, close, and effective monitoring preoperatively and intraoperatively, spinal anesthesia can be safely performed for cesarean section in patients undergoing hemodialysis . Fertility is reduced in patients with renal disease, so pregnancy in patients with end - stage renal failure is uncommon . However, confortini et al in 1971 reported the first case of a 35-year - old end - stage renal failure patient on hemodialysis (hd) who achieved a full - term pregnancy.1 the major issues to be dealt with in this type of patient are the possible complications of pregnancy, including onset or exacerbation of systemic arterial hypertension which may evolve into pre - eclampsia or even eclampsia, premature delivery, or failure of the fetus to grow . Another important point to define is that of full - term pregnancy in patients on dialysis.2 the placenta usually implants in the upper uterine segment . However, in some cases, it implants in the lower uterine segment, either covering the internal cervical os, or lying in close proximity to it . This abnormal implantation into the lower segment, called placenta previa, is an important cause of bleeding in the second half of pregnancy and during labor, and is associated with significant maternal and perinatal morbidity and occasionally mortality.3 there are many reports outlining the safe and successful use of peripheral regional blocks in dialysis - dependent patients if there is no platelet dysfunction or coagulation abnormality . In fact, peripheral regional anesthesia techniques have been used in patients with chronic renal failure (crf) for the creation of arteriovenous fistulae . In this paper we share our experience of spinal anesthesia in a pregnant woman with crf on dialysis and needing an emergency cesarean section due to placenta previa . A 31-year - old woman with seven gravidas, four paras, two abortions, and three live children was referred to us at 37 weeks and five days gestation . She had suffered from crf since the age of 29 years, and had been undergoing hemodialysis three times per week . The patient had had a medical abortion one year earlier when she was five months pregnant due to an intrauterine ex - fetus that developed secondary to her renal disease and the patient was recommended not to become pregnant again . Having noticed the current pregnancy too late, the patient was then undergoing four hours of dialysis three times a week, and increased to four hours per day for the previous seven months . One hour after her last dialysis, she was hospitalized in gynecology services due to vaginal bleeding and cramps . The patient was being monitored for delivery and upon an increase in the amount of vaginal bleeding and detection of placenta previa during ultrasonographic examination, it was decided to perform an emergency cesarean section . Preoperative laboratory values for the patient are summarized in the table . In the preoperative preparation room, central venous catheterization was performed under local anesthesia via the right internal jugular vein . Invasive arterial catheterization was performed from the right radial artery (because an arteriovenous fistula was being used for dialysis on the left side). The patient had been taking methyldopa as an antihypertensive during the preoperative period but had severe hypertension at presentation (180/110 mmhg), so nitroglycerin infusion was started . Her activated partial thromboplastin time being normal (table 1), six hours having elapsed since her last dialysis (ie, the dialysis - dependent antiaggregant effect having receded), and considering the risks of anesthesia, it was decided to use spinal anesthesia in view of its additional antihypertensive effect . Spinal anesthesia with 8 mg of hyperbaric bupivacaine was successfully performed using a 27-gauge spinal needle at the l3/4 intervertebral space in the sitting position . Spinal anesthesia level having reached the t5 and t6 dermatomes, the operation was started at the third minute . A 2220 g baby with an apgar score of 6 in one minute and 8 in five minutes was delivered four minutes after the beginning of surgery . Invasive blood pressure (bp), electrocardiography (ecg), heart rate (hr), and pulse oximetry (spo2) were monitored during surgery . Hypotension, probably related to sympathetic blockage, developed following spinal anesthesia, so the nitroglycerin infusion was terminated without the need for sympathomimetic medication . Thereafter, the patient was taken to the intensive care unit (icu) with a bp of 119/71 mmhg, a hr of 83 beats per minute, and a sensory spinal anesthesia level at the t9 and t10 dermatomes . During the operation, the patient was not given any liquids other than 1 u of erythrocyte suspension due to her anemia . She was given an additional 2 u of erythrocyte suspension while she was kept in the icu . She recovered without any problems, returned to regular hemodialysis on the first postoperative day, and was discharged from the hospital four days after the operation after adjustment of her antihypertensive therapy and on a dialysis program of four - hourly sessions three days a week . Clinical assessment on the day of discharge revealed no complications of spinal anesthesia, such as headache, nausea, change in bp (in particular, hypotension) or neurological deficit . Meanwhile, the baby was taken for ventilator treatment in the neonatal intensive care unit due to respiratory distress syndrome . Having been treated for abo isoimmunization and neonatal jaundice, the baby was discharged from hospital 32 days after delivery . Childbearing is an important issue in women with renal disease.4 although not common, pregnancy in chronic dialysis patients does occur . In fact, the percentage of successful pregnancies in women on chronic dialysis may be increasing.5 however, pregnancy has generally been regarded as very risky in these women.4 maternal complications associated with crf include pre - eclampsia, worsening renal function, preterm delivery, anemia, chronic hypertension, and cesarean delivery.6 hypertension is the most common life - threatening problem in these patients.4 in preparation for elective surgery, patients with crf should receive dialysis the day before the operation . This is essential to achieve a volume status as close to normovolemic as possible, to allow the patient to tolerate fluid loads associated with surgery, and to obtain normal electrolyte concentrations . Otherwise, patients have to be managed medically and receive dialysis after the operation.7 our patient had to be operated on six hours after her last dialysis, at which time her volume and electrolyte balance could not be fully established . The preoperative evaluation of the patient with crf should focus on the comorbidities associated with kidney disease and on the signs and symptoms of uremia, fluid overload, and inadequate dialysis . Laboratory studies should be aimed at assessing electrolyte concentrations, acid - base status, urea and creatinine levels, hematocrit, platelet count, and coagulation.7 blood gas values were also monitored in our patient as part of her preoperative laboratory examination . Particular attention was paid to serum bicarbonate levels, which were in the 1820 meq / l range.8 electrolytes should not be measured immediately after dialysis due to incomplete equilibration between plasma and intracellular fluids . Potassium levels above 5.5 meq / l are usually considered a contraindication to elective surgery because tissue trauma and cell death can cause potassium to increase to life - threatening levels . An ecg is usually obtained as well to screen for changes caused by electrolyte abnormalities.7 no indications of hyperkalemia were observed in our patient s laboratory data or on her ecg . Platelet dysfunction is not related to a low platelet count, and can be detected only by the bleeding time, measured as the time to cessation of hemorrhage after a standardized skin incision.9 however, this test seems to have a limited predictive value for clinical bleeding and is not commonly used . Patients who are receiving adequate dialysis are less likely to have significant platelet dysfunction and their risk of bleeding should not be excessive.7 control of anemia is important because this is a significant cause of left ventricular hypertrophy, heart failure, and angina . A hemoglobin of 11 to 12 g / dl is considered optimal,10 and this value is also used as a target before surgery, although this practice is not supported by clinical evidence . Correction of anemia also helps to improve the platelet dysfunction associated with renal failure.11 our patient was given 1 u of erythrocyte suspension during the operation and 2 u after the operation due to the presence of anemia (see table). She was also given erythropoietin, and was discharged from the hospital on the fourth postoperative day with an acceptable hemoglobin value . If fluid balance is likely to be a problem, central venous monitoring may be useful, but the need for invasive arterial monitoring should be considered carefully before potentially damaging an artery that may be used later to form an arteriovenous fistula . When positioning patients for surgery, care should be taken to protect shunts or fistulae.12 delayed gastric emptying may increase the risk of aspiration during general anesthesia . If anticoagulants have been used during hemodialysis, at least six hours should elapse before siting a regional block . It is wise to confirm that clotting is normal by checking the activated partial thromboplastin time . When performing a block it is essential to use aseptic technique . Presence of any peripheral neuropathy should be documented if regional blocks are to be used.12 intravenous access is usually difficult in patients with crf, and central venous access is often needed . The veins and arteries of the nondominant upper extremity should be spared from vascular cannulation, because they may be needed for dialysis access in the future . Subclavian vein cannulation should also be avoided because this procedure is frequently complicated by thrombosis, which compromises dialysis access . Therefore, we opted to do central venous cannulation via the internal jugular vein in our patient . We also carried out invasive arterial monitoring via the right radial artery because there was an arteriovenous fistula on the left side for dialysis . During all these procedures, we were extremely careful to maintain sterile conditions due to the risk of infection in this type of patient . The choice of hemodynamic monitoring technique should be based on the history and characteristics of the individual patient and should be directed towards specific hemodynamic goals . Patients with renal diseases undergoing high - risk procedures often need invasive hemodynamic monitoring with arterial and central venous catheters.7 we considered it appropriate to carry out preoperative arterial and venous monitoring in view of our patient s clinical condition (ie, anemia, dialysis six hours earlier, and severe hypertension, along with normal bleeding tests). Hypertension in crf patients is usually volume - dependent and responds to adequate dialysis, but most patients will also require pharmacologic therapy . Current recommendations for long - term crf management set a bp goal of lower than 130/80 mmhg.13 preoperative bp in our patient was high (180/110 mmhg). Nitroglycerin infusion was started at the initiation of invasive arterial monitoring, and the infusion rate was adjusted (0.251.0 g / kg / min) until the target bp (130/80 mmhg) was reached . Nitroglycerin infusion was preferred because of its rapid onset and offset, its cardiac protective effect, its lack of vasodilatory properties, and its low risk to the baby . Improvements in dialysis and medical treatment have increased the number of dialysis - dependent crf patients being considered for surgery, including obstetric procedures . In patients with crf necessitating dialysis, mortality rates of 4 to 10% have been reported, with morbidity rates approaching 50%.14 this increased rate of complications is probably due to the low renal reserve of patients with crf and their reduced ability to respond to the stress, fluid load, and tissue trauma caused by surgery . However, additional morbidity is created by organ dysfunction and the coexisting diseases frequently encountered in these patients . Pregnancy adds further risks, including pre - eclampsia, polyhydramnios, intrauterine growth retardation, preterm delivery, low birth weight, and the potential for a stillbirth . We believe that pregnant crf patients requiring dialysis can be urgently and safely operated on using a careful anesthesia method after a detailed renal and hematological preoperative assessment and rapid but detailed preparation.
Bisphosphonates are have been used for many years for a variety of conditions including paget's disease, osteoporosis, multiple myeloma, and hypercalcemia of malignancy . A number of randomized clinical trials have shown that they increase bone density and reduce the incidence of fractures in osteoporosis . Although bisphosphonates are generally safe and effective, concerns have been raised about the potential risk of over - suppression of bone turnover during long - term use . Have reported on nine patients who sustained spontaneous non - spinal fractures while on alendronate therapy . Schneider also described a 59-year - old previously healthy woman who experienced two non - traumatic stress fractures of the femur, 4 years apart, while on alendronate therapy . Reported a 35-year - old man who, after 6 years of treatment with 10 mg / day alendronate, had a sub - trochanteric fracture of the right femur after mild trauma . A bone biopsy showed a lack of osteoid on trabecular surfaces and an absence of tetracycline labeling, suggesting marked suppression of bone turnover . Here, we describe a 63-year - old korean woman who presented with non - traumatic spontaneous diaphyseal fractures of both femurs and delayed fracture healing after 5 years bisphosphonate therapy . A 63-year - old woman was admitted to hospital because of severe pain in both anterior thighs for 5 months . When she presented with osteoporosis and degenerative arthritis 5 years ago, she started to take alendronate 70 mg / wk, alfacalcidol 0.5 g / day and calcium citrate 1,900 mg / day . Three years later, she switched to risedronate 35 mg / wk and calcitriol 0.25 g / day because of dyspepsia . She had total abdominal hysterectomy with bilateral salpingo - oophorectomy 35 years ago due to endometriosis . At the time of admission, her height was 147 cm, and her weight was 54 kg; these had been 158 cm and 55 kg in her fourth decade . There was no history of any trauma, falling, or medication that predisposed to osteoporosis and fracture . Her lifestyle had not been sedentary because she had to work in the fields for her living . Physical examination revealed blood pressure of 100/80 mmhg, pulse rate of 80 beats / min, respiratory rate of 20 breaths / min, and body temperature was 36.5. cardiac and abdominal examinations were normal . Here walking pattern laboratory tests showed hemoglobin 12.2 g / dl, white blood cell count 4,910/mm, platelet count 334,000/l, serum calcium 9.3 mg / dl, serum phosphorus 3.2 mg / dl, total protein 7.5 g / dl, serum albumin 4.7 g / dl, uric acid 5.1 mg / dl, and alkaline phosphatase 72 iu / l . Daily urinary calcium excretion was 369.2 mg (normal, 70 to 180), and daily urinary phosphorous excretion was 959.92 mg (normal, 400 to 1,300). Serum parathyroid hormone (pth) was 18.92 pg / ml (normal, 10 to 65), and 25-(oh)-vitamin d, determined by commercially available radioimmunoassay kit (diasorin, stillwater, mn, usa), was 15.33 ng / ml (normal, 9.0 to 37.6). Osteocalcin was 18.93 ng / ml (normal postmenopausal, 19.6 to 41.2), serum c - telopeptide, 0.301 ng / ml (normal postmenopausal, 1.008), and urine n - telopeptide was 55.7 nbce / mm cr (normal postmenopausal, 6.0 to 125.72). Simple x - rays of the thoracic and lumbar spine showed diffusely decreased vertebral height with osteoporosis and multiple compression fractures of t4, t5, t11, l1, l2, l4, and l5 vertebrae . There was no additional compression spinal fracture compared with x - rays taken 2 years earlier (fig . 1). The most recent bone mineral density (bmd), measured by dual - energy x - ray absorptiometry (qdr-4500a, hologic, waltham, ma, usa), showed a t - score at the third lumbar vertebra of -3.8 and a total hip t - score of -3.3 (table 1). All of the results showed severe osteoporosis, and no improvement in bmd was seen, despite bisphosphonate treatment . Whole spine magnetic resonance imaging revealed multiple compression fractures in the t4, t5, t11, l1, l2, l4, and l5 vertebral bodies and diffuse disk bulging at l2 - 3, l3 - 4, and l4 - 5, with degenerative changes of facet joints and thickening of ligamentum flavum . A whole - body bone scan, which was obtained 2 hours after injecting technetium-99 m hydroxymethylene diphosphonate (hdp) intravenously, showed intense uptake at both distal femurs . Simple x - rays also showed fracture lines in both distal femur shafts, compatible with sites of pain (fig . 2). After spinal nerve block with walking rehabilitation therapy was done for pain control, pain was not improved . Therefore, we speculated that the delayed fracture healing of both distal femurs due to the long - term bisphosphonates therapy was the main cause of the pain . Bisphosphonate therapy was discontinued . However, the pain continued, and the fracture lines seemed to become aggravated and showed delayed fracture healing . However, the patient had surgical internal fixation at both femoral diaphyses because of non - union of the femoral fractures even after 6 months of observation without bisphosphonates (fig . The elapsed time from the first visit to surgical internal fixation was 7 months . During the operation, severe osteoporotic fracture was noted, but structural displacement was mild . Bone biopsy performed with surgical fixation showed decreased trabecular bone with massive fibrosis in the marrow space . Alendronate, risedronate, ibandronate, and zoledronate, which are classified as amino - bisphosphonates, have much higher potency because they contain nitrogen in their side chain . However, concerns have been raised about potential risk of over - suppression of bone remodeling during long - term use . Prolonged use of bisphosphonates may suppress bone turnover to the point that micro - damage persists and accumulates . In experimental animals, alendronate has been shown to inhibit normal repair of micro - damage arising from marked suppression of bone turnover, which results in macro - cracks . Chronic over - suppression of bone turnover by bisphosphonates may allow secondary mineralization to continue, resulting in hard, brittle bone with an increased risk for fracture . Other supportive evidence on over - suppression includes several reports of jaw osteonecrosis in patients treated with high doses of bisphosphonates . Recently, odvina et al . Reported nine patients (eight postmenopausal women and one man) who sustained unusual spontaneous non - spinal fractures while on alendronate therapy (10 mg / day or 70 mg / wk) for 3 - 8 years . Six of the nine patients had delayed or absent fracture healing for 4 months to 2 years during alendronate therapy, and four showed delayed fracture healing for 8 - 12 months even after discontinuation of alendronate . Although co - administration of estrogen or glucocorticoids appears to be a predisposing factor, this apparent complication also occurred with bisphosphonate monotherapy . Our case report described a woman who experienced non - traumatic spontaneous stress fractures while on bisphosphonate therapy and non - union of the femoral shaft fracture despite discontinuation of bisphosphonates . We were able to ensure good adherence to medication by her self - report and serial measurements of urinary n - telopeptide, one of the bone turnover markers, which showed a decrease of 62.5%, from 40.8 to 15.3 nbce / mm cr, during the first 3 months after initiation of bisphosphonate therapy and similar levels afterwards . Therefore, this case had the possibility of delayed fracture healing due to suppression of bone turnover by long - term use of bisphosphonates . Other possible causes of insufficiency or pathological fracture include osteoporosis - include osteomalacia, osteogenesis imperfecta, bone tumors (primary or metastatic), and paget's disease of bone . However, she had no definite clinical and laboratory evidence of such diseases . The residual effect on bone density is known to remain for 3 years after drug withdrawal, and it is possible that the suppressive effect of bisphosphonates on bone resorption might also be cumulative over time . Therefore, close monitoring and assessment of fracture healing after internal fixation in this patient was mandatory, considering the risk of re - fracture . The fixed pins were maintained, and she showed full union status 6 months after internal fixation . In this patient, the biochemical markers such as n- and c - telopetides and serum osteocalcin showed normal levels and did not reveal magnitude of the suppression of bone turnover . The discordance between the histomorphometric and biochemical markers of bone turnover could be related to the variable degree of suppression at different skeletal sites, as the changes in biochemical markers are more reflective of changes in the whole skeleton . Additionally, this patient showed somewhat elevated daily urinary calcium excretion, with normal serum calcium, phosphorus, and pth level . The active vitamin d, calcitriol, was thought to be the cause of this hypercalciuria . Although previous studies have shown the efficacy of bisphosphonates in the first 5 years of therapy in terms of improving bone density and reduced risk of fractures, additional studies are needed to determine how long bisphosphonates can safely be given . Recently, black et al . Have compared the effects of discontinuing alendronate treatment after 5 years versus continuing for 10 years . The results confirmed the safety of alendronate for up to 10 years, including no increased fracture risk with long - term use . However, even those who discontinued alendronate after 5 years showed no higher fracture risk, other than for clinical vertebral fractures, compared with those who continued alendronate . These results suggest that discontinuation of alendronate for up to 5 years does not appear to significantly increase fracture risk, except for the patients at very high risk of vertebral fractures . Furthermore, considering some definitive case reports, we must emphasize the need for awareness of recent controversies about the effect of long - term reduction of bone turnover on bone strength, especially the occurrence of awkward fractures at an unexpected site, as in this patient . The benefits of prolonged use of bisphosphonates must be carefully weighed against the potential adverse effects of over - suppression of bone metabolism.
We report the first french case of r. aeria ie complicated by brain haemorrhage and femoral mycotic aneurysms, in an immunocompetent patient . A 57-year - old man born in nepal was hospitalized in january 2016 for fever reaching 39.4c, chills, diarrhoea and headache . He had a previous history, in 2008, of right sub - thalamic ischaemic stroke and severe mitral insufficiency without ventricular dysfunction for dystrophic mitral valve . He did not have recent dental care and had no history of intravenous drug injections . At admission the patient presented with an already known 4/6 meso - systolic rough mitral murmur without cardiac failure and a painful erythema of the right hand (fig . Two aerobically incubated blood cultures performed at admission were positive with gram - positive branching filamentous bacilli (fig . 1b, c) within 41 and 86 h, as were the seven other blood culture sets . Colonies were identified as r. aeria using matrix - assisted laser desorption - ionization time - of - flight mass spectrometry (see fig . Trans - oesophageal echocardiography revealed mitral ie with several nearby vegetations of the ring base, flail p1 without heart dysfunction or abscess (fig . Pre - operative screening for systemic embolism showed a left frontal subarachnoid haemorrhage on brain magnetic resonance imaging and a hypermetabolic focus in the femoral artery compatible with a mycotic aneurysm on positron emission tomodensitometry (fig . Empiric antibiotherapy was introduced according to european guidelines with intravenous amoxicillin 2 g every 4 h and gentamicin 3 mg / kg once a day . Surgical mitral replacement by bio - prosthesis with annular reconstruction was performed according to the risk of cerebral embolism and cardiac abscess at day 10 after initiation of antibiotics . Blood culture negativity was obtained at day 3 after antibiotics initiation and valvular culture was negative . 16s rrna gene sequence analysis was performed on the mitral sample and confirmed the identification of r. aeria (genbank accession sequence kx270977) with a maximal identity of 100% for r. aeria type strain a1 - 17b (genbank accession number ab071952). Antibiotic combination was maintained for 2 weeks, then amoxicillin alone for a further 4 weeks . Subarachnoid haemorrhage regressed and trans - oesophageal echocardiography showed a good functioning of the prosthesis and the absence of added elements . Initially r. aeria was known as rothia dentocariosa genomovar ii and could be mistaken for nocardia spp . Except for its antibiotic sensitivity . Infective endocardites due to r. aeria (n = 7) and r. dentocariosa (n = 25), are reported to be associated with central nervous system involvement in 13 of the 32 cases (40%) published,, . Antibiotic susceptibility of the r. aeria strains isolated in all case reports showed sensitivity for ampicillin and gentamicin . We report the first infective ie due to r. aeria described in france and confirm that r. aeria ie is serious and associated with mycotic aneurysms and central nervous system involvement . Fast and aggressive management with an antibiotic combination including penicillin for 46 weeks, and valve replacement when necessary, is required and could limit the occurrence of complications and death.
Aneurysms at the origin of the posterior inferior cerebellar artery (pica) remain a challenge for neurosurgeons because the close proximity to the brainstem and lower cranial nerves poses a high risk for surgical clipping.1)14)15) aside from such anatomic unchangeability of adjacent structures, complex anatomic features of pica origin aneurysms themselves also add high risks for both surgical and endovascular treatments.16)19) however, in the past decade, there have been substantial advances in device technologies and endovascular techniques . As a result, the number of patients who are not candidates for endovascular treatment is decreasing . In particular, the " self expanding stent technology " and " supercompliant balloon technology " have revolutionized the methods and results of endovascular treatment.14)22) combining a self expanding stent with a " cross - over technique ", which uses an inter - arterial communication as an access route to the other vascular part,9)12) the authors herein report on a bilateral approach for stent - assisted coiling of two complex pica origin aneurysms . A 71-year - old woman who had no past medical history presented to our emergency room with a sudden thunderclap headache, nausea, and vomiting . On neurological examination, the score on the glasgow coma scale was 11 (e3v3m5) and isocoric pupil response was observed . Initial computed tomography (ct) and ct angiography showed fischer grade 4 subarachnoid hemorrhage (sah) and a ruptured left pica aneurysm (fig . Cerebral angiography and 3d image reconstruction showed a left pica origin aneurysm measuring 7 mm in length with a dome to neck ratio of 0.7 and pica incorporation into the sac (fig . 1c and 1d). As a treatment option, in view of anticipated surgical difficulty and patient's preference, our neurovascular team decided on endovascular treatment . The use of a multi - catheter or balloon remodeling technique is likely to have a high risk of pica occlusion during coil embolization of this complex aneurysm; therefore, coiling with placement of a stent in the vertebral artery (va)-aneurysm - pica path was planned . However, an antegrade approach to the pica appeared to be very difficult and too risky, so a retrograde approach via the contralateral va was decided . For selection of the left pica arising from the aneurysm, a 6f guiding catheter was placed in the right va, an excelsior sl-10 microcatheter (striker, fremont, ca) was introduced into the right va via the guiding catheter to cross over the vertebrovertebral junction to the left va, and the microcatheter over a synchro-14 guidewire (striker, fremont, ca) was able to select the left pica smoothly (fig . After being placed in the left pica, the excelsior microcatheter was exchanged for a rebar-18 microcatheter (ev3 inc, irvine, ca) that was compatible with a stent delivery system . Another 6f guiding catheter was introduced into the left va and an excelsior sl-10 microcatheter was placed in that aneurysm through the guiding catheter for coil delivery . The right vertebral artery was non - dominant and smaller than the left vertebral artery . However, the size of the right vertebral artery was sufficient to accept the rebar-18 microcatheter . After deployment of a solitaireab stent (ev3 inc, irvine, ca) for protection of the pica (fig . 1f), a total of eight detachable coils were packed in the aneurysm . As only a 20-mm - long stent (4 20) was available at that time, it covered the left pica, aneurysm neck, left va, and a short segment of the right va, in order . The patient received dual anti - platelet therapy for 30 days, and continued life - long aspirin . At the four - month follow - up evaluation, a previously healthy 43-year - old man visited our emergency room complaining of a severe headache for six hours . Except for nuchal rigidity, brain ct showed right premedullary sah and minimal intraventricular hemorrhage mainly in the fourth ventricle, three - dimensional ct angiography showed a ruptured right pica origin aneurysm, and cerebral angiography was successively performed for visualization of anatomic details of the aneurysm . It had a maximal size of 11 mm and a low dome - to - neck ratio of 0.9, and gave rise to the pica, which was incorporated into the sac (fig . Aiming at both aneurysm occlusion and pica preservation, coiling with placement of a stent in the va - aneurysm - pica path was considered the best method . All procedural steps were similar to those of case 1, except for the use of two micro - catheters for coil delivery and use of a 4 22 mm enterprise stent . After placement of the first two coils, eliminating contrast filling of the aneurysm dome, systemic heparinization (3,000 iu) was administered intravenously . However, thrombosis occurred in the aneurysm neck during placement of the last coil, which resulted in near occlusion of the pica flow . Tirofiban was given as an intraarterial bolus of 0.5 mg over five minutes, resulting in recanalization of the occluded pica . 2b), no progressive thrombosis was observed and the pica flow was intact, so that the patient was awakened . On neurological examination, no neurological deficit was identified immediately or at one month after the procedure . The patient received dual anti - platelet therapy for 30 days, but was lost to follow - up afterwards . A 71-year - old woman who had no past medical history presented to our emergency room with a sudden thunderclap headache, nausea, and vomiting . On neurological examination, the score on the glasgow coma scale was 11 (e3v3m5) and isocoric pupil response was observed . Initial computed tomography (ct) and ct angiography showed fischer grade 4 subarachnoid hemorrhage (sah) and a ruptured left pica aneurysm (fig . Cerebral angiography and 3d image reconstruction showed a left pica origin aneurysm measuring 7 mm in length with a dome to neck ratio of 0.7 and pica incorporation into the sac (fig . 1c and 1d). As a treatment option, in view of anticipated surgical difficulty and patient's preference, our neurovascular team decided on endovascular treatment . The use of a multi - catheter or balloon remodeling technique is likely to have a high risk of pica occlusion during coil embolization of this complex aneurysm; therefore, coiling with placement of a stent in the vertebral artery (va)-aneurysm - pica path was planned . However, an antegrade approach to the pica appeared to be very difficult and too risky, so a retrograde approach via the contralateral va was decided . For selection of the left pica arising from the aneurysm, a 6f guiding catheter was placed in the right va, an excelsior sl-10 microcatheter (striker, fremont, ca) was introduced into the right va via the guiding catheter to cross over the vertebrovertebral junction to the left va, and the microcatheter over a synchro-14 guidewire (striker, fremont, ca) was able to select the left pica smoothly (fig . After being placed in the left pica, the excelsior microcatheter was exchanged for a rebar-18 microcatheter (ev3 inc, irvine, ca) that was compatible with a stent delivery system . Another 6f guiding catheter was introduced into the left va and an excelsior sl-10 microcatheter was placed in that aneurysm through the guiding catheter for coil delivery . The right vertebral artery was non - dominant and smaller than the left vertebral artery . However, the size of the right vertebral artery was sufficient to accept the rebar-18 microcatheter . After deployment of a solitaireab stent (ev3 inc, irvine, ca) for protection of the pica (fig . 1f), a total of eight detachable coils were packed in the aneurysm . As only a 20-mm - long stent (4 20) was available at that time, it covered the left pica, aneurysm neck, left va, and a short segment of the right va, in order . The patient received dual anti - platelet therapy for 30 days, and continued life - long aspirin . At the four - month follow - up evaluation, a previously healthy 43-year - old man visited our emergency room complaining of a severe headache for six hours . Except for nuchal rigidity, brain ct showed right premedullary sah and minimal intraventricular hemorrhage mainly in the fourth ventricle, three - dimensional ct angiography showed a ruptured right pica origin aneurysm, and cerebral angiography was successively performed for visualization of anatomic details of the aneurysm . It had a maximal size of 11 mm and a low dome - to - neck ratio of 0.9, and gave rise to the pica, which was incorporated into the sac (fig . Aiming at both aneurysm occlusion and pica preservation, coiling with placement of a stent in the va - aneurysm - pica path was considered the best method . All procedural steps were similar to those of case 1, except for the use of two micro - catheters for coil delivery and use of a 4 22 mm enterprise stent . After placement of the first two coils, eliminating contrast filling of the aneurysm dome, systemic heparinization (3,000 iu) was administered intravenously . However, thrombosis occurred in the aneurysm neck during placement of the last coil, which resulted in near occlusion of the pica flow . Tirofiban was given as an intraarterial bolus of 0.5 mg over five minutes, resulting in recanalization of the occluded pica . 2b), no progressive thrombosis was observed and the pica flow was intact, so that the patient was awakened . On neurological examination, no neurological deficit was identified immediately or at one month after the procedure . The patient received dual anti - platelet therapy for 30 days, but was lost to follow - up afterwards . Pica origin aneurysms frequently have unfavorable anatomic findings, including a wide neck and pica incorporation into the sac.1)10)14) endovascular treatment of such aneurysms is associated with risk for pica occlusion or procedural failure . Although special techniques have recently been addressed,6)8) two goals of aneurysm occlusion and pica preservation appear to be mostly secured by stent - assisted coiling, where a self expanding stent successively covers the pica, aneurysm neck, and va . However, in the case of pica incorporation, selecting the pica antegrade through the ipsilateral va is extremely difficult and risky . Anatomy - wise, if the contralateral va were non - interrupted and large enough to accept a catheter, a contralateral and retrograde approach to the pica would be easier than the ipsilateral antegrade one . A few case studies regarding the " cross - over technique " using the communicating artery or vertebrovertebral junction as an access route have been reported in the literature.5)9) moret et al.14) suggested that the anatomic benefits afforded by this technique outweigh the potential risks associated with catheterization of another major cerebral artery . We believe that, in two cases in this study, without stent - assisted coiling using the " cross - over technique ", pica occlusion would have been inevitable . However, long - term follow - up results of this procedure are not available . In particular, the size of the pica is so variable that the issue of long - term stent patency remains unanswered . After far - lateral suboccipital exposure, pica origin aneurysms can be approached between the 11 cranial nerve inferiorly and the ninth and tenth cranial nerves superiorly . Therefore, the 10 and 11 cranial nerves are at risk of injury during the operation, which can result in lower cranial nerve dysfunction.3)7)10)15) in addition, complex aneurysmal geometry may make it difficult for neurosurgeons to complete aneurysm clipping without occlusion of the pica . Occlusion of the pica close to its origin may lead to serious morbidity and mortality, depending on the distribution of the medullary perforators and the presence of collateral supply from other cerebellar arteries . Aneurysm clipping following occipital artery - pica bypass surgery may be a solution to this serious complication,2)19)20) however, it is too early to generalize the staged treatments . Due to recent instrumental and technical developments leading to good results, endovascular treatment is considered the primary treatment for posterior circulation aneurysms . However, complex pica aneurysms, having a wide neck and pica incorporation, remain a challenge for both surgical and endovascular treatments . In such cases, a bilateral approach for
Dengue fever is the most significant arbovirus infection in malaysia and tropical countries, with an upward trend from 44.3 cases/100,000 population in 1999 reaching 181 cases/100,000 population in 2007 . The world health organization (who) ranks dengue as the most crucial mosquito - borne viral disease in the world . This infectious disease affects more than 100 endemic countries including regions of asia, americas, africa, and eastern mediterranean, many of which are rural and developing nations, causing about 50100 million new dengue infections occurring each year . The dengue virus is a global pandemic threat which has increased incidence of 30-fold in the past five decades, with multiple new outbreaks . Although the reported number of patients having intracranial hemorrhage (ich) caused by dengue virus is rare and only 20 cases excluding those in our series have been reported so far, it is likely that its incidence is under - reported or under - diagnosed with the current global disease burden . There were 5866 reported dengue cases and 17 dengue - related deaths in penang, malaysia, from january 2014 till june 2015 . The dengue virus, a member of the flavivirus group in the family flaviviridae, is a single - stranded enveloped rna virus, of which there are four known serotypes, namely denv 14 . Dengue infection understanding has improved tremendously, but still no cure has been devised and the best strategy so far to curb the disease is through prevention and control of its mosquito vectors . Dengue vaccines have been created but most are still undergoing the clinical development stage, and only mexico and philippines have approved its use so far . With better understanding of the disease, who has proposed new classification schemes . The term expanded dengue syndrome was introduced to include unusual presentations of dengue fever with severe organ involvement including severe neurologic complications . The incidence of dengue encephalopathy ranges from 0.5% to 6.2% . Out of the 5400 patients with dengue fever reported by cam et al . The mechanism of which patients with dengue infection may be more prone to ich is complex, but the main contributors are coagulopathy, platelet dysfunction, thrombocytopenia, and vasculopathy . Unfortunately, dengue fever with ich has high morbidity and fatality, but little is known about it . We collected data of all patients with ich diagnosed via a plain computed tomography (ct) of the brain with positive dengue virus type 1 nonstructural protein (ns1) antigen test or positive dengue serology igm with thrombocytopenia from january 2014 till june 2015 at our center . A total of nine patients (patient a to patient i) were analyzed at our center . The data included were basic demographic data, existing comorbid, presenting symptoms, other dengue - related complications, the day ich was diagnosed in relation to onset of fever, glasgow coma scale (gcs), and pupils size and light response on presentation, platelet counts during diagnosis of ich, lowest platelet counts recorded, international normalized ratio (inr), activated partial thromboplastin time (aptt), dengue igm and igg seropositivity, type of ich, volume of ich, presence of midline shift and patency of basal cisterns, surgical intervention, and outcome . The volumes of intracerebral hematomas were calculated using the well - accepted abc/2 ellipsoid method . In cases where there is a subdural hemorrhage, the maximum thickness of the hemorrhage was measured . We defined an intracranial lesion needing surgery as: (1) any intracranial lesion causing a midline shift of 5 mm or more, and/or, (2) effacement of basal cisterns on ct brain . There are three methods of laboratory detection in our center, namely, panbio dengue igm capture elisa (sensitivity of 94.7% for primary infections, 55.7% for secondary infections and specificity of 100%), diaxis dengue ns1 antigen test (sensitivity of 97.9% and specificity of 99%), and acco dengue igg / igm - ns1 combo rapid detection kit (igg / igm: sensitivity 90% and specificity 100%, ns1: sensitivity 96% and specificity 100%). For our literature review, we performed a pubmed and google scholar search using the keywords dengue, intracranial hemorrhage, and intracranial bleed without specifying a time interval . Reports where diagnosis of dengue infection is questionable or very limited information of the case is explained were excluded from the study . Fever and vomiting were seen in all patients, and surprisingly, only 55% of them had a history of headache at presentation . This of course may be due to the low gcs of patients where history from family members may not be accurate . The mean age was 59.3 years (range 4386 years old) and seven of the patients were males . The day of detection of ich from fever onset ranged from 4 to 22 days . The ich of patient i was most likely detected late as the ct of the brain was done only after the patient had poor gcs recovery when sedation was stopped . Four of the patients had other bleeding tendencies and three of the patients were in clinical shock at the time of diagnosis of ich . The mean platelet count, inr, and aptt were 31 10/l (range 280 10/l), 1.2 (range 0.92.3), and 37.3 (range 27.7 more than 180), respectively . The lowest platelet count was the platelet level at diagnosis for three of the patients but was even lower for six of the patients . Seven patients had raised alanine transaminase (alt) levels (range 30784 u / l). Out of the nine patients, eight had positive ns1 test, two had positive igm test, and three had positive igg test . It is interesting that three of the patients with positive igg had ich requiring surgery and all of them died whereas two patients with negative igg did not have ich requiring surgery and survived . Four patients had cerebral convexity subdural hemorrhage, two patients had intracerebral hemorrhage, two patients had subarachnoid hemorrhage, and two had tentorium cerebelli subdural hemorrhage . Presenting symptoms of the patients characteristics of patients from our center all patients with intracranial lesions requiring surgery had gcs of eight or less and died . The other 4 patients had gcs of 14 or 15 and was discharged from the hospital, albeit one patient with a glasgow outcome scale (gos) of 3 . The other three patients were poor surgical candidates in terms of hemodynamic instability or persistent low platelet counts below 100 10/l despite multiple units of platelet transfusions . Patient b's platelet levels remained low with the highest platelet level of 46 10/l during admission and the patient required 4 types of inotropic support for shock [figure 1]. Patient f underwent cardiopulmonary resuscitation on presentation for cardiac arrest and required 2 types of inotropes . Patient g required 3 inotropes and the highest recorded platelet count was 68 10/l [figure 2]. Patient a received platelet and fresh frozen plasma (ffp) transfusion till platelet level reached 101 10/l, inr 12, and aptt 33.8 . The patient then underwent a right decompressive craniectomy, evacuation of subdural hemorrhage, and external ventricular drainage catheter insertion for intracranial pressure monitoring 8 h after admission [figure 3]. Patient c also received multiple units of platelet transfusion till platelet levels reached 109 10/l . The patient then underwent a parietal craniectomy 8 h after admission, but excessive bleeding complicated the procedure and the attempt to evacuate the entire clot was abandoned [figure 4]. Both patients who underwent surgery were nursed in the intensive care unit with cerebral resuscitation care . Computed tomography of the brain of patient b computed tomography of the brain of patient g computed tomography of the brain of patient a computed tomography of the brain of patient c overall, fever, reduced or altered consciousness, vomiting, and headache are the top complaints . Platelet counts on diagnosis ranged from 15 to 100 10/l . As in our series, it is seen again that patients with lower platelet levels did not necessarily have worse outcomes and that higher platelet levels did not seem to protect patients from fatal ich . Dengue igg was detected in two patients where one died and another had outcome of gos of 3 or 4 . Intraparenchymal hemorrhage was seen in 13 patients, pontine hemorrhage in two patients, intraventricular hemorrhage in two patients, medullary hemorrhage in one patient, cerebellar hemorrhage in one patient, and subdural hemorrhage in four patients . All the case reports did not state the volume or thickness of the bleeds, or whether the basal cisterns were effaced or patent . Midline shift was detected in six patients, but the extent of the shift was not specified to determine if surgery was required . It is highly possible these six patients required surgery by reviewing the pictures that were included in the reports . Out of 20 patients, 11 died, six had gos of 24, and three had gos of 5 [table 3]. There are no specific symptoms that clearly differentiate dengue fever from other common febrile infectious diseases such as chikungunya, leptospirosis, malaria, and influenza . While chikungunya infection may be distinguished from dengue fever, as chikungunya may cause significant postillness arthralgia and potentially debilitating polyarthralgia, objective assessment lies with antibody detection . Initial suspicion of dengue fever usually rests upon the finding of leukopenia and thrombocytopenia from a full blood count . The most common tests used routinely are dengue igm, igg, and ns1, but only ns1 seems useful in making an early and accurate diagnosis . Each of these tests has its own disadvantages and cannot be used in isolation for every case . In primary infection, igm antibodies are detectable in 50% of patients by days 35, increasing to 80% by day 5, 99% by day 10, and peak about 2 weeks of illness before declining to undetectable levels over 23 months . Igg antibodies are generally detectable at low titers at the end of the 1 week of illness and increasing slowly thereafter . During a secondary dengue infection, iggs are the main antibodies and are detectable at high levels even in the acute phase . Early convalescent stage, igm levels are significantly lower in secondary infections than in primary ones and may be undetectable . In secondary dengue infections, igm was only detected in 78% of patients after day 7 in one study . If dengue igm was the only test performed, it is reported that 28% of secondary dengue infections would be missed . This drawback limits the usefulness of igm detection for diagnosing dengue infection in the acute phase of the illness when most patients present with complications such as ich . Unfortunately, it is found that its usefulness declines on day 45 of illness but there are studies which show that ns1 antigen may be still be detected until the 14 day of illness . A primary dengue infection invokes the immune system to protect the patient from a secondary infection from the same serotype . However, a phenomenon known as antibody - dependent enhancement of dengue virus infection may actually cause a more severe form of dengue infection . It is well accepted that patients with secondary dengue infections have a higher risk of developing more severe forms of dengue infection . Multiple methods such as using the igm / igg antibody ratios have thus been created to distinguish primary from secondary infections more accurately during the acute phase . In our series, detection of igg early in the course of the disease with a positive ns1 antigen test and negative igm would most likely mean that the patient has a secondary infection . It is unfortunate that we do not have the titers of igm and igg to confirm this observation . In this series, all the patients had a positive ns1 test except for one, which could be due to sampling time after day 5 of illness . It is highly likely that the three patients with positive igg had secondary dengue infection and thus were predisposed to have a more severe ich . Even though sound clinical judgment would still be the best guidance for now, patients with a suspected secondary dengue infection should be monitored more closely and thresholds for diagnostic ct of the brains lowered when suspicion of ich is present . Whether surgery or optimal medical management should be the treatment of choice remains as the age - old question of all ich . Dengue infections causing ich are inherently harder to manage in that the causative factors such as vasculopathy and platelet dysfunction are usually still present and irreversible while surgery is undertaken . Some measures of correcting platelet deficits and coagulation derangements with massive transfusions may be carried out but risks delay of surgery . When the availability of neurosurgical centers is limited and transportation of a critically ill patient entails long - distance hazardous travel, the question of cost to benefit ratio becomes an issue . Considering the fact that the number of reported patients suffering ich due to dengue infection is low, it is too early to advocate if these patients should be treated like any other spontaneous ich . The latest guidelines from the american stroke association recommends that supratentorial hematoma evacuation in deteriorating patients might be a life - saving surgery and that decompressive craniectomy may reduce mortality when there are large hematomas with significant midline shift or elevated intracranial pressure refractory to medical management . Some authors have illustrated that surgical treatment may be of benefit compared to medical management, but it is too premature to conclude with these limited data . The debate remains on whether surgery should be done for these patients immediately with only platelet cover despite the low platelet levels or should adequate platelet levels as recommended by the guidelines be achieved first . The american stroke association advocates that early hematoma evacuation compared to hematoma evacuation when patient deteriorates does not show a clear benefit at the moment . There is conflicting evidence from the available studies such as the stich ii trial that showed no statistical significance in outcome between early surgery compared with initial medical management with delayed surgery if the patient deteriorates and the meta - analysis by another group which showed an improved outcome if surgery was performed within 8 h of hemorrhage . It would be difficult to determine if these recommendations could apply to ich in patients with dengue infection . Since thrombocytopenia and coagulopathy are usually severe in patients with dengue - related ich, neurosurgeons and physicians alike would have difficulty in determining the target levels and amount of blood product transfusion before surgery is undertaken . When ich is present in patients with dengue infection, there is no study as to whether the large amounts of platelets and ffp given would change the outcome . As bleeding in dengue fever is multifactorial and still poorly understood, contributing factors such as vasculopathy and platelet dysfunction may be a cause of the hemorrhage, and the efforts for correcting the deficits may prove to be a waste of precious blood product resources and prolong the time from diagnosis to surgery . Moreover, it is shown that duration of survival of transfused platelets ranges from only a few hours to a day in cases with shock and that would mean that great amounts of blood products would be needed to keep the levels up . A recent randomized control trial on platelet transfusion in dengue fever showed that 53.6% of their patients did not respond to platelet transfusion and these patients were those with a lower baseline platelet count . Unfortunately, current guidelines for platelet transfusion thresholds in thrombocytopenic surgical patients are mainly based on expert opinion and clinical experience . The current recommendation for platelet levels for neurosurgical procedures is 100 10/l . In dengue patients, it is difficult to keep the perioperative platelet levels as recommended and it is shown that a perioperative platelet count below 100 10/l in patients who failed to respond to platelet transfusions had a higher risk of postoperative intracranial hematoma formation . One group suggests prophylactic transfusion of platelets when levels are under a certain threshold, of which the threshold values still vary at the moment . The other group prefers transfusion only when significant hemorrhage occurs . One study showed that a platelet count of <50 10/l and hematocrit of more than 50 are associated with bleeding manifestations . One more study of 225 dengue patients suggested that bleeding occurred more often in patients with platelet counts below 20 10/l and recommended prophylactic transfusion of platelets . In contrast, there was no correlation between bleeding tendencies and platelet counts in dengue patients in other studies . For dengue fever without bleeding, one study showed that prophylactic transfusions of platelet are not recommended as it does not change or reduce the bleeding outcome in patients with dengue hemorrhagic fever and may increase risk of pulmonary edema . Platelet transfusion in adult patients with dengue in the absence of bleeding when the platelet count was <20 10/l did not reduce bleeding or hasten platelet recovery, but there was potential harm by hampering recovery of platelet count to> 50 10/l and prolonging of hospitalization . In a retrospective analysis of 256 dengue - infected patients without prior bleeding and platelet counts <20 10/l, 188 were given platelet transfusion; it was found that subsequent bleeding, platelet increment, and platelet recovery were similar between the two groups . A review article in 2011 strongly concluded that prophylactic platelet transfusion is associated with hazards and wastage, which is not justified by any potential hematological benefit and therefore, should not be adopted as a routine clinical practice . Then again, a salient point that must be considered in patients with ich is the significant morbidity that could be worse than mortality itself at times . The question arises as to whether prophylactic transfusion should be given to patients with very low platelet levels just to avoid devastating consequences of ich even though it is a rare event . The range of lowest recorded platelet levels in our series was 276 10/l and five of the patients had coagulopathy . Our series shows that severe ich may still occur even with a platelet count of 66 (patient f) and a platelet count of 3 does not cause a more severe ich (patient d). It has long been established that there is abnormal hemostasis in patients with dengue fever with platelet dysfunction, coagulopathy, vasculopathy, and immune- or virus - related destruction of platelets . It may be possible that our approach of determining the absolute platelet counts and inr might not be entirely enough as we might be missing out on the actual ability of platelet function and coagulation in vivo . There are laboratory techniques to assess platelet function and blood coagulation, namely, thromboelastography, thromboelastometry, and platelet aggregometry . Although not widely available yet in developing countries, these tests might prove useful in determining a subset of patients that may benefit from prophylactic transfusions to prevent life - threatening hemorrhages . To our knowledge, there has been no trial in the use of thromboelastography, thromboelastometry, or platelet aggregometry in predicting bleeding tendencies in patients with dengue fever . It is important to consider that these symptoms may also be the first symptom of an ongoing ich . Unequivocal symptoms such as seizures, weakness, numbness, altered consciousness, and seizures would warrant ct of the brains, but these presentations may only be present when the ich has expanded beyond treatment . Multiple methods of noninvasive monitoring and detection of raised intracranial pressure have been developed, but most are expensive and require much training . In view of this, a simple noninvasive method to detect increased intracranial pressure is needed . In the setting where dengue infections are highest in developing nations and ct of the brains are not widely available, we can only recommend that fundoscopy to detect papilledema should be a routine assessment for dengue - infected patients with suspected ich . It would seem from our series that prevention of ich in patients with dengue should be the main aim as all patients who had a severe bleed warranting surgery deteriorated very quickly and both surgical and medical managements failed to stop the inevitable . If specific risk factors could be identified, perhaps, prophylactic transfusion and closer monitoring could bring about earlier detection or prevent ich altogether . In terms of risk of bleeding, a study done in malaysia found that prolonged duration of shock and low - normal hematocrit at the time of shock suggesting blood loss in addition to capillary leakage were the strongest predictors of hemorrhage . Only patient g in our series who had upper gastrointestinal bleeding had a low hematocrit and shock . It is possible that patients with only ich as the only bleeding tendency would not show a drop in hematocrit associated with shock as the volume of intracranial blood loss alone would not be sufficient enough to reduce intravascular volume . Other studies found that there was significant association between bleeding tendencies and older patients, secondary dengue infection, high baseline hematocrit levels, low platelet levels, prolonged aptt, female gender, vomiting, high absolute lymphocyte count, and high aspartate aminotransferase level . Even though these may be generalized to include ich although not specific to ich, sahu et al . Outlined the possible predictors for the central nervous system involvement that includes higher mean body temperature, elevated hematocrit, low platelet count, and liver dysfunction . Three of our patients with positive igg had ich requiring surgery and all of them died, whereas two patients with negative igg did not have ich requiring surgery and survived; thus, it is possible that secondary dengue infection is also associated with a tendency to develop severe ich . The mechanisms of which patients with dengue infection may be more prone to ich is complex and are postulated to be caused by coagulopathy, platelet dysfunction, thrombocytopenia, and vasculopathy . To date, there is no proven explanation for ich occurring in only a rare minority of patients with severe dengue . The blood brain barrier and blood cerebrospinal fluid (csf) barrier have been shown to breakdown during dengue virus infection . Dengue igm, igg, and ns1 ag were also detected in csf of patients with dengue . It is plausible that ich follows the same general mechanisms as bleeding elsewhere in the body and focal cerebral vasculopathy is worsened by the presence of igg and ns1 ag immunopathological - related mechanisms . In general, immunopathological events involving t cells, cytokines such as tumor necrosis factor - alpha (tnf-), and cross - reactive antibody that enhances dengue virus replication leading to vasculopathy are still incompletely understood . Thrombocytopenia may be caused by disseminated intravascular coagulation, bone marrow suppression, increased apoptosis, and destruction by the complement system . Interestingly, ns1 antigens are found to induce production of plasminogen cross - reactive antibodies and thus enhance plasminogen activation leading to increased plasmin and promoting fibrinolysis . Platelet dysfunction and destruction have been attributed to the dengue virus and early studies reveal that dengue virus viremia seems associated with the megakaryocytic lineage and replication in platelets . Much have been learned and platelets have been increasingly considered to play a key role in inflammation through inflammasome synthesis of interleukin-1 beta (il-1) which is a cytokine closely associated with endothelial dysfunction and coagulation disorders . Platelets activation by dengue virus through lectin receptor dendritic cell - specific icam-3-grabbing nonintegrin resulting in production of il-1 has been shown using mouse models, chen et al . Observed that high viral titer, macrophage infiltration, and tnf- are three important events that lead to hemorrhage . Chuang et al . Found that dengue virus infection induced macrophage migration inhibitory factor (mif) production may contribute to vascular hyperpermeability and hemostatic abnormality and thus blocking of mif may be an answer for prevention of dengue hemorrhage . There are no specific symptoms that clearly differentiate dengue fever from other common febrile infectious diseases such as chikungunya, leptospirosis, malaria, and influenza . While chikungunya infection may be distinguished from dengue fever, as chikungunya may cause significant postillness arthralgia and potentially debilitating polyarthralgia, objective assessment lies with antibody detection . Initial suspicion of dengue fever usually rests upon the finding of leukopenia and thrombocytopenia from a full blood count . The most common tests used routinely are dengue igm, igg, and ns1, but only ns1 seems useful in making an early and accurate diagnosis . Each of these tests has its own disadvantages and cannot be used in isolation for every case . In primary infection, igm antibodies are detectable in 50% of patients by days 35, increasing to 80% by day 5, 99% by day 10, and peak about 2 weeks of illness before declining to undetectable levels over 23 months . Igg antibodies are generally detectable at low titers at the end of the 1 week of illness and increasing slowly thereafter . During a secondary dengue infection, iggs are the main antibodies and are detectable at high levels even in the acute phase . Early convalescent stage, igm levels are significantly lower in secondary infections than in primary ones and may be undetectable . In secondary dengue infections, igm was only detected in 78% of patients after day 7 in one study . If dengue igm was the only test performed, it is reported that 28% of secondary dengue infections would be missed . This drawback limits the usefulness of igm detection for diagnosing dengue infection in the acute phase of the illness when most patients present with complications such as ich . Unfortunately, it is found that its usefulness declines on day 45 of illness but there are studies which show that ns1 antigen may be still be detected until the 14 day of illness . A primary dengue infection invokes the immune system to protect the patient from a secondary infection from the same serotype . However, a phenomenon known as antibody - dependent enhancement of dengue virus infection may actually cause a more severe form of dengue infection . It is well accepted that patients with secondary dengue infections have a higher risk of developing more severe forms of dengue infection . Multiple methods such as using the igm / igg antibody ratios have thus been created to distinguish primary from secondary infections more accurately during the acute phase . In our series, detection of igg early in the course of the disease with a positive ns1 antigen test and negative igm would most likely mean that the patient has a secondary infection . It is unfortunate that we do not have the titers of igm and igg to confirm this observation . In this series, all the patients had a positive ns1 test except for one, which could be due to sampling time after day 5 of illness . It is highly likely that the three patients with positive igg had secondary dengue infection and thus were predisposed to have a more severe ich . Even though sound clinical judgment would still be the best guidance for now, patients with a suspected secondary dengue infection should be monitored more closely and thresholds for diagnostic ct of the brains lowered when suspicion of ich is present . Whether surgery or optimal medical management should be the treatment of choice remains as the age - old question of all ich . Dengue infections causing ich are inherently harder to manage in that the causative factors such as vasculopathy and platelet dysfunction are usually still present and irreversible while surgery is undertaken . Some measures of correcting platelet deficits and coagulation derangements with massive transfusions may be carried out but risks delay of surgery . When the availability of neurosurgical centers is limited and transportation of a critically ill patient entails long - distance hazardous travel, the question of cost to benefit ratio becomes an issue . Considering the fact that the number of reported patients suffering ich due to dengue infection is low, it is too early to advocate if these patients should be treated like any other spontaneous ich . The latest guidelines from the american stroke association recommends that supratentorial hematoma evacuation in deteriorating patients might be a life - saving surgery and that decompressive craniectomy may reduce mortality when there are large hematomas with significant midline shift or elevated intracranial pressure refractory to medical management . Some authors have illustrated that surgical treatment may be of benefit compared to medical management, but it is too premature to conclude with these limited data . The other question that needs to be answered is the timing of surgery . The debate remains on whether surgery should be done for these patients immediately with only platelet cover despite the low platelet levels or should adequate platelet levels as recommended by the guidelines be achieved first . The american stroke association advocates that early hematoma evacuation compared to hematoma evacuation when patient deteriorates does not show a clear benefit at the moment . There is conflicting evidence from the available studies such as the stich ii trial that showed no statistical significance in outcome between early surgery compared with initial medical management with delayed surgery if the patient deteriorates and the meta - analysis by another group which showed an improved outcome if surgery was performed within 8 h of hemorrhage . It would be difficult to determine if these recommendations could apply to ich in patients with dengue infection . Since thrombocytopenia and coagulopathy are usually severe in patients with dengue - related ich, neurosurgeons and physicians alike would have difficulty in determining the target levels and amount of blood product transfusion before surgery is undertaken . When ich is present in patients with dengue infection, there is no study as to whether the large amounts of platelets and ffp given would change the outcome . As bleeding in dengue fever is multifactorial and still poorly understood, contributing factors such as vasculopathy and platelet dysfunction may be a cause of the hemorrhage, and the efforts for correcting the deficits may prove to be a waste of precious blood product resources and prolong the time from diagnosis to surgery . Moreover, it is shown that duration of survival of transfused platelets ranges from only a few hours to a day in cases with shock and that would mean that great amounts of blood products would be needed to keep the levels up . A recent randomized control trial on platelet transfusion in dengue fever showed that 53.6% of their patients did not respond to platelet transfusion and these patients were those with a lower baseline platelet count . Unfortunately, current guidelines for platelet transfusion thresholds in thrombocytopenic surgical patients are mainly based on expert opinion and clinical experience . The current recommendation for platelet levels for neurosurgical procedures is 100 10/l . In dengue patients, it is difficult to keep the perioperative platelet levels as recommended and it is shown that a perioperative platelet count below 100 10/l in patients who failed to respond to platelet transfusions had a higher risk of postoperative intracranial hematoma formation . One group suggests prophylactic transfusion of platelets when levels are under a certain threshold, of which the threshold values still vary at the moment . One study showed that a platelet count of <50 10/l and hematocrit of more than 50 are associated with bleeding manifestations . One more study of 225 dengue patients suggested that bleeding occurred more often in patients with platelet counts below 20 10/l and recommended prophylactic transfusion of platelets . In contrast, there was no correlation between bleeding tendencies and platelet counts in dengue patients in other studies . For dengue fever without bleeding, one study showed that prophylactic transfusions of platelet are not recommended as it does not change or reduce the bleeding outcome in patients with dengue hemorrhagic fever and may increase risk of pulmonary edema . Platelet transfusion in adult patients with dengue in the absence of bleeding when the platelet count was <20 10/l did not reduce bleeding or hasten platelet recovery, but there was potential harm by hampering recovery of platelet count to> 50 10/l and prolonging of hospitalization . In a retrospective analysis of 256 dengue - infected patients without prior bleeding and platelet counts <20 10/l, 188 were given platelet transfusion; it was found that subsequent bleeding, platelet increment, and platelet recovery were similar between the two groups . A review article in 2011 strongly concluded that prophylactic platelet transfusion is associated with hazards and wastage, which is not justified by any potential hematological benefit and therefore, should not be adopted as a routine clinical practice . Then again, a salient point that must be considered in patients with ich is the significant morbidity that could be worse than mortality itself at times . The question arises as to whether prophylactic transfusion should be given to patients with very low platelet levels just to avoid devastating consequences of ich even though it is a rare event . The range of lowest recorded platelet levels in our series was 276 10/l and five of the patients had coagulopathy . Our series shows that severe ich may still occur even with a platelet count of 66 (patient f) and a platelet count of 3 does not cause a more severe ich (patient d). It has long been established that there is abnormal hemostasis in patients with dengue fever with platelet dysfunction, coagulopathy, vasculopathy, and immune- or virus - related destruction of platelets . It may be possible that our approach of determining the absolute platelet counts and inr might not be entirely enough as we might be missing out on the actual ability of platelet function and coagulation in vivo . There are laboratory techniques to assess platelet function and blood coagulation, namely, thromboelastography, thromboelastometry, and platelet aggregometry . Although not widely available yet in developing countries, these tests might prove useful in determining a subset of patients that may benefit from prophylactic transfusions to prevent life - threatening hemorrhages . To our knowledge, there has been no trial in the use of thromboelastography, thromboelastometry, or platelet aggregometry in predicting bleeding tendencies in patients with dengue fever . It is important to consider that these symptoms may also be the first symptom of an ongoing ich . Unequivocal symptoms such as seizures, weakness, numbness, altered consciousness, and seizures would warrant ct of the brains, but these presentations may only be present when the ich has expanded beyond treatment . Multiple methods of noninvasive monitoring and detection of raised intracranial pressure have been developed, but most are expensive and require much training . In view of this, a simple noninvasive method to detect increased intracranial pressure is needed . In the setting where dengue infections are highest in developing nations and ct of the brains are not widely available, we can only recommend that fundoscopy to detect papilledema should be a routine assessment for dengue - infected patients with suspected ich . It would seem from our series that prevention of ich in patients with dengue should be the main aim as all patients who had a severe bleed warranting surgery deteriorated very quickly and both surgical and medical managements failed to stop the inevitable . If specific risk factors could be identified, perhaps, prophylactic transfusion and closer monitoring could bring about earlier detection or prevent ich altogether . In terms of risk of bleeding, a study done in malaysia found that prolonged duration of shock and low - normal hematocrit at the time of shock suggesting blood loss in addition to capillary leakage were the strongest predictors of hemorrhage . Only patient g in our series who had upper gastrointestinal bleeding had a low hematocrit and shock . It is possible that patients with only ich as the only bleeding tendency would not show a drop in hematocrit associated with shock as the volume of intracranial blood loss alone would not be sufficient enough to reduce intravascular volume . Other studies found that there was significant association between bleeding tendencies and older patients, secondary dengue infection, high baseline hematocrit levels, low platelet levels, prolonged aptt, female gender, vomiting, high absolute lymphocyte count, and high aspartate aminotransferase level . Even though these may be generalized to include ich, it would be ideal if independent risk factors for ich could be determined . Although not specific to ich, sahu et al . Outlined the possible predictors for the central nervous system involvement that includes higher mean body temperature, elevated hematocrit, low platelet count, and liver dysfunction . Three of our patients with positive igg had ich requiring surgery and all of them died, whereas two patients with negative igg did not have ich requiring surgery and survived; thus, it is possible that secondary dengue infection is also associated with a tendency to develop severe ich . The mechanisms of which patients with dengue infection may be more prone to ich is complex and are postulated to be caused by coagulopathy, platelet dysfunction, thrombocytopenia, and vasculopathy . To date, there is no proven explanation for ich occurring in only a rare minority of patients with severe dengue . The blood brain barrier and blood cerebrospinal fluid (csf) barrier have been shown to breakdown during dengue virus infection . Dengue igm, igg, and ns1 ag were also detected in csf of patients with dengue . It is plausible that ich follows the same general mechanisms as bleeding elsewhere in the body and focal cerebral vasculopathy is worsened by the presence of igg and ns1 ag immunopathological - related mechanisms . In general, immunopathological events involving t cells, cytokines such as tumor necrosis factor - alpha (tnf-), and cross - reactive antibody that enhances dengue virus replication leading to vasculopathy are still incompletely understood . Thrombocytopenia may be caused by disseminated intravascular coagulation, bone marrow suppression, increased apoptosis, and destruction by the complement system . Interestingly, ns1 antigens are found to induce production of plasminogen cross - reactive antibodies and thus enhance plasminogen activation leading to increased plasmin and promoting fibrinolysis . Platelet dysfunction and destruction have been attributed to the dengue virus and early studies reveal that dengue virus viremia seems associated with the megakaryocytic lineage and replication in platelets . Much have been learned and platelets have been increasingly considered to play a key role in inflammation through inflammasome synthesis of interleukin-1 beta (il-1) which is a cytokine closely associated with endothelial dysfunction and coagulation disorders . Platelets activation by dengue virus through lectin receptor dendritic cell - specific icam-3-grabbing nonintegrin resulting in production of il-1 has been shown using mouse models, chen et al . Observed that high viral titer, macrophage infiltration, and tnf- are three important events that lead to hemorrhage . Chuang et al . Found that dengue virus infection induced macrophage migration inhibitory factor (mif) production may contribute to vascular hyperpermeability and hemostatic abnormality and thus blocking of mif may be an answer for prevention of dengue hemorrhage . This article highlights the deadly effect of dengue hemorrhagic encephalopathy and the limited weapons and knowledge we have against it . More research is needed in this field to determine possible predictors of ich such as dengue igg in dengue infection.
Percutaneous biopsy is less invasive than surgery, can be performed quickly, does not deform the breast, causes minimal scarring, complications (haematoma and infection) are infrequently found (less than one case in 1,000), fewer surgeries are needed for patients who undergo percutaneous biopsies and therefore the cost of diagnosis is lower [15]. There are two main objectives of percutaneous biopsy techniques: first, achieving the maximum degree of accuracy and second, offering as much information as possible about the tumour (type, grade, invasion, hormonal receptors, her-2 neu, etc . ). To achieve these objectives, the percutaneous biopsy devices have evolved, from fine - needle aspiration cytology towards core - needle biopsy (cnb) and later vacuum - assisted biopsy (vab). Nowadays, ultrasound - guided core needle breast biopsy has become the first choice for performing a percutaneous biopsy for most lesions seen on ultrasound [1, 69]. Virtually any breast lesion that is clearly seen on ultrasound can be sampled under ultrasound guidance . Many surgical biopsies that had to be carried out in the past, because of suspicious radiological findings, are nowadays unnecessary due to the extensive use of ultrasound ncb . In addition, surgical specimens removed after a previously proven malignant result are usually more adequate for the tumour size . Thus, the number of surgical procedures has drastically decreased both for benign and malignant lesions, thanks to the extensive use of ultrasound cnb and the other methods of percutaneous biopsy . The technique was first described by parker and co - workers in the early 1990s, and nowadays ultrasound cnb has become the first choice for performing most breast biopsies [2, 4, 5, 9]. The numerous advantages of ultrasound guidance over stereotactic and mr guidance have been key in explaining the widespread use of this technique . Noteworthy among these advantages are: ultrasound involves non - ionising radiation.full control of the needle position in real time . Neither stereotactic guidance nor mr guidance offers this advantage.ultrasound equipment is cheaper and more readily available than stereotactic or mr units.accessibility of difficult places, such as the axilla or near the nipple . These are limitations for stereotactic or mr guidance.multiple lesions (unilateral or bilateral) can be safely biopsied in one session, usually more quickly than with other imaging techniques.the breast is not compressed.excellent comfort for patients and radiologists, although stereotactic prone tables are also comfortable for both.local anaesthesia and haematoma do not hide the lesion (non - calcified masses can be obscured with use of stereotactic equipment). However, bubbles injected with the local anaesthesia can obscure the lesion.it is a cost - effective technique: liberman et al . Found that ultrasound cnb yielded a 56% decrease in the cost of diagnosis . For masses amenable to either stereotactic or ultrasound guidance, cost savings are greater if the biopsy is performed under ultrasound guidance . According to schueller et al . Ultrasound involves non - ionising radiation . Full control of the needle position in real time . Neither stereotactic guidance nor mr guidance offers this advantage . Multiple lesions (unilateral or bilateral) can be safely biopsied in one session, usually more quickly than with other imaging techniques . Excellent comfort for patients and radiologists, although stereotactic prone tables are also comfortable for both . Local anaesthesia and haematoma do not hide the lesion (non - calcified masses can be obscured with use of stereotactic equipment). . Found that ultrasound cnb yielded a 56% decrease in the cost of diagnosis . For masses amenable to either stereotactic or ultrasound guidance, cost savings are greater if the biopsy is performed under ultrasound guidance . According to schueller et al . The main disadvantage of ultrasound cnb is the limitation of performing a biopsy for lesions not seen on ultrasound . Most clustered microcalcifications, especially if they are not inside a mass, cannot be identified on ultrasound . However, high - resolution transducers can demonstrate some clustered microcalcifications even in the absence of a mass . Although most ultrasound cnb procedures are easy to perform, in some special situations (deeply located lesions, patients with implants, axillary lesions, etc .) A high level of experience is needed to get reliable results . All lesions classified as bi - rads 4 and 5, clearly visible on ultrasound, are amenable to ultrasound cnb . This technique can also be used for some bi - rads 3 lesions under certain circumstances: genetic or family risk, medical or social difficulties for follow - up, pregnancy, extreme anxiety and others, including the patient s decision . Ultrasound cnb can be difficult in patients with severe psychiatric disorders, which makes them impossible to collaborate on, and is contraindicated in some cases of severe blood dyscrasia . However, there are no statistically significant differences in haematoma formation between patients taking anticoagulant therapy daily and non - treated patients . Thus, it is not necessary to stop that therapy to perform an ultrasound cnb . Expert radiologists can perform biopsies of very deep lesions located close to the pectoral muscle in large breasts . Obtaining a biopsy in some patients with silicone implants can be also contraindicated . Although surgeons have been using cnb guided by palpation for a long time, the accuracy is increased with ultrasound guidance . As well as diagnostic objectives, ultrasound guidance allows us to perform other interesting therapeutic procedures such as evacuation of liquid or semi - solid collections and placement of markers or coils for neoadjuvant chemotherapy . More recently, ultrasound guidance has been useful for tumour ablation using radiofrequency, cryoablation, laser therapy or focused ultrasound [1416]. There is a wide spectrum of core - needle devices that can be used under ultrasound guidance (fig . 1). The thickness of the needles can be selected, varying from 18- to 8-gauge; however, 14-gauge conventional tru - cut devices have been the most commonly used . Nowadays there is a trend towards using 10- to 11-gauge vacuum - assisted devices . Fig . 1a small sample of the great variety of biopsy devices that can be used under ultrasound guidance a small sample of the great variety of biopsy devices that can be used under ultrasound guidance it is very important to correlate mammography, ultrasound and mri findings, in order to carry out the puncture using the most suitable method of guidance . Sometimes it is necessary to perform several biopsies in the same patient using different techniques of imaging guidance (i.e. Microcalcifications under stereotactic guidance and a mass, only visible on ultrasound, under ultrasound guidance). Metallic markers can be placed superficially on the skin close to the lesion or inside the lesion to correlate ultrasound and mammography findings . The use of high - frequency (10- to 12-mhz) probes, adjustments in the dynamic range and postprocessing grey scales, as well as correct focus, are important to improve the visibility of breast lesions . The patients should have a complete, thorough level of information about the technique, indications, contraindications, complications and alternative possibilities; therefore, obtaining informed consent is mandatory . Local anaesthesia must be injected superficially and also as deeply as necessary, under sterile conditions . This anaesthesia does not mask the lesion and sometimes can help us to move it to another, more accessible place, deeper or more superficial . However, bubbles mixed with the anaesthesia can mask the lesion; thus, they must be avoided . After localising the lesion with ultrasound, the procedure is performed in an outpatient setting, using the free - hand technique: one hand holds the probe and the other hand holds the needle (fig . 2). One of the main advantages of ultrasound - guided cnb is the full control of the needle position in real time, allowing for corrections in the needle direction . A local anaesthesia is injected, b a small incision is required and c the biopsy device is guided by ultrasound these pictures illustrate how the ultrasound cnb procedure is performed . A local anaesthesia is injected, b a small incision is required and c the biopsy device is guided by ultrasound as a general rule, the shortest route from the skin to the lesion should be used . A vertical approach would be the best, but it is not possible under ultrasound guidance . However, an oblique approach, as parallel to the chest wall as possible, should be used (fig . This approach also enables the best visualisation of the needle, because even large - gauge needles are difficult to visualise if a steep angle is used because of less reflective echoes . However, when the needle is parallel to the probe, the number of needle - generated reflected echoes that are perpendicular to the ultrasound beam is maximised, so the needle can be identified . This horizontal approach can be used to perform a biopsy for cutaneous lesions (fig . Fig . 3the approach to the lesion should be as parallel to the chest wall as possible to avoid pneumothorax . Moreover the transducer should be orientated parallel to the needle in order to facilitate the needle visualisationfig . Note the horizontal approach needed in this case the approach to the lesion should be as parallel to the chest wall as possible to avoid pneumothorax . Moreover the transducer should be orientated parallel to the needle in order to facilitate the needle visualisation an ultrasound cnb is performed in this cutaneous metastasis (arrows). Note the horizontal approach needed in this case it is useful to move the patient to lateral decubitus positions, especially in the case of deeply located lesions or peripheral masses . If possible, an approach through fat is preferable, because the puncture is easier (the fat is soft and the needle can be easily guided; fig . 5a puncture through a fat lobule is easier than through fibrous dense tissue a puncture through a fat lobule is easier than through fibrous dense tissue it is important to insert the tip of the needle inside the mass, because there is a dead space behind the tip . If the tip is not inserted, then part of the specimen will not belong to the lesion but to the perilesional tissue, and there is a risk of lancing the lesion, especially if it is very hard . Pre - fire and post - fire images are important to ensure the correct position of the needle and to rule out the occurrence of complications (fig . 6one of the main advantages of ultrasound cnb is the full control of the needle direction in real time . Pre - fire and post - fire images are useful to ensure the correct position of the needle one of the main advantages of ultrasound cnb is the full control of the needle direction in real time . Pre - fire and post - fire images are useful to ensure the correct position of the needle in the case of very dense breasts, the punction can be difficult . There are some tricks that can be used: coaxial technique: once the coaxial needle is inserted in the lesion, the inner trocar can be removed and replaced by the biopsy needle .a 16-gauge needle instead of a 14-gauge one . The punction is easier and the strength of the shot is greater.stronger devices, such as vacuum - assisted devices.devices with diamond - shaped needle tips, because they transverse the fibrous tissue better than conventional needles (fig . 7diamond - shaped needle tips are better than conventional ones to traverse the fibrous tissue coaxial technique: once the coaxial needle is inserted in the lesion, the inner trocar can be removed and replaced by the biopsy needle . A 16-gauge needle instead of a 14-gauge one . Stronger devices, such as vacuum - assisted devices . Devices with diamond - shaped needle tips, because they transverse the fibrous tissue better than conventional needles (fig . Diamond - shaped needle tips are better than conventional ones to traverse the fibrous tissue in the case of deeply located lesions there is a need to use a parallel approach to the chest wall, so that this structure is not penetrated and pneumothorax is avoided . Other tricks that can be used include: the needle can be used as a lever: the entry of the needle has to be located about 2 cm from the edge of the transducer and the lesion is manually lifted away from the chest wall .local anaesthesia can be used to move the lesion to a more convenient place, especially if it is injected deep to the lesion (fig . 8the injection of local anaesthesia below the lesion can move it to a more superficial position the needle can be used as a lever: the entry of the needle has to be located about 2 cm from the edge of the transducer and the lesion is manually lifted away from the chest wall . Local anaesthesia can be used to move the lesion to a more convenient place, especially if it is injected deep to the lesion (fig . . The injection of local anaesthesia below the lesion can move it to a more superficial position the macroscopic evaluation of the specimens is also important, because it can give additional information about their quality: colour, consistency and grade of immersion of the cylinders in formaldehyde can be useful criteria for knowing their suitability for diagnosis . Intact, white or brown samples that quickly sink are considered more representative and are consequently preferred to fragmented, floating yellow ones, normally containing only adipose tissue . This is the first index of correlation; of course it is not conclusive, but only orientative (fig . If the biopsied lesion is a cluster of microcalcifications, a specimen radiograph is mandatory to confirm the presence of microcalcifications (fig . 9white specimens that sink are usually more representative than floating yellow specimensfig . 10specimen radiograph showing microcalcifications . The radiograph was taken in a digital mammographic unit (inspiration, siemens, erlangen, germany) at 23 kv and 9 mas . The cylinders were collected into a plastic cassette white specimens that sink are usually more representative than floating yellow specimens specimen radiograph showing microcalcifications . The radiograph was taken in a digital mammographic unit (inspiration, siemens, erlangen, germany) at 23 kv and 9 mas . The cylinders were collected into a plastic cassette four specimens may be enough for a reliable diagnosis, according to fishman et al . . Vacuum - assisted biopsy devices under ultrasound guidance have been used as an alternative to conventional ultrasound cnb . When vab is performed, more specimens are removed . However, philpotts et al . Compared the two techniques (181 cnb procedures vs 100 vab procedures) and found no significant differences in false - negative results, underestimation and complications . The reason for such a result is probably the homogeneity of most ultrasound - detected lesions (a small specimen is representative of the whole lesion) and the low frequency of borderline results (such as atypical ductal hyperplasia and others, which are usually associated with microcalcifications and are not seen on ultrasound). However, the number of samples should be greater for those lesions with complex radiological features . In these cases, for example parenchymal distortions or asymmetric densities, more samples and/or thicker needles are recommended . The first cylinders are the most important ones, because later the suspicious lesion can be masked by variable degrees of bleeding . After the initial results by parker and co - workers, with a 100% correlation with surgical results for 49 excised masses and no additional cancers in the remaining 132 cases, all the published series have shown excellent results (table 1) [710, 17, 2126]. Sensitivity of ultrasound - guided cnb is about 97.5%, which makes this technique a very good choice for performing a breast biopsy . In published series 10% of lesions required repeat biopsy, most of the cancers that were not initially diagnosed were biopsied again after new evaluation . Table 1results for breast ultrasound - guided cnb seriesauthorsyeartotal number of cancerscancers diagnosed (sensitivity)parker et al . Despite performing an optimised biopsy procedure, a false - negative result can occur . In the case of microcalcifications, however, the specimen radiograph does not give additional information on non - calcified lesions . Post - biopsy mammography usually shows no substantial alteration in the target lesion with conventional 14-gauge needles . Therefore, the radiological histological correlation is crucial to avoid false - negative results . Five situations of radiological histological correlation can occur: concordant malignancy: a lesion that is radiologically suspicious for malignancy (bi - rads category 4 or 5) is histologically diagnosed as malignant after core biopsy (b4 or b5). Adequate treatment should be performed.discordant malignancy: a radiologically benign lesion (bi - rads 2 or 3) is finally diagnosed as histologically malignant after core biopsy (b4 or b5). Adequate treatment should be performed.concordant benignity: the radiological findings are benign or low intermediate suspicious (bi - rads 2, 3, 4a, 4b), and histological features are benign (b1 or b2 categories). An adequate radiological pathological correlation should be established, and imaging follow - up should be offered to avoid delayed false - negative results . Exceptionally, some of these lesions can be surgically or percutaneously excised because of patient anxiety, patient decision or physician preference.discordant benignity: a radiologically malignant lesion (bi - rads category 4c or 5) is proved to be benign after core biopsy . In this case, both the imaging and the pathological findings should be reviewed again . It is imperative to find a diagnosis; therefore, a new percutaneous biopsy (including vacuum - assisted breast biopsy) or a surgical removal can be offered.borderline findings: atypical ductal hyperplasia, lobular neoplasm, radial scar, papillary lesion and phyllodes tumour, are classified as b3 pathological results and usually require the removal of the whole lesion . It has been demonstrated that the rates of underestimation decrease as the number and calibre of samples increase . In cases of atypical ductal hyperplasia or radial scar with atypia, a surgical biopsy is probably the best option because it is possible to find histological features of ductal carcinoma in situ or even invasive carcinoma in the surrounding tissue . Concordant malignancy: a lesion that is radiologically suspicious for malignancy (bi - rads category 4 or 5) is histologically diagnosed as malignant after core biopsy (b4 or b5). Discordant malignancy: a radiologically benign lesion (bi - rads 2 or 3) is finally diagnosed as histologically malignant after core biopsy (b4 or b5). Concordant benignity: the radiological findings are benign or low intermediate suspicious (bi - rads 2, 3, 4a, 4b), and histological features are benign (b1 or b2 categories). An adequate radiological pathological correlation should be established, and imaging follow - up should be offered to avoid delayed false - negative results . Exceptionally, some of these lesions can be surgically or percutaneously excised because of patient anxiety, patient decision or physician preference . Discordant benignity: a radiologically malignant lesion (bi - rads category 4c or 5) is proved to be benign after core biopsy . In this case it is imperative to find a diagnosis; therefore, a new percutaneous biopsy (including vacuum - assisted breast biopsy) or a surgical removal can be offered . Borderline findings: atypical ductal hyperplasia, lobular neoplasm, radial scar, papillary lesion and phyllodes tumour, are classified as b3 pathological results and usually require the removal of the whole lesion . It has been demonstrated that the rates of underestimation decrease as the number and calibre of samples increase . In cases of atypical ductal hyperplasia or radial scar with atypia, a surgical biopsy is probably the best option because it is possible to find histological features of ductal carcinoma in situ or even invasive carcinoma in the surrounding tissue . Both haematomas and infections are very rare, accounting for less than 1/1,000 biopsies, being similar to the complications of other percutaneous biopsy devices . The possibility of pneumothorax exists, but it is very rare using the free - hand technique and a horizontal approach . It was first described by harter et al . In 1992 . In 1999, diaz et al . Concluded that epithelial displacement was seen in up to 37% of all biopsies . Later, in 2002, chen et al . Compared the recurrences after percutaneous biopsies vs surgical biopsies in patients with breast - conserving therapy, and found no significant differences in recurrence rates . All bi - rads 4 and bi - rads 5 lesions, and some bi - rads 3 ones, should be percutaneously punctured, and in cases with benign results, surgery can be avoided if there is a good radiological pathological correlation and no borderline result is obtained . Ultrasound cnb is a well - known, safe and accurate technique that is currently considered the elective method, whereas stereotaxy and mri should be reserved for lesions that are not clearly seen on ultrasound . Complications are infrequent and not serious . For these reasons, we would encourage all breast radiologists to gain plenty of experience of this technique.

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.