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Primary or secondary neurondocrine (ne) tumours of the female genital tract are rare . Cervical small cell carcinoma and ovarian carcinoids are the most common gynaecological ne tumours . Neuroendocrine tumours comprise a set of neoplasms that arises from the diffuse neuroendocrine cell system . These tumours are more commonly identified in the gastrointestinal tract, pancreas, lung and thymus . Recently, a simplified terminology has been proposed, dividing ne tumours in two groups: poorly differentiated neuroendocrine carcinomas (necs) and well - differentiated neuroendocrine tumours (nets). Necs include small cell carcinoma and large cell neuroendocrine carcinoma, while nets account for typical and atypical carcinoids [14]. The clinical features of gynaecological ne tumours generally depend on the organ of origin and are non - specific . However, some patients may present with paraneoplastic syndromes owing to the production of several peptides and hormones . The absence of endocrine clinical syndromes in most patients with ne tumours is possibly related to the release of insufficient amounts or inactive hormones . The purpose of this article is to review the epidemiological, clinical and imaging features with pathological correlation of primary gynaecological ne tumours, emphasising the importance of differentiating them from metastatic disease and other carcinomas with an ne component . For the classification of ne differentiation, tumour cells have to express at least two ne markers such as chromogranin a, synaptophysin or neuron - specific enolase . A great variety of other peptides and hormones may be found, including calcitonin, gastrin, serotonin, substance p, vasoactive intestinal peptide, pancreatic polypeptide, somatostatin and adrenocorticotrophic hormone [1, 3]. Some cancers display a combination of ne and non - ne features, usually either glandular or squamous components . The amount of ne component within a non - ne carcinoma may range from a single ne cell to a well - identifiable ne tumour cell population . In this set, two terms are widely accepted: mixed exocrine - endocrine carcinoma (meec) and (adeno)carcinoma with (focal) ne differentiation . The finding of a focal non - ne differentiation in almost pure ne tumours is less common . According to the world health organisation (who) classification of ne tumours, the diagnosis of a mixed exocrine - endocrine tumour should take into account at least two major diagnostic parameters: an extension of at least 30% for each component and the recognition of structural ne features such as well - differentiated organoid or solid / diffuse growth patterns . In fact, no reasonable explanation is provided for the limit of 30%, and consequently morphological criteria should also be considered [7, 8]. Carcinomas with focal ne differentiation have been identified in several non - ne tumours of the gynaecological tract, such as endometrial endometrioid adenocarcinomas, endocervical adenocarcinomas and adenosquamous carcinomas, and even ovarian surface epithelial neoplasms of mucinous, endometrioid and serous types . These tumours display less than one - third of ne cells and a non - ne growth pattern, and the finding of these ne cells has not been shown to affect outcome [5, 7]. Few data are currently at our disposal regarding the prognosis of mixed exocrine - endocrine carcinoma (meec) in comparison to pure ne or non - ne tumours . However, the therapeutic schemes are different and the accurate histological diagnosis is mandatory the differentiation of small cell ne carcinomas from poorly differentiated squamous cell carcinoma (scc) with ne features may be especially difficult . Sometimes, and particularly for cervical cancers, the recognition of the ne component is only possible after the hysterectomy, since the tissue obtained on cervical biopsy may be insufficient, leading to the diagnosis of poorly differentiated cervical cancer . Due to the rarity of these tumours, data in the literature concerning their imaging features are scarce . We know that small cell carcinoma and large cell ne carcinoma tend to behave aggressively, in contrast to carcinoids, which are typically confined to the organ . Nevertheless, these are non - specific features and the available data regarding these extremely rare tumours are limited and discourage scientific generalisations . Consequently, no reliable guidelines, for either diagnosis or treatment, are established . For staging purposes, physicians and radiologists should consider the currently used revised international federation of gynecology and obstetrics (figo) staging system [911]. The imaging approach to gynaecological ne tumours is similar to that for other histological types . For the cervix, uterus and adnexa, ultrasound (us) using both transabdominal and transvaginal probes plays a critical role in the initial diagnosis . The use of computed tomography (ct) or positron emission tomography - computed tomography (pet - ct) is established in cervical and endometrial tumours for detection of nodal and distant metastases, which may be particularly useful for poorly differentiated ne carcinomas, typically invasive and metastatic . In ovarian cancers, ct is also indicated to evaluate the extent of disease and detect nonresectable metastases . Magnetic resonance imaging (mri) may be used for characterising indeterminate adnexal masses and for pre - treatment staging purposes, essentially in cervical and endometrial tumours . Mri may replace ct in the staging of adnexal tumours in young females, pregnancy, renal insufficiency and allergy to iodinated contrast [10, 12, 13] (table 1).table 1histological classification of ne tumours . Table created by the authors based on the literature review [14, 7]poorly differentiated neuroendocrine carcinomas (necs)small cell carcinomalarge cell neuroendocrine carcinomawell - differentiated neuroendocrine tumours (nets)typical carcinoidatypical carcinoidcombination of ne and non - ne featuresmixed exocrine - endocrine carcinoma (meec) (adeno) carcinoma with (focal) ne differentiation histological classification of ne tumours . Table created by the authors based on the literature review [14, 7] ne tumours of the vagina and vulva are much rarer . Us plays a limited role in the diagnosis, but ct and mri may be used for staging purposes . Mri is primarily used for local staging of vaginal tumours and should be performed after vaginal filling with gel . Particularly for typical and atypical carcinoids (well - differentiated neuroendocrine tumours), patients with negative octreotide scans should not be considered for somatostatin analogue therapy and typically show better responses to chemotherapy schemes . Primary carcinoid tumours of the ovary are rare and account for less than 5% of all carcinoid tumours and for less than 0.1% of all ovarian neoplasms . These are germ cell tumours in origin and classified as teratomas with a predominance of ne features . The great majority is asymptomatic and incidentally found on cross - sectional or ultrasound imaging . The median age of diagnosis is 55 years of age (range from 17 to 83 years), with most of the patients being peri- or postmenopausal [1, 1417]. These tumours are typically unilateral, slow growing and diagnosed in an early stage . In a 5-decade analysis performed by modlin et al ., 66% of 113 carcinoid ovarian tumours were localised lesions, while 22% 31% presented with distant spread . They may exist in pure forms, predominantly solid with small cysts or as a nodule within a mature teratoma, mucinous cystadenoma or even brenner tumour . Ovarian carcinoids contain well - differentiated ne cells and some subtypes are similar to the gastrointestinal carcinoids . They are immunoreactive to at least one neuroendocrine marker, including chromogranin, synaptophysin or leu-7, and may also be present in peptide hormones such as pancreatic polypeptide, gastrin, vasoactive intestinal peptides and glucagon . Insular carcinoid is the most common type of ovarian carcinoid, typically presenting with a pelvic mass . Near 30% of patients will have signs and symptoms of carcinoid syndrome, including facial flushing, diarrhoea, bronchospasm and oedema . It happens because serotonin - like substances are directly released into the systemic circulation through the ovarian venous system, bypassing hepatic deactivation . Carcinoid syndrome is less common in the other subtypes (13% in trabecular and 3.2% in strumal carcinoids). Trabecular carcinoid constitutes the second most common ovarian carcinoid tumour and 25% of patients will present with constipation due to peptide yy production, which inhibits intestinal motility [15, 17]. Some cases of hirsutism are also thought to be related to peptide yy production, which is known to inhibit peripheral steroid - producing cells [14, 19]. Mucinous carcinoids are rare (1.5%), may be pure or associated with mature teratomas . In these cases, the differential diagnosis must include metastasis from a gastrointestinal tumour and primary mucinous appendiceal carcinoid . We should also consider strumal carcinoids, tumours that contain normal thyroid and carcinoid tissues . Eight per cent of the patients may present with thyroid hormone - related symptoms and some ovarian strumal carcinoids are also reported to cause severe constipation owing to peptide yy production [15, 22]. Primary carcinoid tumours typically behave in a benign fashion, and the prognosis is generally favourable . If the tumour presents with stage i, survival may be greater than 90%; however, for women with advanced stage disease, the prognosis is poor and the 5-year survival is approximately 33% . Attending to the more favourable prognosis of ovarian carcinoid tumours, the differentiation of such lesions from other highly malignant ovarian neoplasms is mandatory in order to choose the most appropriate therapeutic options . Particular note should be taken in relation to mucinous carcinoid, which behaves more aggressively and may present with extra - ovarian spread and lymph node involvement . While performing the diagnosis of an ovarian carcinoid, it is important to rule out ovarian metastases, essentially from the gastrointestinal tract . The insular and trabecular subtypes most commonly have metastases in the ovary simulating a primary tumour . Metastatic carcinoid is frequently bilateral, while primary ovarian carcinoids tend to be unilateral [1, 23]. Compared the features of primary insular carcinoids and metastatic midgut carcinoids and concluded that the presence of extra - ovarian tumour, bilaterality, multinodularity, vascular invasion and the absence of teratomatous elements are suggestive of an extra - ovarian origin . Both small cell carcinoma and large cell ne carcinoma of the ovary are highly malignant and aggressive, regardless of the stage . Ovarian small cell carcinoma resembles the pulmonary type and is often associated with common epithelial tumours, typically an endometrioid carcinoma, suggesting an origin from ovarian surface epithelium . Its immune profile includes positivity for neuron - specific enolase and less commonly for chromogranin [1, 5]. Large cell ne carcinomas have been associated with benign and malignant surface epithelial - stromal tumours, and immunohistochemical markers for chromogranin are typically positive [25, 26]. We review three cases of neuroendocrine ovarian tumours, one primary carcinoid and two secondary carcinoid tumours (figs . 1, 2 and 3). Once again, we emphasise that few data are provided in the literature regarding the imaging features of these tumours all the cases show well - circumscribed, predominately solid ovarian tumours . However, fig . 1 exemplifies a typical benign behaviour, while fig . Figure 2 represents an atypical presentation of metastatic disease, with overlap between benign and malignant features.fig . 1 a e struma carcinoid of the right ovary . A 49-year - old female complaining of pelvic and lower abdominal pain . Ultrasound (a) and axial enhanced ct image (b). Large, heterogeneous, mixed tumour of the ovary, predominantly solid, with necrotic areas and calcifications . The solid portions are moderately echogenic on us and hyperdense on enhanced ct, while cystic areas are anechogenic on us and hypodense on ct . This tumour is composed of areas of thyroid tissue and carcinoid (h&e), the latter having acinar and trabecular architecture (c). The tumour cells show positive cytoplasmatic staining for chromogranine (d) and for synaptophysin (not shown). The thyroid area with compact folicular structure presents intense immunoreactivity for thyroglobulin (e)fig . 2 a d metastatic ovarian carcinoid of unknown primary origin in a 23-year - old female complaining of pelvic pain and amenorrhoea . Sagittal t2-weighted mr image (a) and axial t1-weighted mr image with fat saturation, after intravenous gadolinium administration (b). Large, well - defined solid lesion of the left ovary, cranially and anteriorly located in relation to the uterus . On t2wi, it displays intermediate signal intensity and shows a central high signal region . The lesion shows peripheral, intense contrast enhancement and a large central area of necrosis . H&e section of the ovary occupied by a tumour with insular pattern composed by neuroendocrine cells (c). These cells have intense cytoplasmatic staining for synaptophysin (d) and have small chromogranine - positive granules in the cytoplasm (not shown)fig . 3 a b metastatic, bilateral ovarian carcinoid, from a primary jejunal tumour in a 28-year - old female complaining of pelvic pain and dyspareunia . Multiple liver metastases were also seen (not shown) a e struma carcinoid of the right ovary . Large, heterogeneous, mixed tumour of the ovary, predominantly solid, with necrotic areas and calcifications . The solid portions are moderately echogenic on us and hyperdense on enhanced ct, while cystic areas are anechogenic on us and hypodense on ct . This tumour is composed of areas of thyroid tissue and carcinoid (h&e), the latter having acinar and trabecular architecture (c). The tumour cells show positive cytoplasmatic staining for chromogranine (d) and for synaptophysin (not shown). The thyroid area with compact folicular structure presents intense immunoreactivity for thyroglobulin (e) a d metastatic ovarian carcinoid of unknown primary origin in a 23-year - old female complaining of pelvic pain and amenorrhoea . Sagittal t2-weighted mr image (a) and axial t1-weighted mr image with fat saturation, after intravenous gadolinium administration (b). Large, well - defined solid lesion of the left ovary, cranially and anteriorly located in relation to the uterus . On t2wi, it displays intermediate signal intensity and shows a central high signal region . The lesion shows peripheral, intense contrast enhancement and a large central area of necrosis . H&e section of the ovary occupied by a tumour with insular pattern composed by neuroendocrine cells (c). These cells have intense cytoplasmatic staining for synaptophysin (d) and have small chromogranine - positive granules in the cytoplasm (not shown) a b metastatic, bilateral ovarian carcinoid, from a primary jejunal tumour in a 28-year - old female complaining of pelvic pain and dyspareunia . Coronal (a) and axial (b) enhanced ct images . Large cell ne carcinoma and carcinoid types are rarer than the former and few data can be found in the literature . Staging and treatment strategies for both small and large cell cervical carcinoma globally follow the same criteria used for common histological types of cervical carcinomas . Small cell carcinoma accounts for 16% of cervical carcinomas [6, 27]. Median age of diagnosis is in the 5th decade, ranging from 21 to 87 years . Some patients present with a cervical mass and an abnormal pap smear may be found . This tumour is strongly associated with human papilloma virus 18 (hpv-18), but hpv-16-positive tumours have also been demonstrated . The most commonly found are hypercalcaemia (parathormone), hypoglycaemia (insulin), carcinoid syndrome (serotonin), cushing s syndrome (corticotropin), siadh (vasopressin) and myasthenia gravis . Most tumours are voluminous, exceeding 6 cm, and show deep cervical infiltration and necrosis . Often, growth may be exophytic, which appears to be associated with a better prognosis . Nevertheless, it is generally a highly aggressive tumour with a worse prognosis than that of poorly differentiated squamous cell carcinomas . The rates of lymphovascular space invasion and extra - pelvic recurrences for bone, supraclavicular lymph nodes and lung are particularly high [6, 27, 28, 30]. According to mccusker et al ., females with small cell tumours have 1.84 times greater risk of death compared to patients with squamous cell carcinomas . It appears to have a similar outcome to small cell carcinoma and its treatment remains challenging because of the high rate of recurrence and distant metastases even with early stage disease [2, 32]. We review three cases of cervical small cell carcinoma (figs . 4, 5 and 6). Cervical necs resemble cervical squamous cell carcinoma, displaying the same mri features: low signal intensity on t1-weighted images (t1wi) and high signal intensity on t2-weighted images (t2wi). No histological distinction can be provided regarding only imaging features . However, according to the literature, cervical necs are generally highly aggressive and expected to be diagnosed in advanced figo stages . Concordantly, we illustrate necs in iv - a (invasion of adjacent organs) (fig . 4) and iii - b (invasion of the pelvic side wall) (fig . 6) stages.fig . 4 a b cervical neuroendocrine tumour (figo iv - a; invasion of adjacent organs). A 32-year - old female complaining of vaginal bleeding after intercourse and fetid vaginal discharge . Sagittal t2-weighted mr image (a) and axial t2-weighted mr image (b). The parametria are circumferentially infiltrated and the tumour extends anteriorly to the bladder, with transmural invasion and fistulae (arrow). . 5 a e cervical small cell carcinoma (figo ii - a; invasion of the upper two - thirds of vagina with no parametrial invasion). Sagittal t2-weighted mr image (a), axial t2-weighted mr images (b) and sagittal diffusion weighted image (dwi), b(c) retroverted uterus with an anterior cervical tumour, highly restrictive on dwi . No parametrial invasion is noted, but huge, bilateral obturator lymph nodes are found (arrows). The left ovary shows follicular activity and is posteriorly displaced (arrowhead). In this h&e section the cervix is occupied by a solid tumour, composed of small cells, with uniform nuclei and scarce cytoplasm (d). Some tumour cells show positive cytoplasmatic staining for synaptophysin (e) and for chromogranin (not shown)fig . 6 a b cervical small cell carcinoma (figo iii - b; extension to the pelvic side wall). A 64 year - old female complaining of menorrhagia . Axial t2-weighted mr image (a) and axial t1-weighted mr image with fat saturation after intravenous gadolinium administration (b). Cervical tumour extending posteriorly to the pelvic wall (asterisk) and superiorly to the left adnexal area, where it forms a huge solid, heterogeneous mass with central necrotic areas (arrow). This mass shows intense enhancement after gadolinium administration and is adherent to the uterine corpus, which may be found inferiorly . Bilateral internal iliac lymph nodes are also noted (arrowheads) a b cervical neuroendocrine tumour (figo iv - a; invasion of adjacent organs). A 32-year - old female complaining of vaginal bleeding after intercourse and fetid vaginal discharge . Sagittal t2-weighted mr image (a) and axial t2-weighted mr image (b). Large cervical cancer with infiltration of the lower segment of the uterine corpus . The parametria are circumferentially infiltrated and the tumour extends anteriorly to the bladder, with transmural invasion and fistulae (arrow). Bilateral, nodal metastases of the external iliac groups are also seen (arrowheads) a e cervical small cell carcinoma (figo ii - a; invasion of the upper two - thirds of vagina with no parametrial invasion). Sagittal t2-weighted mr image (a), axial t2-weighted mr images (b) and sagittal diffusion weighted image (dwi), b(c) retroverted uterus with an anterior cervical tumour, highly restrictive on dwi . No parametrial invasion is noted, but huge, bilateral obturator lymph nodes are found (arrows). The left ovary shows follicular activity and is posteriorly displaced (arrowhead). In this h&e section the cervix is occupied by a solid tumour, composed of small cells, with uniform nuclei and scarce cytoplasm (d). Some tumour cells show positive cytoplasmatic staining for synaptophysin (e) and for chromogranin (not shown) a b cervical small cell carcinoma (figo iii - b; extension to the pelvic side wall). Axial t2-weighted mr image (a) and axial t1-weighted mr image with fat saturation after intravenous gadolinium administration (b). Cervical tumour extending posteriorly to the pelvic wall (asterisk) and superiorly to the left adnexal area, where it forms a huge solid, heterogeneous mass with central necrotic areas (arrow). This mass shows intense enhancement after gadolinium administration and is adherent to the uterine corpus, which may be found inferiorly . Well - differentiated neuroendocrine tumours (nets) include typical and atypical carcinoid tumours . Among the uncommon sites of primary carcinoid tumours, the uterine cervix constitutes one of the rarest locations, representing only 0.5% to 5% of cervical cancers . Carcinoid syndrome is very rare and even in the lack of clinically evident carcinoid syndrome, many patients present with elevated urinary 5-hydroxyindoleacetic acid (5-hiaa). However, the diagnosis of a cervical carcinoid is frequently postoperative and laboratorial investigation is typically not helpful since it is not performed in the absence of symptoms . These tumours display a very aggressive, malignant behaviour, and their prognosis is poor (2- or 3-year survival rates, ranging from 12.5 to 33%, according to some studies). Atypical carcinoids of the cervix are also very rare and, according to a review of yoshida et al ., only 13 cases have been reported in the literature . The reported endometrial neoplasms with ne differentiation essentially include small cell carcinoma and endometrial adenocarcinoma with ne cells . Small cell carcinoma of the endometrium resembles that of the lung and is uncommon, accounting for less than 1% of all endometrial carcinomas . Like in other endometrial carcinomas, abnormal bleeding is the primary presenting symptom, but some patients present with advanced disease, sometimes with pain from metastases . These tumours usually present as bulky intraluminal masses, and deep myometrial invasion (3350%) and extra - uterine spread (near 50%) are common . Pelvic lymph nodes, adnexa and the peritoneum constitute the typical sites of extra - uterine involvement . The overall prognosis is poor [5, 35]. In 50 to 66%, endometrial small cell carcinoma displays an admixed component of grade 1 or 2 endometrioid adenocarcinoma, and few cases showed a malignant mixed mullerian tumour within a predominant small cell carcinoma . We review two cases of poorly differentiated endometrial carcinoma with ne differentiation (figs . 7 and 8). Like in cervical carcinoma, imaging does not allow histological distinction, since all endometrial cancers tend to present similarly: low to moderate signal on t2wi and hypointensity relative to the hyperintense enhancing myometrium on dynamic contrast - enhanced mri sequences . However, when a highly aggressive endometrial tumour is found, a neuroendocrine nature may be suspected . There are no consistent data regarding the prognosis of non - pure ne endometrial tumours; however, the cases we illustrate were diagnosed in advanced figo stages, iv - b (distant metastases) (fig . 7) and iii - c1 (involvement of pelvic nodes) (fig . 7 a d poorly differentiated endometrial carcinoma with ne differentiation (figo iv - b; distant metastases). Axial t2-weighted mr image (a) and axial enhanced ct image (b). There is no invasion of the cervical stroma or bladder (not shown), but a right obturator node (arrow) is found . Ct evaluation shows that the disease has disseminated outside the pelvis, revealing para - aortic lymph nodes (arrowhead) as well as retroperitoneal and peritoneal metastases (asterisks). Vagina biopsy (h&e) infiltrated by a solid tumour composed by sheets of uniform cells, with small oval nuclei and scarse cytoplasm (c). These cells have an intense cytoplasmatic staining for neuron - specific enolase (d) and have small chromogranine - positive granules in the cytoplasm (not shown). 8 a d undifferentiated endometrial carcinoma with ne differentiation (figo iii - c1; involvement of pelvic nodes). Endometrial tumour prolapsing into the cervical canal, without invasion of the cervical stroma and myometrium . The tumour extends bilaterally through the lumen of the tubes to the parametrial tissues (arrows). H&e section of the tumour with a solid pattern composed by large cells with pleomorphic nuclei, mitoses and scarce cytoplasm (c). These cells have small chromogranine - positive granules (d) and small synaptophysin granules in the cytoplasm (not shown) and are strongly positive for pancytokeratin cam 5.2 (not shown) a d poorly differentiated endometrial carcinoma with ne differentiation (figo iv - b; distant metastases). Axial t2-weighted mr image (a) and axial enhanced ct image (b). There is no invasion of the cervical stroma or bladder (not shown), but a right obturator node (arrow) is found . Ct evaluation shows that the disease has disseminated outside the pelvis, revealing para - aortic lymph nodes (arrowhead) as well as retroperitoneal and peritoneal metastases (asterisks). Vagina biopsy (h&e) infiltrated by a solid tumour composed by sheets of uniform cells, with small oval nuclei and scarse cytoplasm (c). These cells have an intense cytoplasmatic staining for neuron - specific enolase (d) and have small chromogranine - positive granules in the cytoplasm (not shown). This tumour was synaptophysin negative a d undifferentiated endometrial carcinoma with ne differentiation (figo iii - c1; involvement of pelvic nodes). Endometrial tumour prolapsing into the cervical canal, without invasion of the cervical stroma and myometrium . The tumour extends bilaterally through the lumen of the tubes to the parametrial tissues (arrows). H&e section of the tumour with a solid pattern composed by large cells with pleomorphic nuclei, mitoses and scarce cytoplasm (c). These cells have small chromogranine - positive granules (d) and small synaptophysin granules in the cytoplasm (not shown) and are strongly positive for pancytokeratin cam 5.2 (not shown) neuroendocrine tumours of the fallopian tube, vagina and vulva are very rare and only a few cases have been reported . In the fallopian tube, these tumours encompass carcinoids and small cell carcinoma, and they are not well known . In the vagina, almost ten cases of small cell carcinoma have been reported, occurring in females between 41 and 78 years of age . Like in other vaginal tumours, the most frequent complaint was vaginal bleeding or discharge . Metastatic disease was common in this small cohort, so the prognosis is thought to be poor . In the vulva, neuroendocrine differentiation may be found in small cell carcinoma of the bartholin gland and merkel cell tumours, which tend to present as vulvar masses . Two groups are currently considered: necs, including small cell carcinoma and large cell ne carcinoma, and nets, which encompass typical and atypical carcinoids . The clinical features of theses tumours are non - specific and depend on the organ of origin and on the extension and aggressiveness of the disease . The imaging evaluation should follow the same criteria used for other tumours, and figo remains the preferred staging system . The diagnosis is obviously histological, and the role of imaging tools essentially includes initial detection, staging and biopsy guidance . However, some points should be taken into account when imaging studies are performed: cervical small cell carcinoma and ovarian carcinoid are the most common gynaecological ne tumours.cervical small cell carcinomas usually behave aggressively.ovarian carcinoids tend to behave in a benign fashion.while managing an ovarian carcinoid, extra - ovarian extension, bilaterality and multinodularity should raise the suspicion of metastatic disease.endometrial ne tumours are very rare and are usually small cell carcinoma or adenocarcinoma with ne differentiation.as for other tumours, us, ct, pet - ct and mri remain the most commonly used imaging tools, but octreotide scans may be useful for typical and atypical carcinoids . Ovarian carcinoids tend to behave in a benign fashion . While managing an ovarian carcinoid, extra - ovarian extension, bilaterality and multinodularity should raise the suspicion of metastatic disease . Endometrial ne tumours are very rare and are usually small cell carcinoma or adenocarcinoma with ne differentiation . As for other tumours, us, ct, pet - ct and mri remain the most commonly used imaging tools, but octreotide scans may be useful for typical and atypical carcinoids.
Adenoid cystic carcinoma (adcc) is a malignant salivary gland tumor that was first described by billroth in 1859 under the name cylindroma attributing to its cribriform appearance formed by the tumor cells with cylindrical pseudolumina or pseudospaces . The term adenoid cystic carcinoma was introduced by ewing (foote and frazell) in 1954 . Adcc is a relatively rare malignant salivary gland tumor comprising less than 1% of all malignancies of head and neck . It is the 5 most common malignancy of salivary gland origin, representing 5 - 10% of all salivary gland neoplasms . The parotid and submandibular glands are the two most common sites for adcc accounting for 55% of the cases . Among the major glands, adcc accounts for 8.3% of all palatal salivary gland tumors and 17.7% of malignant palatal salivary gland tumors (afip series). The other less common sites are lower lip, retromolar tonsillar pillar area, sublingual gland, buccal mucosa and floor of the mouth . The nose and paranasal sinuses represent the next most common sites for minor gland adccs . The tumor is most often clinically deceptive by its small size and slow growth, which actually overlies its extensive subclinical invasion and marked ability for early metastasis making the prognosis questionable . The three recognized histological variants of adcc are cribriform, tubular and solid although, cribriform is the most commonest and solid is the least commonest . It is not uncommon to have more than one histopathologic pattern in a single neoplasm and rather all three patterns can be observed in the majority of tumors . The main reason for histological typing is to assess the prognostic difference between histologic types . Tubular pattern (well differentiated) is believed to have the best prognosis compared to the cribriform pattern (moderately differentiated) and solid pattern (poorly differentiated). A 60-year - old male patient reported to the department of oral and maxillofacial surgery with a chief complaint of pain and discharge in the upper left back region of the jaw since one and half years . According to the patient one year prior to our consultation, he got the maxillary molars extracted from a local dentist due to pain and mobility of the teeth in that area . Upon consultation patient revealed that the pain continued to exist even after the extraction . The patient is a known hypertensive and is on medication since three years for the same . The patient had smoking habit, which he quit only a couple of years ago . Intraoral examination revealed a solitary erythematous swelling with diffuse borders involving the left posterior portion of hard palate and was also seen involving the soft palate on the same side . The swelling was seen extending from mesial aspect of first premolar to 1 cm posterior to tuberosity on left side, involving the palatal area but not crossing the midline [figure 1]. Intraorally, the lesion was seen involving the hard and soft palate but not crossing the midline the swelling was nontender . The clinical differential diagnosis included a benign or malignant neoplasm of minor salivary glands, a neoplasm of maxillary sinus . Ct scan showing complete obliteration of left maxillary sinus an incisional biopsy of palate and sinus lining was performed . Islands of uniform cells arranged in cord - like pattern with hyperchromatic nuclei were seen enclosing round to oval pseudocystic spaces in the stroma . The tumor cells are also seen arranged in the form of solid islands [figure 4] and ductal pattern [figure 5]. Focal areas showed small cords and longitudinal tubules of isomorphic cells set in a background of densely hyalinized stroma [figure 6]. Variable - sized cribriform spaces formed by small deeply basophilic cells imparting swiss cheese pattern solid pattern composed of nests of basaloid cells and mitotic figures tubular pattern characterized by ductal structures formed by layers of isomorphic basaloid cells tubules of isomorphic tumor cells set in a densely hyalinized stroma a diagnosis of adcc (cribriform pattern) was established . The patient was treated by wide surgical excision with clear margins and hemi - maxillectomy of left maxillary region with post - radiotherapy . The present case was staged as t4n0m0 based on american joint committee on cancer as a guide to prognosis . The patient was under regular follow - up and is free of the disease at one - year follow - up . Adenoid cystic carcinoma most often presents a diagnostic and treatment challenge owing to the rarity of the lesion . Most findings regarding adcc are actually based upon studies with a small number of patients and there is a need for further information regarding its clinical behaviour as well as treatment modalities and their results . Adcc may occur at any age although in most cases the patients age ranged from 24 to 78 years . The age of patients affected with major salivary gland tumors has been shown to be younger (mean 44 years) compared to the age of those who developed tumors of the minor glands (mean 54 years) and shows female predilection (f: m 1.2:1). Pain is a common and important finding, occurring early in the course of the disease before there is a noticeable swelling . Palate is more commonly involved generally the area of greater palatine foramen . Among the malignant neoplasms of minor salivary glands, the most common was mucoepidermoid carcinoma (21.8%) followed by polymorphous low grade adenocarcinoma (plga) (7.1%) and adccs of the minor glands have been reported to have worse prognosis than those of the major salivary glands . Tumors involving the nose, paranasal sinuses and maxillary sinus have the worst prognosis as they are usually detected with higher stages at the time of diagnosis . Tumors of minor salivary glands usually have the tendency to infiltrate extra glandular soft tissues and bone thereby allowing increased dissemination of the tumor . Lymph node involvement is uncommon (<5% of cases) and is usually due to contiguous spread rather than lymphatic permeation or embolization . The differential diagnosis of adcc includes plga, basal cell adenoma (bca), mixed tumor and basaloid squamous cell carcinoma (bsc). The cribriform pattern so typical of adcc may also rarely be seen in bca, mixed tumor and plga, and the distinction between plga and adcc is more challenging . A polymorphous architecture characterizes plga whereas adcc has a more limited range of histologic patterns with no more than three patterns . Plga shows uniform cell population with cytologically bland, round or oval vesicular nuclei and pale eosinophilic cytoplasm where as cells in adcc have clear cytoplasm, angular, hyperchromatic nuclei and may show mitotic activity . The ki-67 index is reported to be 10 times higher in adcc compared to plga . Smooth muscle markers of myoepithelial differentiation are positive in adcc but negative in plga . Though plga may form solid areas, they lack the overall high - grade feel associated with adcc . Occasional foci in pleomorphic adenoma (pa) can resemble adcc but the presence of typical myxochondroid matrix and plasmacytoid or spindle shaped cells helps to avoid confusion . Cribriform structures may sometimes be observed in bca and such cases can be differentiated from adcc on the basis of gradual structural alteration from areas typical of bca . The peripheral palisading and focal squamous differentiation with whorling pattern present in bca are not usually encountered in adcc . The basement membrane material secreted by bsc tends to dissect between tumor cells rather than form crisp cribriform spaces seen in adcc . Focal keratinization, attachment to rete pegs, and presence of surface squamous dysplasia or carcinoma in situ helps to distinguish it from adcc . Immunohistochemical studies demonstrated that the pseudocysts are positive for periodic acid schiff reagent (pas) and alcian blue and contain basement membrane components such as type iv collagen, heparin sulfate and laminin isoforms . Epithelial cells are positive for carcinoembryonic antigen (cea) and epithelial membrane antigen (ema). Duct lining cells are positive for c - kit (cd117) and myoepithelial cells are positive for s-100 protein, calponin, p63, smooth muscle actin and myosin . Expression of s-100, glial fibrillary acidic protein (gfap) and neural cell adhesion molecule have been correlated with the presence of perineural invasion . Molecular genetic data: alterations in chromosomes 6q, 9p and 17p12 - 13 are the most frequent cytogenetic alterations reported . Hypermethylation of the promoter region of the p16 gene was associated with higher histologic grades of malignancy . Possible treatment modalities available for the treatment of adcc are surgery, radiotherapy, chemotherapy and combined therapy . It requires excision with the widest surgical margins possible as tumor cells extend well beyond the clinical and radiographic margins . Neutron therapy, which involves larger particles of greater energy can achieve reasonable local control as a primary therapeutic modality . Main factors associated with patient survival are anatomic location, primary lesion size, presence or absence of metastasis at the time of diagnosis, perineural invasion and histological variant . Distant metastasis occurs in 25%-50% of patients and lung is the most common involved site . The 5-year survival rate after effective treatment is 75%, but long - term survival rates are low (10 years-20% and 15 years-10%). The present patient was recalled at every 6-month interval in view of the sinus involvement and propensity for distant metastasis . Lesions involving the sinus tend to have a poor prognosis due to its infiltrative growth and distant metastasis.
The quantity of trauma victims and the management involved in attending them leads to a great workload in most hospitals and places an enormous amount of pressure on the health care system . Furthermore, traumatic injuries have been reported as the leading cause of death in the first 40 years of life and appear to be on the increase . During the last decade, interpersonal violence (ipv) was one of the main etiological factors of fractures of the maxillofacial complex . Road traffic accidents remain the most important cause of maxillofacial trauma worldwide, although falls and assaults have become more significant in north america, brazil and europe . Blunt and cutting wounds on the head and neck are also common . Legal underreporting is common due to associations with alcohol abuse, illicit drug use, firearms and acts of violence against women, children, and the elderly, which generate fear, shame, low self - esteem and a sense of powerlessness . Facial fractures associated to ipv have functional, aesthetic and psychological effects on the victims, as well as a socioeconomic impact on the health systems of many countries .these victims are more likely to suffer from depression, anxiety and post - traumatic stress disorder . Epidemiological studies help to identify demands, develop prevention programs and structure an integrated care system . The aim of this retrospective study was to compare the peculiarities of maxillofacial injuries caused by interpersonal violence with other etiologic factors . This retrospective study involved data collected from clinical notes and surgical records of patients treated at seven different hospitals located in three different cities in so paulo state, brazil . The sample was selected based on analysis of medical records of facial trauma patients attended by the department of oral and maxillofacial surgery in the piracicaba dental school at the university of campinas from january 1999 to december 2012 . A standardized form was designed to investigate the epidemiologic features of maxillofacial traumas, considering aspects such as age, gender, diagnosis, soft tissue lesions, facial injuries and general injuries . The exclusion criterion was incomplete information about the type of trauma on the medical chart . The origin of the facial trauma was classified as follows: traffic accidents (automobile and motorcycle); work accidents; sport accidents; falls; interpersonal violence; cycling accidents; pedestrian accidents and others . Facial injury sites were classified as follows: upper (frontal bone); middle (maxilla, nasal bones, zygomatic - orbital complex, naso - orbital ethmoidal) and lower (mandible). Mandibular fractures were described according to the anatomic site: condylar fractures; angle fractures; body fractures and fractures of symphysis . The present study was approved by the research ethics committee of piracicaba dental school, state university of campinas (fop / unicamp) under protocol number 75/2013 . Data were submitted to statistical analysis (simple descriptive statistics and chi - squared test) with the support of the statistical package for social sciences (spss 18.0 for windows, spss inc, chicago, il). Binary logistic regression and the chi - square test were performed and the results were considered relevant when p <0.05 . These included absolute frequencies (raw counts) and relative frequencies (proportions or percentages of the total number of observations). Based on the 13-year study period, 3,724 patient referrals to the maxillofacial surgery department were enrolled in the present study . The median age at the time of admission was 29.2 years, with a standard deviation of 17.06 years . A total of 612 patients (16.4%) exhibited facial injuries related to ipv . In this group, male victims prevailed with 81% (n = 496) and the mean age was 31.28 years, with a standard deviation of 13.33 years . Weekly consumption of alcohol was reported by 32.4% and tobacco use was 43.8% . In total, 79 patients (12.8%) reported using non - injecting illicit drugs and 14 (2.2%) stated that they currently used injecting drugs . In 176 cases, isolated alveolar - dental injuries were observed in 35 patients . With regards to maxillofacial fractures, table 2 shows the distribution of diagnoses in the general sample and among patients who were victims of physical assault . Distribution of patients with facial injuries according to etiological factors n = number of patients . Distribution of facial injury diagnoses among a general sample and patients whose facial injury was associated with interpersonal violence (ipv) n = number of patients . In the cases of mandibular fractures, the anatomic regions most affected were the body and the mandibular angle, with 36 cases in each region, followed by the condyle (25 cases), symphysis (9 cases) and isolated mandibular ramus (2 cases). In 41 patients, fractures involving more than one anatomic region of the mandible lacerations and abrasions on the face were identified in 181 and 105 patients, respectively . Table 3 displays the distribution of body injuries in these patients, when compared to the general sample . Distribution of body injuries among patients with facial trauma and patients whose facial injury was associated with interpersonal violence (ipv) the calculation is based on whole sample (n = 3724). The calculation is based on patients that involved with ipv (n = 612). Comparison of features of facial injury of victims of interpersonal violence (ipv) and other etiologic factors n = number of patients; = chi - square . A greater number of fractures and lacerations of the face was observed, although with fewer facial abrasions . With regards to the area of bone fractures, fewer injuries occurred in other regions of the body, mainly due to fewer injuries of the upper and lower limbs . However, patients that were associated with ipv exhibited a greater number of injuries to the neurocranium . Binary logistic regression was performed to determine whether fractures in facial injuries were affected by gender, age (median 30 years), drug use, the presence of lacerations or the presence of abrasions . Fractures were associated with gender: male patients are more at risk than females (or = 1.61, p <0.002, 95% ci: 1.05 to 2.46). Although motor vehicle accidents are still the main cause of maxillofacial fractures in some developing countries, recent data from developed countries have shown that ipv is the dominant cause . Ipv is one of the leading causes of maxillofacial fractures and its prevalence in a population varies according to geographic region and appears to be associated with factors such as socioeconomic status, population density and age characteristics . The prevalence of male victims has been reported in most studies, ranging from 61% to 92%, as well as a mean age of approximately 30 years [10 - 14]. However, children are also affected and several studies have reported the patterns and severity of these injuries . This age group is susceptible to craniofacial trauma due to their greater cranial mass - to - body weight ratio . Compared to adults, less 65 patients were under 18 years of age, although most of them (36 patients) were aged between 16 and 18 years . These patients mainly exhibited nasal bone (21 patients) and mandibular fractures (12 patients), results which are in accordance with the literature . The prevalence of male victims is influenced by cultural models of gender, defined as the set of attributes, values, roles and behavior patterns that are expected from a man in a particular culture . Generally, the traits valued in this socialization are aggression, domination and exploitation of danger, which could contribute to their association with ipv . Ipv among females is mainly associated with domestic violence . In the present study, it was not possible to determine the pattern of violence suffered . When the victim is female, the case should be investigated regardless of the site of the injury, as there is a greater possibility that they are the victim of physical aggression due to the vulnerability of the gender . Another relevant factor is the differences in female participation in social activities, making them more (or less) prone to urban violence . Patients who were victims of physical assault, unlike other etiologies such as sports accidents, are often affected by problems such as drug use and poverty . In the present study, we found high consumption rates of alcohol, tobacco and non - injecting drugs . Its consumption results in an increase in confidence, followed by the subjective feeling of increased mental and physical capacity, as well as impaired judgment and coordination . . Found that alcohol was involved in 87% of maxillofacial fractures caused by ipv, compared with 58% for motor vehicle accidents . Studies have reported that fractures of the mandible are the most prevalent, followed by zygomatic complex fractures [17 - 19]. Furthermore, we showed that the prevalence of mandibular and nasal bone fractures was higher in this group of patients . The literature indicates that trauma associated with ipv are less severe than those associated with other etiological factors due to the magnitude and speed of impact . Despite this fact, patients in which ipv was the etiologic factor exhibited a higher prevalence of bone fractures when compared to all other patients treated . Possibly, some patients with minor injuries do not seek medical care due to shame or fear . A high frequency of soft tissue lesions were found on the face, particularly lacerations . In the literature, the most common orofacial injuries are lip injuries, followed by other soft tissue injuries on the face, gingival and oral mucosa injuries, and teeth and/or periodontal injuries . The pattern of facial fractures is related to the type, direction and force of impact, as well as the anatomical features of the area . Ipv injuries are usually caused by punches or kicks, for which the face is the main target region . The face is probably the target for most acts of physical aggression as it is easily reached, due to its location at the same height as the aggressor s raised arm . It has also been suggested that the aggressor either consciously or unconsciously wishes to affect the victim s self - esteem . Males appear to be injured by more dangerous mechanisms and are more frequently injured by objects than females . The mandible, which is mobile and prominent on the face, is more susceptible to trauma . In the present study, the region of the mandibular angle has been considered the least resistant, mainly due to the presence of the mandibular third molar . These accidents often cause pan - facial, cranial, orthopedic, abdominal and cervical spine injuries, which may require multidisciplinary input, with significant interventions such as intubation, craniotomy and tracheostomy . Conversely, ipv tends to be associated with isolated fractures, particularly in the mandibular region . The present study confirmed that most patients were affected by a fracture in only one region of the face . A lower prevalence of injuries was recorded for other parts of body . Although the world health organization considers the response of health - care services to victims of violence to be an international priority, health professionals are generally not qualified to identify the injuries or council victims . The role of health professionals is to identify the etiology of the injury and, in cases of physical aggression, provide the victim with information on where to seek help . Moreover, health professionals are not well prepared to ask sensitive questions and are unaware of victim - support institutions . The findings of the present study demonstrated that facial trauma caused by interpersonal violence seem to be associated with a higher rate of facial fractures and lacerations when compared to all other patients with facial injuries . Male patients are more likely to exhibit severe injuries and exhibited a greater amount of bone fractures . Prominent areas of the face and neurocranium should be carefully examined as they tend to be more prone to injuries . Glaucia maria bovi ambrosano, associate professor, department of community dentistry, campinas state university piracicaba dental school, for statistical analysis assistance.
The influence of protecting groups on the reactivity and selectivity of glycosyl donors is a widely recognized and exploited phenomenon . The corresponding influence of protecting groups on the anomeric equilibrium, while evident for many years, is less widely studied and exploited . Recent examples of the latter include the recognition that 2n,3o - oxazolidinones both facilitate anomerization in the 2-amino-2-deoxyhexopyranosides and stabilize the axial over the equatorial anomer, with similar effects being apparent in hexopyranosides carrying a 2,3-o - cyclic carbonate group, and the realization that cyclic 4,6-o - acetals modulate the anomeric effect in the hexopyranoses . Our interest in the use of the n - acetyl-4o,5n - oxazolidinone protected sialyl donors, and their n - desacetyl counterparts, which have emerged as some of the most powerful and equatorially selective systems for the preparation of sialyl o - glycosides, as well as their c- and s - counterparts, prompted us to examine the effect of these systems on the anomeric equilibrium of sialyl glycosides . The magnitude of the anomeric effect is typically assessed either by mutarotation of anomeric hemiacetals or by the brnsted or lewis acid - mediated equilibration of glycosides, and such methods have been used to determine the position of the anomeric equilibrium in both n - acetyl neuraminic acid itself and its methyl glycoside, leading to the conclusion that the axial glycoside is significantly more strongly favored in the sialic acids (figure 1) than in glucose and that mutarotation of the hemiacetal takes place via a ring - opening mechanism . The attempted synthesis of a n - acetyl-4o,5n - protected sialyl hemiacetal 2 by hydrolysis of the corresponding thioglycoside 3 resulted in the formation of a complex mixture of the two anomers of 2 and of the acyclic form 4, and its hydrate 5, as determined by nmr spectroscopy, in which the latter two species predominated (figure 2). The complexity of the spectra together with the predominance of the open - chain forms revealed the strain placed on the pyranose forms by the presence of the trans - fused oxazolidinone ring, even if no distortion of the pyranose ring is evident from x - ray structures, and precluded use of the standard methods in this investigation . Compounds employed in and arising from the mutarotation of an oxazolidinone - protected sialose derivative . Accordingly, we turned our attention to the use of neutral methods for the equilibration of sialic acid glycosides such as would not involve the intermediacy of the anomeric hemiacetal and would be compatible with the readily cleaved activated oxazolidinone ring . Precedent for the equilibration of anomeric stereochemistry through the reversible homolytic cleavage and reformation of anomeric c h, c co, and c te bonds, suggested that the answer might lie in radical reactions . The need to approximate as closely as possible the properties of o - glycosidic bonds suggested the synthesis and equilibration of o - glycosyl derivatives of sterically hindered n, n - disubstituted hydroxylamines by the fischer albeit less substituted than the systems required for the radical equilibration process, glycosyl hydroxylamines are found in nature in the enediyne antibiotics calicheamicin 16 and esperamicin 7, and simple hydroxylamines have been employed as glycosidic bond surrogates in neoglycoconjugates (figure 3). Moreover, the distinct conformational preferences of the o - glycosyl hydroxylamine linkage have been advanced as a design element used by nature to assist in the binding of calicheamicin to the minor groove of dna.o - glycosyl hydroxylamines have been identified as metabolites of various drugs, including glucosides and glucuronides of the antioxidant 2,2,6,6-tetramethylpiperidin-1-ol . Natural products featuring the o - glycosyl hydroxylamine moiety . In a preliminary communication we established the concept with sialyl glycosides of 2,2,4,4-tetramethylpiperidin-1-ol in a single solvent . In this article we report in full on the synthesis of the o - sialyl glycosides of two further hydroxylamines of differing steric bulk and conduct equilibration studies in three solvents spanning a broad range of polarities . The influence of the sialyl protecting groups, solvent polarity, and hydroxylamine steric bulk on the thermal equilibration reactions of the sialyl hydroxylamines are presented and afford insight into the influence of the trans - fused oxazolidinone group in sialic acid chemistry . The lack of kinetic selectivity of sialyl anomeric radicals is discussed from the viewpoint of their conformation, which is based on literature electron spin resonance data of cognate radicals . Despite the presence of o - glycosyl hydroxylamines in various enediyne antibiotics and their use in neoglycoconjugates, literature methods for their synthesis are limited to the glycosylation of (i) various n - acylated hydroxylamines, e.g., n - hydroxyphthalimide and n - carboethoxy hydroxylamine, (ii) oximes, and (iii) nitrones, each with subsequent manipulation of the functionality at nitrogen . These methods are, however, not suitable for the synthesis of the sterically hindered o - glycosyl n, n - dialkylhydroxylamines required for this investigation, and we therefore turned to the design of alternative routes . A brief investigation of the direct glycosylation of n - hydroxy-2,2,6,6-tetramethylpiperidine with efficient sialyl donors such as developed previously in our laboratory was unfruitful, perhaps for reasons of steric hindrance, and we turned therefore to radical reactions and their relative indifference to steric constraints . Previous workers have generated sialyl anomeric radicals for the purposes of c - sialoside formation by the action of stannyl radicals on sialyl chlorides, but we have preferred simple photolysis of a readily available s - sialyl xanthate ester 8 . We note in passing that attempted photochemical equilibration of xanthate 8 resulted, in a process reminiscent of the barton fortunately, 254 nm photolysis of 8 through pyrex in a rayonnet photoreactor in the presence of the stable nitroxyl radicals tempo (10), tmio (11), and sg1 (12) resulted in the formation of the desired o - glycosyl hydroxylamines 1416 as reported in scheme 2 . Attempted application of this method to dbno 13(107) resulted only in the formation of the glycal 9 . That the photolysis of 8 can be interrupted with appropriate radical traps before the formation of glycal 9 indicates that it takes place via a two - step process giving an initial radical pair followed by disproportionation (scheme 1). This observation, which is critical to the success of the project, differs from the original conclusion of the barton and porter laboratories regarding the photoelimination of thiobenzoate esters . Those workers preferred a concerted elimination from a photochemically excited state of the thiocarbonyl ester on the basis of (i) the observation of a short - lived transient on irradiation of the thiobenzoate, and (ii) the inability to trap radical intermediates with (unspecified) radical traps . We also note, however, and more in line with the results observed here, that the photolysis of s - acyl xanthate esters is known to proceed with homolytic scission of the s - acyl bond resulting in acyl radical formation . Returning to the synthesis of the o - glycosyl hydroxylamines, when the photolysis of 8 was conducted in the presence of tempo (10) the product 14 was obtained as an approximately 1:2 mixture, favoring the isomer (-) with the axial hydroxylamino residue, in 69% yield together with 10% of the glycal . With tmio (11), the adduct 15 was obtained in 72% yield as an approximate 1:2.2: mixture . Photolysis in dichloroethane in the presence of racemic sg1 (12) gave only the glycal 9, but the anomeric radical could be intercepted in 45% yield when neat sg1 (12 equiv) was used as solvent . Finally, photolysis in the presence of di(t - butyl)nitroxide in dichloroethane afforded only the glycal 9 . Given the use of racemic sg1, there are four potential diastereomers of 16, but the product was isolated as a mixture of two predominant but inseparable isomers, the major one of which we tentatively assign as an equatorial (-) adduct and the minor as an axial (-) adduct without commenting on the configuration at the stereogenic center adjacent to the phosphoryl group . The anomeric stereochemistry in each of the adducts 1416 is assigned on the basis of the j heteronuclear coupling constant between the anomeric carboxyl carbon and the axial methylene proton at c3 in the pyranose ring, which is diagnostic in the sialic acid glycosides . These assignments are also supported by noe measurements for the axial (-) anomer of 14 and subsequent members of the same series (vide infra), which reveal the spatial proximity of the axial h6 in the pyranose ring and one or more of the methyl singlets in the hydroxylamine moiety . Resubmission of 14 to the photolysis conditions did not result in any change in the anomeric ratio consistent with the lack of a suitable chromophore, and indicating the products 1416 to be kinetic mixtures . Rate constants for the trapping of alkyl radicals by nitroxyl radicals, while high (10 to 10 s), are nevertheless below the diffusion controlled limit for stabilized alkyl radicals and are dependent on steric bulk in both the radical and the trap . The stereoselectivities observed in the formation of 1416 therefore must factor in the inherent face selectivity of the intermediate radical 18 (figure 4) and the steric bulk of the trap, which according to bowry and ingold, follows the trend dbno> tempo> tmio on the basis of kinetic trapping data (sg1 not being included in the study). We conclude therefore that radical 18 shows a modest axial selectivity that can be overridden by the use of the presumably more bulky trap sg1 . This modest axial selectivity is consistent with the work of paulsen and matschulat, who recorded an axial / equatorial trapping preference of 1.8:1 for the reaction of 18 with allyltributylstannane at 60 c in thf, but not with the work of nagy and bednarski, who reported an approximate axial / equatorial ratio of 1:1 for the same reaction conducted at room temperature under photochemical conditions . We depict radical 18 as a close to planar -radical with extensive delocalization onto the carboxyl oxygen resulting in two distinct conformers about the exocyclic c1c2 bond (figure 4) on the basis of literature esr data for simple model radicals . Thus, esr studies of the tri - o - acetyl-2-deoxy-1-glucosyl radical 19 show it to adopt a chair conformation with an out of plane bend of 6 for the anomeric carbon consistent with a radical that is 90% sp hybridized . The methoxycarbonylmethyl radical 20 is a planar -radical with extensive delocalization onto the carboxyl oxygen and a barrier to rotation about the ch2co2me bond of 11 kcal mol, and the methoxy(methoxycarbonyl)methyl radical 21 displays esr parameters that closely mirror those of 20, including the hyperfine splitting ah indicative of coupling to the ester methyl hydrogens, which is diagnostic of extensive delocalization . Finally, the close model radicals 22 and 23 also show esr spectral parameters consistent with those for 20, including hyperfine splitting by the ester methyl hydrogens and two distinct rotamers about the c5co2me bond . The two radicals 22 and 23, isomeric at the -position, adopt different conformations so as in each case to maximize the overlap of the -acetoxy group with the singly filled orbital, a phenomenon known as quasi - homoanomeric stabilization . It follows that when the -position is unsubstituted as in radicals 18 and 19, and the constraint of the quasi - homoanomeric interaction absent, the chair conformation will be retained . Predicted structure of the intermediate radical 18 and literature structures of the model radicals 1923 with key esr parameters (hyperfine splitting constants a in mt). Presumably, it is the close to -type, approximately planar structure of radical 18 and the presence of the anomeric carboxyl group that accounts for the low kinetic stereoselectivities in its reactions with radical traps, which contrast with the high axial selectivity seen in the chemistry of simple per acetylated glycosyl radicals, including 19, and their 1-alkoxy derivatives . Thus, trapping of radical 18 along the axial direction, while favored on stereoelectronic grounds and leading directly to the chair conformation of the product, involves a 1,3-diaxial interaction between the axial h4 and h6 and the partially formed c2o bond, while equatorial trapping results in 1,3-diaxial interaction of h4 and h6 with the fully formed c2-co2me bond albeit while placing the incoming bulky hydroxylamine in the sterically more accessible equatorial position (figure 5). This latter mode only begins to predominate with the most bulky nitroxide trap, sg1 (12) and the formation of 16 (scheme 2). A similar rationale has been previously invoked for exo endo - selectivity, with competing steric interactions between a full covalent bond to a substituent at the radical center and partially formed covalent bond to an incoming trap for the bicyclic radical 24 . Competing transition states for the trapping of radical 18 showing 1,3-diaxial interactions with h4 and h6, and structure of the radical 24 . With a series of glycosyl hydroxylamines in hand, and with a view to examining the influence of protecting groups on the anomeric equilibrium, a set of standard protecting group manipulations were applied to 14 and 15, as illustrated in scheme 3, giving a number of derivatives for subsequent equilibration studies . A series of equilibration reactions were then conducted by heating individually (0.050.1 m) solutions of substances 14, 15, and 3033 to 90 c in each of deuteriobenzene, deuterio-1,2-dichloroethane, and deuterioacetonitrile, with periodic monitoring by nmr spectroscopy until no further change was observed (table 1). Attempted equilibration of the sg1 derivative 16 resulted only in the formation of the elimination product 9, for which reason, coupled with the problem of extra diastereomers arising from the stereogenic center in the aglycone, the sg1 series was not pursued further . A further compound, the n, n - diacetyl tempo glycoside 25 was also subjected to equilibration in deuterio-1,2-dichloroethane (table 1); however, further work with this compound was not conducted in other solvents, nor was the tmio analogue prepared . The mechanism of the equilibration process involves reversible homolytic scission of the glycosyl - hydroxylamine c o bond to an anomeric radical and a persistent aminoxyl radical (scheme 4). Ingold radical effect that the recombination is intermolecular rather than intramolecular within the confines of the initial radical pair . The intermolecular nature of the process enables its use in combinatorial library generation, and for the present purposes it was readily demonstrated by a simple crossover experiment . Thus, heating of 15 in the presence of an equimolar amount of tempo in 1,2-dichloroethane to 90 c for nine days, after which no further change was observed, resulted in the formation of an approximately 1:2 mixture of 14 and 15, both as a mixture of anomers favoring the -anomers . The predominace of the tmio sialosides 15 over the tempo sialosides 14 in this equilibrated mixture indicates the greater thermodynamic stability of 15 over 14, which is consistent with the less hindered, more tied back nature and therefore less persistent nature of the tmio radical 11 in comparison to the tempo radical 10 . Successful achievement of the equilibration process required rigorous exclusion of oxygen, which was achieved by copious sparring with argon, and possibly moisture, as with some batches of deuterioacetonitrile a further type of byproduct exemplified by 34 was observed (scheme 5). While this thermal oxidative decarboxylation process of a sialic acid glycoside is certainly interesting in view of other sialic acid decarboxylation processes, both oxidative and reductive, we have not pursed it further at present, especially as it could be avoided by degassing and use of fresh deuterioacetonitrile . First, as compared to sialic acid itself (figure 1) and to simple methyl sialosides the o - sialyl hydroxylamines studied all show an inverted preference for the equatorial over the axial glycosides . Second, there is a solvent effect on the position of the anomeric equilibrium with the equatorial (-) anomers generally being more highly favored in the higher polarity solvents . Third, the magnitude of this solvent effect is on the whole greater in the tmio series (table 1, entries 2, 5, and 7) than in the tempo series (table 1, entries 1, 4, and 6). Fourth, the anomeric ratio at equilibrium is a function of the protecting groups employed with the same trends being seen for the both the tempo and tmio series of compounds . The general preference of the equatorial over the axial glycosides seen in table 1, which contrasts sharply with the strong anomeric effect in simple sialosides, reflects the significant steric bulk of the hindered hydroxylamine aglycones and the attendant destabilization of the axial glycosides through classical 1,3-diaxial interactions . This reversal of the anomeric preference between simple sialosides and those of hindered hydroxylamines does not detract from the validity of comparisons within the later series of compounds that form the core of this article . The observed solvent effect, which is consistent with the well - known general trend whereby the anomeric effect is greatest in less polar solvents, simply reflects the more polar nature of equatorial versus axial glycosides . The greater solvent dependence of the tmio series as compared to the tempo series of compounds presumably arises from the more polar nature of the tmio group relative to the tempo group, which is a function of the presence of the electron - withdrawing arene in the tmio moiety . The most consistent trend, which holds for both the tempo and tmio series of compounds and for all three solvents assayed, involves the change in anomeric ratio as a function of protecting groups at the o4 and n5 positions . Thus, there is generally a greater preference for the equatorial glycoside, or a smaller anomeric effect, in the 4o,5n - oxazolidinone series (table 1, entries 4 and 5), than in the n - acetyl-4o,5n - oxazolidinone series (table 1, entries 6 and 7), which in turn show a greater equatorial preference than the 4o,5n - diacetyl series (table 1, entries 1 and 2). Although less data is available, the 4o,5n,5n - triacetyl compounds (table 1, entry 3) appear to fall between the diacetyl and the n - acetyloxazolidinones . Decreasing preference for the axial (-) sialoside as a function of protecting group . Turning to the reasons underlying the general greater preference for the equatorial glycosides (figure 6) in the oxazolidinone series, we discount steric arguments based on a smaller steric clash between the aglycone and the o4 protecting group in the oxazolidinones because the usual exoanomeric effect conformation about the glycosidic bond will orient the aglycone toward the rear of the molecule and away from o4 (figure 7). Indeed, the typical exoanomeric effect conformation with an n o anomeric c ring oxygen torsion angle of -60 is evident in the x - ray crystal structure of a calicheamicin derivative, in the x - ray structures of a simple o - glycosyl hydroxylamine, and of various o - glycosyl oximes and hydroxyimides, albeit a molecular mechanics - based computational paper appears to suggest a n o anomeric c ring oxygen torsion angle of 170. newman projection about the hydroxylamine n o bond showing the typical exoanomeric effect conformation of the o - glycosyl hydroxylamines . Rather we suggest that the influence of the oxazolidinone on the anomeric effect is rooted in the highly polar nature of the oxazolidinone system with its strong dipole moment aligned with the axis of the carbonyl bond (figure 8). Thus, as illustrated in figure 9, the oxazolidinone moiety exhibits a powerful electron - withdrawing effect in the mean plane of the pyranose ring due to the alignment of the carbonyl dipole with the polar c4o4 and c5n5 bonds . This is to be contrasted with the 4o,5n - diacetyl system in which, assuming the standard minimum energy conformations of the ester and amide groups with the latter apparent from the 180 h5c5n5nh torsion angle with jnh, h5 = 10 hz, the two carbonyl dipoles are oriented in opposite directions and approximately perpendicular to the c4o4 and c5n5 bonds resulting overall in a smaller electron - withdrawing effect . The electron - withdrawing effect of the n - acetyloxazolidinone system, while still powerful because of the alignment of the oxazolidinone carbonyl with the c4o4 and c5n5 bonds in the mean plane of the pyranose ring, is moderated by the orientation of the acetyl group, which, as is clear from the available x - ray crystal structures and literature data with simple models, is oriented so as to minimize the overall dipole (figure 8) at least in weakly polar environments . Dipole moments (debye units) of open chain and cyclic carbonyl functionalities in nonpolar solvents . Orientations of the key o4 and n5 protecting group carbonyl dipole moments in the o - sialyl hydroxylamines (side chains omitted for clarity). If the anomeric effect is understood in terms of donation of electron density from a lone pair (n) on the ring oxygen into the synperiplanar c o * orbital of the axial glycosidic bond, the influence of the oxazolidinone can be attributed to the lowering of the energy of the lone pair due to the presence of the strongly electron - withdrawing group, which in turn reduces the n * interaction and so diminishes the anomeric effect . In valence bond terms this equates to the destabilization of the double bond no bond resonance form owing to the increased energy of the oxocarbenium ion arising from the presence of the strongly electron - withdrawing protecting group (figure 10). It can also be argued in view of the high dipole moment of the oxazolidinone group that there is a dipolar component to the stabilization of the equatorial glycosides . Thus, in the axial glycosides the vectors of the oxazolidinone dipole and the anomeric carbon oxygen bond dipole subtend an angle of approximately 90, whereas in the equatorial glycoside the angle subtended is approximately 60, suggesting that the equatorial glycosides will be more highly stabilized in less polar media, as is the case (table 1). A new method of equilibration of sialyl anomeric c o bonds has been developed on the basis of the fischer ingold persistent radical effect . This method enables the investigation of protecting groups on the anomeric equilibrium and is compatible with acid and base sensitive functionality . The trans - fused oxazolidinone moiety is found by this method to stabilize equatorial glycosides over their axial counterparts, i.e., to reduce the magnitude of the anomeric effect . This effect, which adds to a series of recent observations on the influence of cyclic protecting groups on the anomeric effect, is a manifestation of the strongly electron - withdrawing nature of the trans - fused oxazolidinone, (and by extrapolation trans - fused cyclic carbonates), which is a direct consequence of the highly dipolar nature of such heterocycles . The strongly electron - withdrawing nature of these groups manifest by the present study more than likely also plays an important role in the high kinetic selectivity observed in the trans - fused oxazolidinone (and cyclic carbonate)-directed -sialoside synthesis methods . The unusually poor kinetic diastereoselectivity of anomeric radicals in the sialic acid series is a function of the -type planar nature of these radicals coupled with the existence of competing 1,3-diaxial interactions in the diastereomeric transition states for the formation of both anomers.
Familial hemiplegic migraine (fhm) is an autosomal dominant rare type of migraine with aura characterized by some degree of hemiparesis . Fhm1, fhm2 and fhm3 are caused by mutations in the ion - channel genes cacna1a, atp1a2 and scn1a, respectively [24]. The majority of fhm families have pure fhm, but cerebellar ataxia is frequent in fhm families with mutation in the cacna1a gene [5, 6]. We present a norwegian family with a v628 m mutation in the atp1a2 gene (fig . 1). 1the pedigree, the arrow indicate the proband, squares are men and circles are women, filled symbols are affected, and forward slash indicate deceased the pedigree, the arrow indicate the proband, squares are men and circles are women, filled symbols are affected, and forward slash indicate deceased a 16-year - old male was referred to the neurological ward on a hot summer day due to a prolonged attack of fhm . The patient had played soccer and shortly after heading a ball he experienced a left - sided homonymous hemianopsia, followed by left - sided sensory symptoms and motor weakness, and then a contralateral migrainous headache . From the age of 2 years he experienced several attacks of sudden drowsiness, nausea and headache . Most attacks were preceded by head trauma and were interpreted as commotio . At the age of 6 years he also experienced motor weakness and reduced coordination of the hand during attacks, and at the age of 9 years this was followed by dysphasia and somnolence . Several ct - scans of the brain were normal . At the age of 10 years he had the diagnosis fhm after a prolonged attack with visual disturbances, scotoma, sensory symptoms, motor weakness, aphasia, somnolence and contralateral headache . He was discharged with 75 mg acetylsalicyl acid daily, and 160 mg propranolol daily . After initiation of the prophylactic treatment, the attack frequency 12 times per year was unchanged, but the duration of the aura symptoms and headache was reduced to 12 and 2436 h duration, respectively . Prior to hospital admission the patient barely drank or ate anything for 5 days due to nausea and severe migrainous headache . At first, he received treatment with metoclopramide, paracetamole and ketobemidon and 1,000 ml i.v . Blood test including blood count, b-12, creatine kinase, electrolytes, liver status, tsh, antinuclear antibodies, anticardiolipin antibodies and rheumatoid factor were all normal . Due to fear of vasoconstriction and migraine infarction, several measures were evaluated including continuous conservative management and verapamil treatment . After external advice continuous infusion of intravenous nimodipine 510 ml / h (dose) was initiated . The patient s symptoms worsened within hours, and the following morning he had a generalized tonic total infusion dose was then 16 mg . An acute mri showed mild swelling of the right parietal lobe (figs . 2, 3). He continued to have some degree of hemiparesis along with apraxia, homonymous hemianopsia and mild cognitive failure, i.e., 25 of 30 points on the mini mental state examination (mmse), and mildly impaired memory score and moderately impaired construction score on the cognistat . At that time a single photon emission computed tomography (spect) showed reduced perfusion in the right hemisphere, predominantly in the temporal and parietal lobe (fig . 4). The patient was discharged after 3 weeks hospitalization and re - admitted 4 weeks later on day 55 . At that time he suffered from dizziness and memory deficits . Clinical examination disclosed a partial homonymous hemianopsia, and the mmse score was 29 due to one point lost on the memory task . This provoked an attack of 1-week duration, and he was advised to refrain from intensive physical activity.fig . 2t2-weighted mri on day 10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regionsfig . 3apparent diffusion coefficient (adc) map on day 10 showing restricted diffusion in the right temporal and parietal regionsfig . 499 m tc - hmpao - spect on day 22 showing cerebral blood flow hypoperfusion in the right hemisphere, predominantly in the temporal and parietal regions t2-weighted mri on day 10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regions apparent diffusion coefficient (adc) map on day 10 showing restricted diffusion in the right temporal and parietal regions 99 m tc - hmpao - spect on day 22 showing cerebral blood flow hypoperfusion in the right hemisphere, predominantly in the temporal and parietal regions grandmother . The proband s deceased grandmother on the mother s side was reported to have had attacks of hemiplegia, but never had epileptic seizures . It usually started with unilateral sensory symptoms followed by unilateral hemiparesis and then she got a contralateral headache . Initially the attacks lasted up to 4 days and occurred from two to four times every month . The attack duration and frequency declined during adolescence, and she experienced her last attack at age 38 years . The attacks were often provoked by physical activity and the affected side varied between attacks . He experienced his first attack at the age of 13 year and had not more than 15 attacks since then . The attacks usually start with dizziness followed by homonymous visual disturbances, unilateral sensory symptoms, aphasia and unilateral weakness and then a contralateral headache . The aura symptoms usually last from 1 to 6 h, while the headache last up to 24 h. the aura symptoms change side from attack to attack . The nucleotide 1987 g> a substitution causes a change of the amino acid valine to methionine (v628 m). Both 16-year - old patients and mother had the mutation, while the elder brother was not tested . A 16-year - old male was referred to the neurological ward on a hot summer day due to a prolonged attack of fhm . The patient had played soccer and shortly after heading a ball he experienced a left - sided homonymous hemianopsia, followed by left - sided sensory symptoms and motor weakness, and then a contralateral migrainous headache . From the age of 2 years he experienced several attacks of sudden drowsiness, nausea and headache . Most attacks were preceded by head trauma and were interpreted as commotio . At the age of 6 years he also experienced motor weakness and reduced coordination of the hand during attacks, and at the age of 9 years this was followed by dysphasia and somnolence . Several ct - scans of the brain were normal . At the age of 10 years he had the diagnosis fhm after a prolonged attack with visual disturbances, scotoma, sensory symptoms, motor weakness, aphasia, somnolence and contralateral headache . He was discharged with 75 mg acetylsalicyl acid daily, and 160 mg propranolol daily . After initiation of the prophylactic treatment, the attack frequency 12 times per year was unchanged, but the duration of the aura symptoms and headache was reduced to 12 and 2436 h duration, respectively . Prior to hospital admission the patient barely drank or ate anything for 5 days due to nausea and severe migrainous headache . At first, he received treatment with metoclopramide, paracetamole and ketobemidon and 1,000 ml i.v . Blood test including blood count, b-12, creatine kinase, electrolytes, liver status, tsh, antinuclear antibodies, anticardiolipin antibodies and rheumatoid factor were all normal . Due to fear of vasoconstriction and migraine infarction, several measures were evaluated including continuous conservative management and verapamil treatment . After external advice continuous infusion of intravenous nimodipine 510 ml / h (dose) was initiated . The patient s symptoms worsened within hours, and the following morning he had a generalized tonic total infusion dose was then 16 mg . An acute mri showed mild swelling of the right parietal lobe (figs . 2, 3). He continued to have some degree of hemiparesis along with apraxia, homonymous hemianopsia and mild cognitive failure, i.e., 25 of 30 points on the mini mental state examination (mmse), and mildly impaired memory score and moderately impaired construction score on the cognistat . At that time a single photon emission computed tomography (spect) showed reduced perfusion in the right hemisphere, predominantly in the temporal and parietal lobe (fig . 4). The patient was discharged after 3 weeks hospitalization and re - admitted 4 weeks later on day 55 . At that time he suffered from dizziness and memory deficits . Clinical examination disclosed a partial homonymous hemianopsia, and the mmse score was 29 due to one point lost on the memory task . This provoked an attack of 1-week duration, and he was advised to refrain from intensive physical activity.fig . 2t2-weighted mri on day 10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regionsfig . 3apparent diffusion coefficient (adc) map on day 10 showing restricted diffusion in the right temporal and parietal regionsfig . 499 m tc - hmpao - spect on day 22 showing cerebral blood flow hypoperfusion in the right hemisphere, predominantly in the temporal and parietal regions t2-weighted mri on day 10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regions apparent diffusion coefficient (adc) map on day 10 showing restricted diffusion in the right temporal and parietal regions 99 m tc - hmpao - spect on day 22 showing cerebral blood flow hypoperfusion in the right hemisphere, predominantly in the temporal and parietal regions the proband s deceased grandmother on the mother s side was reported to have had attacks of hemiplegia, but never had epileptic seizures . Mother . It usually started with unilateral sensory symptoms followed by unilateral hemiparesis and then she got a contralateral headache . Initially the attacks lasted up to 4 days and occurred from two to four times every month . The attack duration and frequency declined during adolescence, and she experienced her last attack at age 38 years . The attacks were often provoked by physical activity and the affected side varied between attacks . He experienced his first attack at the age of 13 year and had not more than 15 attacks since then . The attacks usually start with dizziness followed by homonymous visual disturbances, unilateral sensory symptoms, aphasia and unilateral weakness and then a contralateral headache . The aura symptoms usually last from 1 to 6 h, while the headache last up to 24 h. the aura symptoms change side from attack to attack . The nucleotide 1987 g> a substitution causes a change of the amino acid valine to methionine (v628 m). Both 16-year - old patients and mother had the mutation, while the elder brother was not tested . The phenotype genotype correlation of the identified mutation (v628 m) is strengthened by the fact that it has also been reported in a turkish family . The norwegian family had attacks of pure fhm, with pronounced intra - familial variation in frequency, severity and duration of attacks . Physical activity provoked attacks in the affected alive, and mild head trauma provoked attacks in the proband . Mutations in the atp1a2 gene usually cause pure fhm, but alternating hemiplegia of childhood, benign familial infantile convulsions, cerebellar symptoms, severe episodic neurological deficits and mental retardation have also been reported [818]. The hypovolemia caused by dehydration and arterial hypotension probably added to sustain the prolonged fhm attack, but these factors alone has not previously been sufficient to cause a generalized tonic the seizure is most likely caused by nimodipine, as the proband and his mother both noticed worsening of the aura symptoms after initiation of the intravenous nimodipine infusion . Nimodipine is a calcium - channel blocker that dilates cerebral vessels and it is used to prevent delayed cerebral ischemia following subarachnoid hemorrhage . In theory nimodipine might prevent the vasospasm of fhm by lowering ca - influx . However, the continuous intravenous infusion of nimodipine probably aggravated the arterial hypotension, a known side effect . This caused additional hypoperfusion and hypoxia in the right hemisphere, which lead to a generalized tonic clonic seizure and a secondary edema as shown on mri and spect (for details see above). We conclude that nimodipine is contraindicated in the management of prolonged fhm attack, and recommend conservative management except from supplement of sufficient intravenous fluid in nauseated patients in order to avoid hypovolemia.
A few days later, four more cases occurred among postal workers at the brentwood mail processing facility in the washington area . The appearance of new cases in different locations around the country suggested contamination with b. anthracis of several postal facilities . In each location, the population potentially exposed was large . Public health, criminal investigation, and postal authorities at local, state and federal levels investigated the suspected bioterrorism event . Within hours of the discovery of clusters of cases, the centers for disease control and prevention(cdc) deployed teams to four different locations to support state and local investigations . Four letters containing anthrax spores were known to have been postmarked and processed in the hamilton mail processing center (hmpc) located in hamilton township, a suburb of trenton, new jersey . The hmpc is part of a complicated network of stations and buildings designed to move millions of pieces of mail per day . It is not a typical post office, but a very large facility located in a 281,387 square foot building with many operations areas containing mechanized equipment . It was typically staffed by 250 employees per shift and visited by numerous others in the course of processing 2 million items per day . On october 19,,,,, field investigation in trenton was initiated by a cdc team composed of an epidemiologist, environmental experts, occupational physicians, epidemic intelligence service (eis) officers, a public health informatics fellow, and support personnel . The team was headquartered in the new jersey department of health and senior services (njdhss) and had access to the local area network and computing environment of the department . This article describes the experience of the informatics fellow and the new jersey geographic information system(gis) coordinator during the first two weeks of the investigation in assessing information system needs and organizing on - site computer support for the new jersey arm of the investigation . Early in the investigation, the number of people exposed to anthrax was estimated to be large . The response team planned to sample for anthrax contamination in the facility; identify the exposed population; locate, culture, and give prophylactic antibiotics to those most likely to be exposed; and monitor the exposed and treated persons over time for potential long - term effects . Information on each person and sample was to be obtained, stored, analyzed, and augmented over time . For epidemiologic investigation and management purposes, summary reports had to be generated frequently . Because of the large number of agencies involved and the rapid progress of events, a system of document management for the many drafts of guidelines and other communications was also needed . Immediate needs for output included lists of employees, clients, visitors, and other potentially exposed persons . The lists were used by eis officers for individual follow - up and were linked to the map of floor plans of the postal facility and environmental sampling results to help identify persons with the greatest likelihood of exposure . Investigators and managers needed summary information, including the percentage of exposed patients receiving medication and the distribution of exposed persons by county . The media relations' officer of the team produced reports as often as twice a day for the public . Information needs of the emergency response team in new jersey not all potential data sources were available during the investigation . The database system in the building was unavailable, as the computers had been turned off due to concern that fans in the computers might create air currents that would jeopardize safety or alter environmental sampling results . The list of employees in the facility computers was therefore unavailable to the investigators and had to be assembled from other sources . Digital autocad drawing files of the facility were also stored on a workstation within the facility, and were likewise unavailable a situation that complicated the production of accurate and representative floor - plan maps . Laboratory results included cultures of both human and environmental samples performed by the new jersey state laboratory and cdc laboratories . Managing reports from samples processed in different laboratories required linking of multiple unique identifiers . Electronic documents that were produced or revised during the investigation included survey results, background documents, guidelines, antibiotic stockpile logistics, and press releases . Frequent revisions made it necessary to identify and provide access to the latest documents and to track document revisions . The information system developed consisted of three main components: a database of possibly exposed persons, a gis of the physical plant and equipment, and a document repository . The exposed - person database required merging of files in multiple formats, manual data entry, rapid analysis, and frequent production of reports . Gis work was done using floor plans produced in arcview gis 3.2a (environmental systems research institute, inc ., redlands, ca) by screen - digitizing scanned not - to - scale xerographic copies of autocad drawings of the postal building . Documents were made available to the team by using web pages (html) on the new jersey department of health intranet to link to the latest copies of the files . Building outlines, walls, work areas, equipment, furniture, the ventilation system and diffusers, and environmental sampling locations were digitized and stored as shapefiles, a format used by both the arcview gis and epi map software . The geographical features were then linked to environmental sampling data to specific locations in the building using epi map . A unique sample identification number was used as the key for linking the shapefile to the environmental sample database (figure 1). As the map creation process progressed over the 2-week period, additional, larger size paper drawings were provided to which a complete floor plan and heating, ventilation, and air conditioning systems components could be added . Another version of the floor plan has been published and is available on the internet . (environmental.png) preliminary sampling at the mail facility 1 to support investigations in other facilities where the letters were manipulated, digitized floor plans of the carteret and west trenton post offices were produced from rudimentary sketches . Since epi map is public domain software, it was installed on a number of computers without need for licensing making a gis tool freely available to the investigators . The document repository was created using html on an intranet site inside the firewall of the njdhss . The web page included up - to - date information such as press releases and a list of activities for the day . It was updated twice a day or more often when important information had to be communicated to the rest of the investigation team . Each link was checked on a daily basis to ensure that the correct document was being accessed . Early in the investigation, the number of people exposed to anthrax was estimated to be large . The response team planned to sample for anthrax contamination in the facility; identify the exposed population; locate, culture, and give prophylactic antibiotics to those most likely to be exposed; and monitor the exposed and treated persons over time for potential long - term effects . Information on each person and sample was to be obtained, stored, analyzed, and augmented over time . For epidemiologic investigation and management purposes, summary reports had to be generated frequently . Because of the large number of agencies involved and the rapid progress of events, a system of document management for the many drafts of guidelines and other communications was also needed . Immediate needs for output included lists of employees, clients, visitors, and other potentially exposed persons . The lists were used by eis officers for individual follow - up and were linked to the map of floor plans of the postal facility and environmental sampling results to help identify persons with the greatest likelihood of exposure . Investigators and managers needed summary information, including the percentage of exposed patients receiving medication and the distribution of exposed persons by county . The media relations' officer of the team produced reports as often as twice a day for the public . Information needs of the emergency response team in new jersey not all potential data sources were available during the investigation . The database system in the building was unavailable, as the computers had been turned off due to concern that fans in the computers might create air currents that would jeopardize safety or alter environmental sampling results . The list of employees in the facility computers was therefore unavailable to the investigators and had to be assembled from other sources . Digital autocad drawing files of the facility were also stored on a workstation within the facility, and were likewise unavailable a situation that complicated the production of accurate and representative floor - plan maps . Laboratory results included cultures of both human and environmental samples performed by the new jersey state laboratory and cdc laboratories . Managing reports from samples processed in different laboratories required linking of multiple unique identifiers . Electronic documents that were produced or revised during the investigation included survey results, background documents, guidelines, antibiotic stockpile logistics, and press releases . Frequent revisions made it necessary to identify and provide access to the latest documents and to track document revisions . The information system developed consisted of three main components: a database of possibly exposed persons, a gis of the physical plant and equipment, and a document repository . The exposed - person database required merging of files in multiple formats, manual data entry, rapid analysis, and frequent production of reports . Gis work was done using floor plans produced in arcview gis 3.2a (environmental systems research institute, inc ., redlands, ca) by screen - digitizing scanned not - to - scale xerographic copies of autocad drawings of the postal building . Documents were made available to the team by using web pages (html) on the new jersey department of health intranet to link to the latest copies of the files . Building outlines, walls, work areas, equipment, furniture, the ventilation system and diffusers, and environmental sampling locations were digitized and stored as shapefiles, a format used by both the arcview gis and epi map software . The geographical features were then linked to environmental sampling data to specific locations in the building using epi map . A unique sample identification number was used as the key for linking the shapefile to the environmental sample database (figure 1). As the map creation process progressed over the 2-week period, additional, larger size paper drawings were provided to which a complete floor plan and heating, ventilation, and air conditioning systems components could be added . Another version of the floor plan has been published and is available on the internet . (environmental.png) preliminary sampling at the mail facility 1 to support investigations in other facilities where the letters were manipulated, digitized floor plans of the carteret and west trenton post offices were produced from rudimentary sketches . Since epi map is public domain software, it was installed on a number of computers without need for licensing making a gis tool freely available to the investigators . The document repository was created using html on an intranet site inside the firewall of the njdhss . The web page included up - to - date information such as press releases and a list of activities for the day . It was updated twice a day or more often when important information had to be communicated to the rest of the investigation team . Each link was checked on a daily basis to ensure that the correct document was being accessed . Recent publications on informatics and bioterrorism have focused on detection of bioterrorist events . Some have reviewed the informatics contribution to past emergencies and formulated proposals for various kinds of surveillance and/or response [6 - 10]. The goal of this paper is to present needs, difficulties, and solutions encountered in the first 2 weeks of a single field investigation . Every epidemic investigation is unique, but a comprehensive review of activities carried out during an actual investigation may focus discussion on understanding and improving the informatics response in such events . At the beginning of this investigation, although the njdhss provided a secure computer environment in which to work, many elements of the system had to be designed and set up during the investigation . Much of the base information did not exist (e.g., gis of the hamilton facility) or had been made unavailable by the event under investigation (e.g., hamilton facility autocad digital drawing files and employee lists). As in most field investigations, both data sources and hypotheses shifted and evolved over the course of the investigation . During an investigation, information is critical for decision - making processes and for detection of cases, identification of risk factors, and managing prevention and control measures . In the first hours or days, the emergency response team is required to design and implement an information system capable of providing reliable, timely information . Experience in developing such " emergency information systems " and with the software being used plays an important role in determining the time needed to complete the task . In this event, close collaboration between the epidemiologist and informaticians was supported by software designed for field investigation and for exchange of data with other commercial products . The presence of trained professionals from a gis center was an important element in being able to map the site of the investigation . Use of industry standard shapefiles and microsoft access databases allowed working copies to be distributed and used in public domain software as needed . A database system was operational within two days of the beginning of operations, and the gis elements evolved over a two - week period as floor plans became available . Potential problems identified initially included lags in entry of clinical data, lack of access to data sources because computer systems were disrupted or inaccessible, and the variety of formats and platforms that were designed for other purposes . At least one source produced data in different formats on different days . In field investigation, the informatics environment is defined by the agencies, networks, and individual computers from which data are obtained and with which exchange of data occurs . Unless the entire investigation is internal to a single agency or company, each of the partners state, county, health care facility, law enforcement agency, industry, or other entity has its own informatics environment . The standards most likely to be common to all the partners are those of the computer industry, such as the internet, microsoft windows, and popular gis standards . Those conducting the investigation must be prepared to adapt to the standards used by the data source partners rather than the reverse . In this investigation, existing database systems provided useful information, but the data items were available on paper, in excel files, in text files, in access files, and in arcview shapefiles . The informatician is responsible for merging and integrating elements stored in different database formats to make the information available to the emergency response team . The need for a speedy public health response is a challenge for informaticians because the steps that are typically time consuming, such as requirements generation, analysis, design, implementation, and testing of a new system, must be completed in hours rather than in weeks or months . In this experience, some parts of the system were functional within two days after arrival at the investigation site . Epi info 2000 was used to set up databases for manual data entry and updating and for importing and merging data provided by collaborating sources in a variety of microsoft windows formats . The system developed took advantage of several features available in epi info through its use of commercial component software and computer industry standards: rapid development of a relational database, in which identifying information was localized to a single table for security purposes importation of data from several of the 20 different file formats that can be read in epi info 2000 the ability to perform data management interactively but to preserve and replay the steps through automatically generated programs or scripts the ability to link and display microsoft access data with arcview shapefiles to create maps rapid development, with minimal coding, simplifying the testing and debugging process the ability to add or delete variables from a database merely by revising a form on the screen . The availability of analytic output and epidemiologic statistics in the same program used for data management interaction with high end gis programs and transition of database management to a commercial program through epi info's incorporation of gis and windows standards public domain status of the program allowed downloading the latest version from the internet and distributing it to any number of partners without licensing difficulties geographical and spatial analysis has gained importance in public health during the last few years . The availability of maps during the investigation can be attributed to the fact that the njdhss had made resources available from its gis center . The gis professional was able to create the shapefiles while the rest of the emergency response team (ert) focused on data collection and data management . Once created, maps were widely available for use among all members of the team, and epi map 2000 could be used to link data in microsoft access files to the shapefiles of the building floor plans . The main use of gis in this investigation was to create a relatively detailed base map and to locate environmental sampling and provide spatial analysis and visual support to the investigation . We identified and located all samples taken in the different facilities . In a matter of days we connected the database system with the map files to provide up to date information regarding sample processing and results . The maps created provided a new perspective of the magnitude of contamination inside the facility and also identified non - contaminated areas . We extended the mapping capabilities to illustrate the area of coverage of the active and passive surveillance systems implemented in surrounding communities for the detection of new cases (figure 2). The surveillance system was originally restricted to nine counties and was latter extended to others . Changes in the number of centers included in the surveillance system were reflected in different versions of the same map to document changes over time . (surveillance.png) surveillance of new cases in new jersey because of the diversity of the data sources during the investigation, the database system required frequent maintenance . We were able to automate most of the data management routines using epi info program (scripting) files and to produce automatic reports . Laboratory results were available through the local area network in the new jersey state health department where the team worked . Although we might have been able to connect to the laboratory system to obtain real - time updates, for security reasons, the laboratory results for the investigation were exported and became available to the emergency response team at the end of each day . There was therefore a modest, but significant delay in availability of laboratory information . During emergency field situations, any system intended for use by more than one group of people should be easy to use, compatible with other software, and well documented . The database was documented using entity relationship (er) diagrams (figure 3). Epi info has a command to display the structure of a table, making it easy to document the system for the replacement team that arrived after two weeks . This second team preferred to work with microsoft access rather than epi info, but the fact that epi info 2000 stores data in generic access 97 tables facilitated the transition . (databasemodel.eps) example of the entity relationship diagram for the database system implemented document tracking and version control are important when geographically separated teams are working on the same set of problems . In this case each one of the field sites had a set of experts developing documents and recommendations that applied to the whole event and not only to the geographic area of investigation . It was important to keep the most recent set of recommendations available to all members of the emergency response team for decision - making and information dissemination . During the investigation, we were able to access the most recent documents with minimal effort, using links in a web page . A more sophisticated version could be developed to allow tracking changes in a document over time . Emergency situations require rapid design and implementation of databases to provide reliable information in the field . Having the ability to respond rapidly to emergency situations requires planning and preparedness . Key elements in preparation are in paying sufficient attention to informatics support and having both staff and software that can adapt quickly to new situations and computing environments . The likelihood of success is greater if the same people and software provide support for more routine epidemic investigations in this field investigation, the informatics environment included excel spreadsheets, files in several other formats, paper questionnaires and computer printouts, an existing gis capability using arcview, and the need for frequent revisions in database structure, frequent summary reports, and protection of personal identifiers . Software for field use must be flexible enough to interact with and unite numerous data sources and computing environments . Investigators must be prepared for new situations, and be ready to adapt new techniques and software as the needs of an investigation unfold . The traditional steps in systems development of requirements gathering, analysis, design, implementation, and testing must be traversed in rapid sequence during the first few hours or days of an investigation and repeated again as the system evolves and capabilities are added . More formalized approach to systems development needs to be either extensively modified or compressed to account for the time - critical nature of the situation . The use of commercial software standards such as windows file formats, web pages, and mainstream gis programs facilitates the rapid implementation of systems in the field . The design of systems for emergencies should include methods for storing and maintaining version control of documents and images in addition to more structured data . A floor plan of the contaminated hamilton facility was available only as an incomplete and poor quality 8 1/2 11 inch photocopy of the original' e' sized autocad drawing . It was scanned as a tiff image, imported into arcview gis 3.2 software and used as a backdrop to create shapefiles of the building outline, walls, operational areas and equipment placements, and niosh and fbi sampling locations . Shapefiles were developed using a scanned copy of the floor plan of each facility and digitized using arcview . The elements of information to be displayed were divided into several layers showing samples taken by different agencies and their location . After completion, the shapefiles were linked to the data using epi map 2000, a component of epi info that is arcviewcompatible . Elements of information stored in databases created in epi info 2000 were sent directly to epi map for displaying (figure 1). The process was automated by using scripts or programs . To obtain data on persons possibly exposed, a liaison between the u.s . He was able to obtain ms - excelfiles or computer printouts listing personnel for each of the facilities under investigation . Additional information was added to those lists to record presence in the facility during the exposure period, sex of the employee, and post exposure prophylaxis (pep). Most of the clinical follow - up services, including pep, were provided by the local hospital . The informatics department of the hospital extracted relevant clinical data from the database system and made them available to the investigation team as excel tables on some days and comma delimited text files on other days . Primary data items collected by eis officers and other staff were entered into epi info 2000 or into excel and then imported into epi info 2000 (microsoft access 97) format . Potentially exposed populations such as regular visitors and relatives of postal workers were identified and manually entered into the main database as the investigation proceeded . Demographic characteristics of potentially exposed persons were assembled from various sources, including the local hospital where pep was administered . When pep was offered to a large group of people, tracking of different treatment protocols and their results became necessary . Paper forms were designed not to become obsolete if protocols are modified during the follow up, and similar revisions in the database were expected . Database management was done in epi info 2000, the windows version of epi info . Epi info 2000 was developed at cdc from commercial component software and visual basic and offers compatibility with microsoft access files and 20 other standard database formats . It includes a mapping program that uses the arcview compatible shapefile format and allows the display of data from access tables on shapefiles . Most of the basic statistics needed for reporting and analysis are built in so that we did not have to spend time coding or debugging algorithms . The data collection and data management tools in epi info do not require expert database managers . The database system consisted of a central database designed and maintained in epi info 2000 . The unique key at first contained information (was " intelligent "), but we designed a mechanism to replace intelligent keys by non - intelligent keys . The intelligent key contained facility code, deployment place (nj) exposed unique identifier, and postal facility involved . We included the deployment site in the key assuming that the information would be linked with other sites at a latter time . From the beginning of the operation, security was a concern . Because of the complexity of the investigation we decided that all sensitive information defined as information that would allow identification of specific individuals would be handled separately from the rest of the database . We created a single flat table with all identifiers and related the table to the rest of the database system . With that structure identifiers were not sent to cdc headquarters in atlanta, for example, and sensitive data were protected by the security features of the njdhss computing environment . A commercial html editor (microsoft front page 2000) was used for the maintenance of the document repository web page . Web pages included in the system did not contain any special scripting that required an advanced web programming techniques . The level of html coding was basic, and mostly inserted automatically by front page . The document repository allowed coding and storing versions of documents during the progress of the investigation . The document check - in process included a simple computer - based form with the name of the document, the author, and a time stamp . The first two elements were used to create a catalog, and the timestamp was used for versioning control . A floor plan of the contaminated hamilton facility was available only as an incomplete and poor quality 8 1/2 11 inch photocopy of the original' e' sized autocad drawing . It was scanned as a tiff image, imported into arcview gis 3.2 software and used as a backdrop to create shapefiles of the building outline, walls, operational areas and equipment placements, and niosh and fbi sampling locations . Shapefiles were developed using a scanned copy of the floor plan of each facility and digitized using arcview . The elements of information to be displayed were divided into several layers showing samples taken by different agencies and their location . After completion, the shapefiles were linked to the data using epi map 2000, a component of epi info that is arcviewcompatible . Elements of information stored in databases created in epi info 2000 were sent directly to epi map for displaying (figure 1). To obtain data on persons possibly exposed, a liaison between the u.s . Postal service and the epidemiologic team he was able to obtain ms - excelfiles or computer printouts listing personnel for each of the facilities under investigation . Additional information was added to those lists to record presence in the facility during the exposure period, sex of the employee, and post exposure prophylaxis (pep). Most of the clinical follow - up services, including pep, were provided by the local hospital . The informatics department of the hospital extracted relevant clinical data from the database system and made them available to the investigation team as excel tables on some days and comma delimited text files on other days . Primary data items collected by eis officers and other staff were entered into epi info 2000 or into excel and then imported into epi info 2000 (microsoft access 97) format . Potentially exposed populations such as regular visitors and relatives of postal workers were identified and manually entered into the main database as the investigation proceeded . Demographic characteristics of potentially exposed persons were assembled from various sources, including the local hospital where pep was administered . When pep was offered to a large group of people, paper forms were designed not to become obsolete if protocols are modified during the follow up, and similar revisions in the database were expected . Database management was done in epi info 2000, the windows version of epi info . Epi info 2000 was developed at cdc from commercial component software and visual basic and offers compatibility with microsoft access files and 20 other standard database formats . It includes a mapping program that uses the arcview compatible shapefile format and allows the display of data from access tables on shapefiles . Most of the basic statistics needed for reporting and analysis are built in so that we did not have to spend time coding or debugging algorithms . The data collection and data management tools in epi info do not require expert database managers . The database system consisted of a central database designed and maintained in epi info 2000 . The unique key at first contained information (was " intelligent "), but we designed a mechanism to replace intelligent keys by non - intelligent keys . The intelligent key contained facility code, deployment place (nj) exposed unique identifier, and postal facility involved . We included the deployment site in the key assuming that the information would be linked with other sites at a latter time . From the beginning of the operation, security was a concern . Because of the complexity of the investigation we decided that all sensitive information defined as information that would allow identification of specific individuals would be handled separately from the rest of the database . We created a single flat table with all identifiers and related the table to the rest of the database system . With that structure identifiers were not sent to cdc headquarters in atlanta, for example, and sensitive data were protected by the security features of the njdhss computing environment . A commercial html editor (microsoft front page 2000) was used for the maintenance of the document repository web page . Web pages included in the system did not contain any special scripting that required an advanced web programming techniques . The level of html coding was basic, and mostly inserted automatically by front page . The document repository allowed coding and storing versions of documents during the progress of the investigation . Members of the team were instructed to check in documents on a regular basis . The document check - in process included a simple computer - based form with the name of the document, the author, and a time stamp . The first two elements were used to create a catalog, and the timestamp was used for versioning control . Jz performed the analysis of informatics needs, set up the epi info portions of the field system and the document management system, and drafted the manuscript . Rs developed the arcview gis system and shapefiles used in the investigation, and contributed to the manuscript . Ad provided advice on the analysis of informatics needs and the development of the epi info applications through daily telephone conversations and wrote portions of the manuscript . Esri (environmental systems research institute) provided a special license for the free distribution of mapobjects software within epiinfo / epimap 2000.
Duodenal ulcer perforation is still a common complication of chronic peptic ulcer disease . Despite the wide spread use of anti - secretory and h. pylori eradication therapy most of the literature pertaining to the situation in the developed world, showed that the disease is largely confined to the elderly patients taking ulcerogenic medications. [15] the situation in the developed nations is summed up by johnson: the surgeon s major role in the management of peptic ulcer disease will be the performance of life - saving emergency operations in the elderly unfit patient . In contrast to the developing world, the patients are younger and have a long life - time of potentially useful activity ahead of them[18] as anti - secretory and anti - h . Pylori eradication drugs are been freely used, we had expected an improvement in this disease condition . Hence, we set out in this review to look at the prevalence, pattern of presentation, risk factors and management outcome in the study environment and we also set out to look at the management pattern if sufficient for a semi - urban area in a developing world . This review was conducted at the federal medical centre ido - ekiti, southwest nigeria . The case records of all patients with clinically established diagnosis of pdus, managed in this center from january 2004 to december 2011 were reviewed . The diagnostic protocol for every patient at presentation included: the clinical findings, abdominal ultrasound, and chest x - rays which in some cases gave further credence to the diagnosis . Full blood count, urea and electrolytes, creatinine, and urinalysis results were documented . The patients were optimized for surgery with intravenous fluids, antibiotics, and urethral catheterization . Clinical and intra - operative findings, treatment outcome and follow - ups were all evaluated . None of the patients had a prior diagnosis of their ulcer by an upper gastro - intestinal endoscopy (ugie) before presentation . Although, 20 (66.6%) of them had peptic ulcer symptoms with, inadequate or no medical treatment . The remainder, 10 (33.3%) presented for the first time with perforation, with no prior treatment for peptic ulcer disease . The notable risk factor was the free abuse of herbal concoction (admixture of local gin, spices, roots and bitters) for body pains . Fifteen patients took alcohol and only two took non - steroidal anti - inflammatory drugs [table 1]. A good proportion of the patients 76.6% were referred to us from private health institutions, while the rest came to our center from their homes . After adequate resuscitation, all the 30 patients had emergency surgery of simple closure of their perforations, re - enforced with an omental patch and thorough peritoneal larvage . Most of the 26 managed patients had a favorable outcome; were discharged home and followed - up at the clinic for 4 - 6 weeks but later lost to follow - up . Pdu is still a major complication of chronic peptic ulcer disease seen quite often in the study center, as well as other centers of the world as a frequent cause of acute abdomen . Although experience from some parts of nigeria showed that gastric outlet obstruction has overtaken pdu in incidence, it is still frequently seen in many centers in nigeria, as well as many centers in the developed world. [17] this study has also highlighted that the perforation affected mostly the younger age group (in their 3 -6 decade) [table 2], which is in keeping with studies in many centers in the developing world. [111] much has been written about non - operative management of pdu in the western world . Since the early works of croft et al ., there has been considerable interests in the non - operative management of pdus . In a randomized trial comparing surgical and non - surgical therapy, for pdu, they showed a mortality rate of 5% in each group and no difference in morbidity ., have also shown that non - operative therapy could be effective in selected patients . They showed that as much as 81% of their patients were treated conservatively, but 43% later had surgery . Some writers have claimed that virtually all patients with pdu, could be managed conservatively . However, conservative management has not been accepted by many workers; whereas, most surgeons have difficulty in understanding how a patient who has wide spread peritonitis with food debris widely distributed throughout the abdominal cavity can improve without operation . Undoubtedly, there are patients with small leaks, from a perforation and with relatively mild peritoneal contamination who may be managed conservatively; these are in a minority . We did not consider this treatment option because of the delay in presentation, 2 - 7 days as well as the socio - economic implications of long hospitalization . All patients were operated as soon as they were resuscitated and optimized for surgery . In the pre h. pylori era, in the developed world, it has been shown that only 30% of patients with pdu treated with simple closure and thorough peritoneal larvage had good long - term result . In the present era, (h. pylori era), studies have shown, that operation may achieve long - term satisfactory results, only when h. pylori infections, present in 50 - 70% of patients with pdu is totally eliminated . However, all have agreed that it is sometimes difficult to determine the h. pylori status of patients having emergent operation for perforated d.u . In this study, none of the patients had an ugie done before presentation and surgery; and in all of them, their h. pylori status was not known: although, 20 (66.6%) of them were erratically treated at one time or the other for peptic ulcer disease symptoms . All patients had simple closure of their perforation with omental patch reinforcement, thorough peritoneal larvage and h. pylori eradication therapy was instituted . This is only palliative and is sufficient for the elderly patients in the developed world . Patients who have suffered one perforation may suffer another one if anti - secretory drugs are not maintained; as it were, for life . This life - long treatment is obviously not within the reach of an average patient in the developing world . In this study, most workers, from the developing world have also shown that pdu, affects mostly the younger age group. [18] all have attested to the fact that this group are not likely to be anti eradication drug compliant for life . The costs of these drugs, re - infection by h. pylori organisms even after the eradication and dyspeptic symptoms after surgery,[18] shows that this palliative surgery (omental patch) is obviously not appropriate for a resource poor center, vis - a - vis for the developing world; that a shift towards the free use of definitive ulcer surgery should be the goal . Dempsey summed it all, using the possible h. pylori infection eradication as an excuse not to do a definitive ulcer operation in any patient with perforated d - u is irrational . Therefore the various surgical options that have been proposed by workers in the field include: omental patch closure + highly selective vagotomyomental patch closure + bilateral truncal vagotomy (btv)resectional surgery + truncal vagotomypyloroplasty + btv . Dakubo et al ., writing from a west african sub - region had shown statistically that age greater than 60 years, alcohol intake and resectional surgery are key factors in predicting post - operative morbidity and mortality . It stands therefore to reason that since the majority of the patients in the study center vis - a - vis developing world are below 60 years, they stand to benefit from a curative definitive ulcer surgery . Pyloroplasty (inco - operating the perforation) and btv is quite safe and highly recommended because of its short operative time, simplicity and easily reproducible by all, even trainee surgeons . Omental patch closure + highly selective vagotomy omental patch closure + bilateral truncal vagotomy (btv) resectional surgery + truncal vagotomy pyloroplasty + btv . Pdu disease is still a frequent cause of acute abdomen in many centers of the developing world where it affects mostly the youths . The relative high costs of triple regime therapy for h. pylori eradication, fear of re - perforation and easy loss to follow - up, call for an urgent re - appraisal of the present method of surgical treatment . Therefore, a more liberal role for wide spread definitive ulcer surgery is suggested for most of our young patients.
Hemophilia b is a chronic hereditary disease characterized by a deficiency in factor ix (fix) activity . It is less common than hemophilia a which is due to factor viii (fviii) deficiency . According to the world federation of hemophilia global survey of 2014, 178,500 persons were identified with hemophilia across 106 countries, of whom only 16.1% had hemophilia b.1 hemophilia b is an x - linked disease . Missense, frameshift, and nonsense mutations are the most frequently seen, and deletions are rarely described.2 the severity of the disease is related to clotting factor levels . The disease is classified as severe, moderate, or mild when the clotting factor levels are <1, 15, or> 5, respectively.3 people with mild - to - moderate hemophilia rarely bleed unless after trauma or invasive procedures . Frequency and severity of bleeding is greatest in severe hemophilia b. data from the us show that only 36% of patients with hemophilia b have the severe form of the disease.4 recurrent and spontaneous bleeding into joints and/or soft tissues since early infancy are hallmarks of severity . Gastrointestinal and intracranial bleeding can be life threatening.3 repeated bleeding into joints without adequate treatment can result in crippling chronic joint disease, pain, and reduced quality of life.3,58 the introduction and availability of factor concentrate has dramatically improved the treatment of hemophilia with a significant decrease in morbidity and mortality, and an increase in quality of life.9 early on - demand treatment of acute bleeding episodes decreases the number of joint deformities compared to untreated or minimally treated patient.10,11 nevertheless, long - term musculoskeletal follow - up of on - demand treatment in hemophilia showed a progressive deterioration of the joint functions in these patients . As on - demand treatment appears to be clearly suboptimal, prophylaxis meaning preventive use of clotting factor given at regular intervals was proposed and adopted since several decades in sweden.12 in 1994, the medical and scientific advisory council (masac) of the us national hemophilia foundation issued guidelines stating that prophylaxis with twice- or thrice - weekly fix infusion, at a dose of 2540 iu / kg, should be considered the optimal treatment for hemophilia b.13 the european pediatric network for haemophilia management define primary prophylaxis as regular treatment started after first episode of bleeding and secondary prophylaxis as regular or intermittent regular treatment started after several episodes of bleeding.14,15 the world federation of hemophila (wfh) guidelines recommend prophylaxis to prevent bleed and joint destruction and preserve normal musculoskeletal function . Moreover, it is advisable to give it to all patients before performing any activities associated with an increased risk of trauma . Prophylaxis should be the state - of - art treatment.3 despite the guidelines, prophylaxis has not been universally adopted . Medical, psychosocial, and cost controversies limit the implementation of prophylaxis.16 in the canadian 2006 survey, only 32% of patients with severe hemophilia b received prophylaxis, while 69% of patients with hemophilia a received the treatment . This difference was particularly notable in the 02 years age - group (17% in hemophilia b versus 53% in hemophilia a) and also present in patients> 18 years of age (20% versus 55%). This difference could be explained by a less severe phenotype, less frequent joint bleeds, later presentation in life, paucity of clinical trials studying the use of prophylaxis, or simply a current treatment tradition.17 moderate hemophilia b is diagnosed much more frequently than the severe (37% versus 27%) form . Positivity to cross - reacting material, which corresponds to measurable level of fix antigen in plasma, is frequently observed in hemophilia b. this could be explained by the frequency of non - null mutation . Most mutations in hemophilia b are missense mutations and these result in mild - to - moderate form of the disease and account for variable factor levels in plasma.18 studies reporting prophylactic use of fix in hemophilia b patients are limited in number, and concern mainly small populations within larger clinical trials.1922 benefits of prophylaxis remain questionable even if intuitively the tendency is in favor.23,24 there is currently no evidence to suggest or refute prophylaxis.25 following a 6-month regimen of prophylaxis with recombinant fix (rix), patients achieved significantly meaningful improvements in physical health - related quality of life (hrqol). Those who switched from intermittent prophylaxis to on - demand prophylaxis experienced improvements in physical and mental hrqol.26 there is no general agreement on the optimal prophylaxis regimen, and some schemes differ from those proposed by the consensus perspectives on prophylactic therapy for hemophilia, held in london, september 2021, 2002.14 given the paucity of controlled clinical trials regarding prophylaxis in subjects with hemophilia b, choice of treatment approach is limited to clinical judgment, clinical experience, and interpretation of currently available data based on pharmacokinetics (pk), bleeding phenotype, and type of fix used . In recent years, greater attention has been paid to the pk of coagulation factor, dosing intervals, and trough levels . Pk parameters had an increasing role in management choice even if there was no clear evidence on clinical efficacy . They are routinely measured in clinical practice to guide treatment dosing, particularly for primary prophylaxis, and in connection with surgery.27 in 2011, the european medicines agency considered appropriate incremental recovery, half - life, area under the curve (auc), and clearance to be the most important surrogate end points for efficacy of fix products.28 pk parameters of fix are not well characterized or widely investigated . The pk of fix are more complicated than those of fviii, and also differ between plasma - derived and recombinant forms resulting in variation between studies . Fix has a longer half - life in the circulation than fviii.29 according to sampling time, the half - life of fix could be different . After administration of 75 iu / kg of nonacog alfa to patients aged 1261 years, the half - life was reported as 2224 hours when sampled at 72 hours,30 and after administration of 50 iu / g, the half - life was reported as 34 hours when sampled at 96 hours.31 a sampling schedule at 48 hours gives shorter half - life . In powell s study, the residual fix observed 1 week after administration of fix is supportive of longer half - life.31 strategy to achieve prophylaxis is to maintain the plasma level of factor activity at or above 1 u / dl.3 in current bio - assays, accuracy can be expected to be rather poor at the conventional target of 1 u / dl, which is the lower limit of the assays.32 this issue with assaying was clearly demonstrated by the discrepancies in factor levels in patients with mild - to - moderate and severe hemophilia in the uk national external quality assessment service data.33 furthermore, measuring plasma fix activity may not fully reflect the hemostatic efficacy of infused fix . Fix is a small protein (55 kda) with access to both intravascular and extravascular compartments.34 a number of clinical observations in hemophilia b patients suggest the presence of extravascular pool of fix . Continuous infusion of fix results in reduced dose requirement of fix to maintain a level of 100%.35 furthermore, there are experimental data demonstrating that fix binds to collagen iv . This may be a source of hemostatically active fix which is not measurable by plasma assays.34,36 experiments on baboons showed that perfusion of excess bovine fix increased the circulating fix proportionally . Reanalysis of these data suggests that the extravascular component contains at least threefold more fix than that present in the circulation.34 two experiments on mouse demonstrated the affinity of fix to collagen iv and its impact on hemostasis . In a knock - in mouse model expressing a fix variant with reduced affinity to collagen iv, hemostasis was delayed despite high levels of fix . Bolus infusion of fix variant with enhanced collagen affinity demonstrates a prolonged hemostatic effect in hemophilia b mouse . This hemostatic effect persists days after plasma levels reach less than 1%.37 fix (and fixa) binds rapidly and reversibly to vascular endothelium and subendothelial extracellular matrix . This is mediated by the interaction of specific residues in the fix gla domain with collagen iv, located predominantly in the basement membrane.38,39 the clinical significance of maintaining a 1% trough level is mainly applicable in fviii deficiency . Such evidence does not exist in fix deficiency and is widely debated.40 in a cohort of 64 patients (51 hemophilia a and 13 hemophilia b) described by ahnstrom et al, some patients did not bleed with a trough level less than 1%, while others bled with a trough level higher than 3% . They found that there is no relationship between factor level and incidence of bleed and that the correlation was very weak . Authors suggest that dosing in prophylactic treatment should be individualized.41 due to interindividual variations in pk parameters, targeting a particular trough level may not be appropriate for every individual.41 relationship between fix trough levels and therapeutic outcomes has not been confirmed in clinical trials.40 baseline factor levels are not the only determinants of bleeding phenotype in hemophilia . With the same factor level, the severity and frequency of bleeding may be different for people with hemophilia.42 in hemophilia b, where limited data exist, there is a need to strike a balance between clinical and pk end points in the evaluation of clinical efficacy.43 in hemophilia, bleeding frequency is considered a key indicator of the efficacy of the treatment regimen . The hemophilia severity score (hss) has been developed as a method to predict bleeding score in hemophilia.44 it includes in its assessment the annual joint bleeding rate, annual factor consumption, and wfh orthopedic score . This score was used by vyas et al to evaluate 178 hemophilia patients without inhibitors in a single us center (hemophilia a [n=139], hemophilia b [n=39]). They found heterogeneity of hemophilia phenotype and widespread variability in the hss values of patients with the same baseline factor activity.45 data from a single - center cohort study of 171 patients with severe hemophilia a and b in the netherlands demonstrated the importance of clinical issues in determining the phenotype . They found that age at first joint bleed was an indicator of bleeding pattern.46 there was higher annual clotting factor consumption in subjects who experienced a joint bleed at an early age comparing to those who experienced later in life.47 according to the united kingdom haemophilia centres doctors organisation (ukhcdo) annual report, a large variation in rates of clotting factor concentrate consumption in patients with same diagnosis was also widely observed.48 clinical assessment of the frequency and severity of bleeds remain an important measure of the efficacy of treatment for hemophilia b. the role of pk - guided therapy and its relationship to clinical efficacy remain open issues to be established . Historically, effective treatment of hemophilia b started with fresh frozen plasma and prothrombin complex concentrate . In the early 1990s, highly purified plasma - derived factor concentrate was introduced.49,50 since then, large numbers have become commercially available.51 improvement in sourcing and purification procedure of blood components and the introduction of different steps of viral inactivation increased the safety of plasma - derived concentrates.52 despite these improvements, concerns remain and transfusion - transmitted emerging pathogens like prion and unencapsulated virus should not be overlooked.5356 in 1997, the first purified rfix product, nonacog alfa, received us food and drug administration (fda) approval followed in 2014 by another rfix product, bax326 . These rfix products have a lower risk of pathogenic contamination and have low activated fix activity, which confers low thrombogenic potential in humans.2 traditionally, in vivo recovery (ivr) is the parameter used to characterize the pk properties of coagulation factors.57 following bolus injection, recombinant protein has lower ivr compared to plasma - derived fix . Terminal half - life is similar for the two products (1719 hours).40,58,59 pk studies that enrolled 308 subjects with hemophilia b in three comparative studies showed that the ivr of rfix is approximately one - third to one - half that of plasma - derived fix.58,60,61 this disparity in ivr was also noted by kisker et al in a double - blind crossover study of plasma - derived fix and rfix in 15 subjects older than 5 years with severe hemophilia b.62 this disparity could be related to baseline fix levels, body weight, and age.58,63 while some individuals have identical recoveries with both types of concentrates, others have pronounced differences.64 a recent survey in italy indicated that plasma - derived fix and rfix are used in similar doses with similar outcomes.65 despite disparity in recovery, recombinant coagulant factors are nowadays the preferred treatment option for hemophiliacs and, in particular, for pediatric patients mainly in light of the wide margin of safety and efficacy . Wfh who and nnh propose the twice- or thrice - weekly prophylaxis dose of 50 iu / kg . This regimen was extrapolated from the fviii prophylaxis experience, early pk data, and the few studies done on fix . In the original phase 3 nonacog alfa study in previously treated subjects aged 12 years, the mean dose was 40.3 iu / kg administered 23 times weekly.22 in the phase 3 trial of reformulated nonacog alfa in previously treated subjects, the median dose was 51.7 iu / kg administered 23 times weekly.30 frequency and dose of prophylaxis were determined by the investigator in these two studies . Both the studies demonstrated safety and efficacy in persons with hemophilia b. however, rigorous regimens of prophylaxis are difficult and adherence remains a problem.66 only 45% of individuals with severe hemophilia b are currently receiving prophylaxis in the us.67 cost of clotting factor is certainly a main obstacle for the implementation of such management . It is the largest predictor of overall cost in the care of people with hemophilia.68,69 however, the leading reasons for lack of adherence to the prescribed regimen are time consumption, convenient access to peripheral vein, forgetfulness, and the feeling of being healthy with the disappearance of symptoms with time.70,71 many patients find it very difficult to spend 1520 minutes every morning to mix and infuse intravenous factor . This is certainly more difficult in children where venous access could be difficult and where central venous access devices are associated with the concomitant risk of infection and thrombosis . In an european survey carried out by de moerloose between october 2005 and september 2006, an interview was conducted with 30 patients in each of six european countries (france, germany, italy, spain, sweden, and the uk), resulting in a total of 180 patients . The feeling of reduction, fluctuation, or disappearance of symptoms was cited in 38% of cases, forgetfulness in 36%, lack of time for the treatment in 30%, and convenience in 30% . Forgetfulness was the reason most often cited by patients on prophylaxis (46%).72 therefore, new approaches of management must be proposed to achieve adherence and make patients lives easier . Despite recent promising success in gene therapy for hemophilia b, a cure for hemophilia is not yet available.73,74 novel clotting formulations with longer half - life represent a major advance but at a high financial cost . It has the potential of increasing convenience to patient, owing to fewer infusions, less preparation and infusion time, and preservation of venous access in those for whom venous access is a challenge.75 there is some evidence to support such a regimen . According to powel s study, residual fix was observed 1 week after administration of fix.31 maintaining a 1% trough level is widely debated in fix deficiency.40 experimental data demonstrate that fix has access to both intravascular and extravascular compartments with potential availability of clinically significant extravascular stores of fix.34 patients with hemophilia b seem to have less severe phenotype and less frequent joint bleed than those with hemophilia a. vyas et al found heterogeneity of hemophilia phenotype and widespread variability in the hss values of 178 patients with the same baseline factor activity.45 in 1976, morfini et al published the first randomized trial comparing once - weekly and twice - weekly prophylaxis with on - demand treatment in ten subjects with hemophilia b over 1 year.20 a total of 7.5 iu / kg was administered to the twice - weekly group and 15 iu / kg to the once - weekly group . The two prophylactic groups had a significant reduction in bleeding episodes when compared to the observed 1-year period preceding the trial (p<0.005). Patients having measurable fix in plasma for a higher number of days bleed less.20 in 2014, valentino et al published a phase 4 multicenter, randomized, open - label, four - period crossover study that evaluated the efficacy and safety of nonacog alfa as a prophylaxis regimen (50 iu / kg twice - weekly or 100 iu / kg once - weekly) compared with on - demand administration.75 inclusion criteria were being male, aged 665 years, with severe - to - moderately severe hemophilia (fix 2), and 12 or more bleeding episodes, including six or more hemarthroses episodes . The primary end point was the annual bleeding rate (abr) of two prophylactic regimens compared to on - demand treatment . The abr was 35.1, 2.6, and 4.6 for the on - demand treatment, 50 iu / kg twice a week, and 100 iu / kg once a week, respectively . A significant difference was observed between the two prophylaxis groups and the on - demand group (p<0.0001) with a reduction of bleed of 89.4% in favor of the prophylaxis group . No patient discontinued the prophylactic treatment because of inefficacy or less - than - expected effect . There was no serious adverse event, or report of thrombogenicity, or occurrence of inhibitors.75 the use of high dose of fix (100 iu / kg per dose) was of concern as it resulted in thrombogenicity . In a review conducted by rendo et al of five clinical trials in patients with hemophilia b, patients who received 100 iu / kg per dose of nonacog alfa reported no thrombotic events based on clinical findings and laboratory markers such as thrombin antithrombin (tat) and d - dimers.77 this finding was also confirmed in a study conducted by kavakli et al . This study evaluated the efficacy and safety of once - weekly prophylaxis with 100 iu / kg nonacog alfa compared with on - demand treatment in 25 adolescent and adult patients for a period of 52 weeks.76 at visits 2 and 4 of the study, assessment of thrombosis was done with monitoring of d - dimers and tat iii complex . None of the patients developed clinical thrombotic event and there was no inhibitor occurrence . Otherwise, mean abr was significantly lower in the prophylactic group comparing to the on - demand treatment group . There was 3.6 bleeding events for the prophylactic group and 32.9 events for the on - demand group (p<0.0001). Most interestingly, eight of 17 patients had a fix superior to 2 u / dl 1 week after dosing . No severe adverse event was observed and once - weekly prophylaxis was well tolerated.77 the studies of valentino75 and kavakli76 showed efficacy of once - weekly prophylaxis with no unexpected safety issues, no thrombotic event, no occurrence of inhibitors, and significant reduction in bleeding episodes . The population size of the studies was small, although this is quite frequent in hemophilia b studies . Forty - one patients were evaluated in the twice - weekly prophylaxis group of the valantino study and 25 patients in the kavakli study . Besides, the study conducted by valantino was of short duration . This is relatively a too short period to confirm safety and efficacy of a product . The kavakli study was conducted over a 52-week period and supports safety and efficacy of this regimen in a better way . Otherwise, the two studies were conducted in previously treated patients and 92% of the patients included in the kavakli study had tertiary prophylaxis . The benefit of such regimen in untreated patient or in primary prophylaxis or secondary prophylaxis remains to be demonstrated . Effect of switching from twice - weekly to once - weekly prophylaxis is not yet known . In 1994, the medical and scientific advisory council (masac) of the us national hemophilia foundation issued guidelines stating that prophylaxis with twice- or thrice - weekly fix infusion, at a dose of 2540 iu / kg, should be considered the optimal treatment for hemophilia b.13 the european pediatric network for haemophilia management define primary prophylaxis as regular treatment started after first episode of bleeding and secondary prophylaxis as regular or intermittent regular treatment started after several episodes of bleeding.14,15 the world federation of hemophila (wfh) guidelines recommend prophylaxis to prevent bleed and joint destruction and preserve normal musculoskeletal function . Moreover, it is advisable to give it to all patients before performing any activities associated with an increased risk of trauma . Prophylaxis should be the state - of - art treatment.3 despite the guidelines, prophylaxis has not been universally adopted . Medical, psychosocial, and cost controversies limit the implementation of prophylaxis.16 in the canadian 2006 survey, only 32% of patients with severe hemophilia b received prophylaxis, while 69% of patients with hemophilia a received the treatment . This difference was particularly notable in the 02 years age - group (17% in hemophilia b versus 53% in hemophilia a) and also present in patients> 18 years of age (20% versus 55%). This difference could be explained by a less severe phenotype, less frequent joint bleeds, later presentation in life, paucity of clinical trials studying the use of prophylaxis, or simply a current treatment tradition.17 moderate hemophilia b is diagnosed much more frequently than the severe (37% versus 27%) form . Positivity to cross - reacting material, which corresponds to measurable level of fix antigen in plasma, is frequently observed in hemophilia b. this could be explained by the frequency of non - null mutation . Most mutations in hemophilia b are missense mutations and these result in mild - to - moderate form of the disease and account for variable factor levels in plasma.18 studies reporting prophylactic use of fix in hemophilia b patients are limited in number, and concern mainly small populations within larger clinical trials.1922 benefits of prophylaxis remain questionable even if intuitively the tendency is in favor.23,24 there is currently no evidence to suggest or refute prophylaxis.25 following a 6-month regimen of prophylaxis with recombinant fix (rix), patients achieved significantly meaningful improvements in physical health - related quality of life (hrqol). Those who switched from intermittent prophylaxis to on - demand prophylaxis experienced improvements in physical and mental hrqol.26 there is no general agreement on the optimal prophylaxis regimen, and some schemes differ from those proposed by the consensus perspectives on prophylactic therapy for hemophilia, held in london, september 2021, 2002.14 given the paucity of controlled clinical trials regarding prophylaxis in subjects with hemophilia b, choice of treatment approach is limited to clinical judgment, clinical experience, and interpretation of currently available data based on pharmacokinetics (pk), bleeding phenotype, and type of fix used . In recent years, greater attention has been paid to the pk of coagulation factor, dosing intervals, and trough levels . Pk parameters had an increasing role in management choice even if there was no clear evidence on clinical efficacy . They are routinely measured in clinical practice to guide treatment dosing, particularly for primary prophylaxis, and in connection with surgery.27 in 2011, the european medicines agency considered appropriate incremental recovery, half - life, area under the curve (auc), and clearance to be the most important surrogate end points for efficacy of fix products.28 pk parameters of fix are not well characterized or widely investigated . The pk of fix are more complicated than those of fviii, and also differ between plasma - derived and recombinant forms resulting in variation between studies . Fix has a longer half - life in the circulation than fviii.29 according to sampling time, the half - life of fix could be different . After administration of 75 iu / kg of nonacog alfa to patients aged 1261 years, the half - life was reported as 2224 hours when sampled at 72 hours,30 and after administration of 50 iu / g, the half - life was reported as 34 hours when sampled at 96 hours.31 a sampling schedule at 48 hours gives shorter half - life . In powell s study, the residual fix observed 1 week after administration of fix is supportive of longer half - life.31 strategy to achieve prophylaxis is to maintain the plasma level of factor activity at or above 1 u / dl.3 in current bio - assays, accuracy can be expected to be rather poor at the conventional target of 1 u / dl, which is the lower limit of the assays.32 this issue with assaying was clearly demonstrated by the discrepancies in factor levels in patients with mild - to - moderate and severe hemophilia in the uk national external quality assessment service data.33 furthermore, measuring plasma fix activity may not fully reflect the hemostatic efficacy of infused fix . Fix is a small protein (55 kda) with access to both intravascular and extravascular compartments.34 a number of clinical observations in hemophilia b patients suggest the presence of extravascular pool of fix . Continuous infusion of fix results in reduced dose requirement of fix to maintain a level of 100%.35 furthermore, there are experimental data demonstrating that fix binds to collagen iv . This may be a source of hemostatically active fix which is not measurable by plasma assays.34,36 experiments on baboons showed that perfusion of excess bovine fix increased the circulating fix proportionally . Reanalysis of these data suggests that the extravascular component contains at least threefold more fix than that present in the circulation.34 two experiments on mouse demonstrated the affinity of fix to collagen iv and its impact on hemostasis . In a knock - in mouse model expressing a fix variant with reduced affinity to collagen iv, hemostasis was delayed despite high levels of fix . Bolus infusion of fix variant with enhanced collagen affinity demonstrates a prolonged hemostatic effect in hemophilia b mouse . This hemostatic effect persists days after plasma levels reach less than 1%.37 fix (and fixa) binds rapidly and reversibly to vascular endothelium and subendothelial extracellular matrix . This is mediated by the interaction of specific residues in the fix gla domain with collagen iv, located predominantly in the basement membrane.38,39 the clinical significance of maintaining a 1% trough level is mainly applicable in fviii deficiency . Such evidence does not exist in fix deficiency and is widely debated.40 in a cohort of 64 patients (51 hemophilia a and 13 hemophilia b) described by ahnstrom et al, some patients did not bleed with a trough level less than 1%, while others bled with a trough level higher than 3% . They found that there is no relationship between factor level and incidence of bleed and that the correlation was very weak . Authors suggest that dosing in prophylactic treatment should be individualized.41 due to interindividual variations in pk parameters, targeting a particular trough level may not be appropriate for every individual.41 relationship between fix trough levels and therapeutic outcomes has not been confirmed in clinical trials.40 baseline factor levels are not the only determinants of bleeding phenotype in hemophilia . With the same factor level, the severity and frequency of bleeding may be different for people with hemophilia.42 in hemophilia b, where limited data exist, there is a need to strike a balance between clinical and pk end points in the evaluation of clinical efficacy.43 in hemophilia, bleeding frequency is considered a key indicator of the efficacy of the treatment regimen . The hemophilia severity score (hss) has been developed as a method to predict bleeding score in hemophilia.44 it includes in its assessment the annual joint bleeding rate, annual factor consumption, and wfh orthopedic score . This score was used by vyas et al to evaluate 178 hemophilia patients without inhibitors in a single us center (hemophilia a [n=139], hemophilia b [n=39]). They found heterogeneity of hemophilia phenotype and widespread variability in the hss values of patients with the same baseline factor activity.45 data from a single - center cohort study of 171 patients with severe hemophilia a and b in the netherlands demonstrated the importance of clinical issues in determining the phenotype . They found that age at first joint bleed was an indicator of bleeding pattern.46 there was higher annual clotting factor consumption in subjects who experienced a joint bleed at an early age comparing to those who experienced later in life.47 according to the united kingdom haemophilia centres doctors organisation (ukhcdo) annual report, a large variation in rates of clotting factor concentrate consumption in patients with same diagnosis was also widely observed.48 clinical assessment of the frequency and severity of bleeds remain an important measure of the efficacy of treatment for hemophilia b. the role of pk - guided therapy and its relationship to clinical efficacy remain open issues to be established . Historically, effective treatment of hemophilia b started with fresh frozen plasma and prothrombin complex concentrate . In the early 1990s, highly purified plasma - derived factor concentrate was introduced.49,50 since then, large numbers have become commercially available.51 improvement in sourcing and purification procedure of blood components and the introduction of different steps of viral inactivation increased the safety of plasma - derived concentrates.52 despite these improvements, concerns remain and transfusion - transmitted emerging pathogens like prion and unencapsulated virus should not be overlooked.5356 in 1997, the first purified rfix product, nonacog alfa, received us food and drug administration (fda) approval followed in 2014 by another rfix product, bax326 . These rfix products have a lower risk of pathogenic contamination and have low activated fix activity, which confers low thrombogenic potential in humans.2 traditionally, in vivo recovery (ivr) is the parameter used to characterize the pk properties of coagulation factors.57 following bolus injection, recombinant protein has lower ivr compared to plasma - derived fix . Terminal half - life is similar for the two products (1719 hours).40,58,59 pk studies that enrolled 308 subjects with hemophilia b in three comparative studies showed that the ivr of rfix is approximately one - third to one - half that of plasma - derived fix.58,60,61 this disparity in ivr was also noted by kisker et al in a double - blind crossover study of plasma - derived fix and rfix in 15 subjects older than 5 years with severe hemophilia b.62 this disparity could be related to baseline fix levels, body weight, and age.58,63 while some individuals have identical recoveries with both types of concentrates, others have pronounced differences.64 a recent survey in italy indicated that plasma - derived fix and rfix are used in similar doses with similar outcomes.65 despite disparity in recovery, recombinant coagulant factors are nowadays the preferred treatment option for hemophiliacs and, in particular, for pediatric patients mainly in light of the wide margin of safety and efficacy . Wfh who and nnh propose the twice- or thrice - weekly prophylaxis dose of 50 iu / kg . This regimen was extrapolated from the fviii prophylaxis experience, early pk data, and the few studies done on fix . In the original phase 3 nonacog alfa study in previously treated subjects aged 12 years, the mean dose was 40.3 iu / kg administered 23 times weekly.22 in the phase 3 trial of reformulated nonacog alfa in previously treated subjects, the median dose was 51.7 iu / kg administered 23 times weekly.30 frequency and dose of prophylaxis were determined by the investigator in these two studies . Both the studies demonstrated safety and efficacy in persons with hemophilia b. however, rigorous regimens of prophylaxis are difficult and adherence remains a problem.66 only 45% of individuals with severe hemophilia b are currently receiving prophylaxis in the us.67 cost of clotting factor is certainly a main obstacle for the implementation of such management . It is the largest predictor of overall cost in the care of people with hemophilia.68,69 however, the leading reasons for lack of adherence to the prescribed regimen are time consumption, convenient access to peripheral vein, forgetfulness, and the feeling of being healthy with the disappearance of symptoms with time.70,71 many patients find it very difficult to spend 1520 minutes every morning to mix and infuse intravenous factor . This is certainly more difficult in children where venous access could be difficult and where central venous access devices are associated with the concomitant risk of infection and thrombosis . In an european survey carried out by de moerloose between october 2005 and september 2006, an interview was conducted with 30 patients in each of six european countries (france, germany, italy, spain, sweden, and the uk), resulting in a total of 180 patients . The feeling of reduction, fluctuation, or disappearance of symptoms was cited in 38% of cases, forgetfulness in 36%, lack of time for the treatment in 30%, and convenience in 30% . Forgetfulness was the reason most often cited by patients on prophylaxis (46%).72 therefore, new approaches of management must be proposed to achieve adherence and make patients lives easier . Despite recent promising success in gene therapy for hemophilia b, a cure for hemophilia is not yet available.73,74 novel clotting formulations with longer half - life represent a major advance but at a high financial cost . It has the potential of increasing convenience to patient, owing to fewer infusions, less preparation and infusion time, and preservation of venous access in those for whom venous access is a challenge.75 there is some evidence to support such a regimen . According to powel s study, residual fix was observed 1 week after administration of fix.31 maintaining a 1% trough level is widely debated in fix deficiency.40 experimental data demonstrate that fix has access to both intravascular and extravascular compartments with potential availability of clinically significant extravascular stores of fix.34 patients with hemophilia b seem to have less severe phenotype and less frequent joint bleed than those with hemophilia a. vyas et al found heterogeneity of hemophilia phenotype and widespread variability in the hss values of 178 patients with the same baseline factor activity.45 in 1976, morfini et al published the first randomized trial comparing once - weekly and twice - weekly prophylaxis with on - demand treatment in ten subjects with hemophilia b over 1 year.20 a total of 7.5 iu / kg was administered to the twice - weekly group and 15 iu / kg to the once - weekly group . The two prophylactic groups had a significant reduction in bleeding episodes when compared to the observed 1-year period preceding the trial (p<0.005). Patients having measurable fix in plasma for a higher number of days bleed less.20 in 2014, valentino et al published a phase 4 multicenter, randomized, open - label, four - period crossover study that evaluated the efficacy and safety of nonacog alfa as a prophylaxis regimen (50 iu / kg twice - weekly or 100 iu / kg once - weekly) compared with on - demand administration.75 inclusion criteria were being male, aged 665 years, with severe - to - moderately severe hemophilia (fix 2), and 12 or more bleeding episodes, including six or more hemarthroses episodes . The primary end point was the annual bleeding rate (abr) of two prophylactic regimens compared to on - demand treatment . The abr was 35.1, 2.6, and 4.6 for the on - demand treatment, 50 iu / kg twice a week, and 100 iu / kg once a week, respectively . A significant difference was observed between the two prophylaxis groups and the on - demand group (p<0.0001) with a reduction of bleed of 89.4% in favor of the prophylaxis group . No patient discontinued the prophylactic treatment because of inefficacy or less - than - expected effect . There was no serious adverse event, or report of thrombogenicity, or occurrence of inhibitors.75 the use of high dose of fix (100 iu / kg per dose) was of concern as it resulted in thrombogenicity . In a review conducted by rendo et al of five clinical trials in patients with hemophilia b, patients who received 100 iu / kg per dose of nonacog alfa reported no thrombotic events based on clinical findings and laboratory markers such as thrombin antithrombin (tat) and d - dimers.77 this finding was also confirmed in a study conducted by kavakli et al . This study evaluated the efficacy and safety of once - weekly prophylaxis with 100 iu / kg nonacog alfa compared with on - demand treatment in 25 adolescent and adult patients for a period of 52 weeks.76 at visits 2 and 4 of the study, assessment of thrombosis was done with monitoring of d - dimers and tat iii complex . None of the patients developed clinical thrombotic event and there was no inhibitor occurrence . Otherwise, mean abr was significantly lower in the prophylactic group comparing to the on - demand treatment group . There was 3.6 bleeding events for the prophylactic group and 32.9 events for the on - demand group (p<0.0001). Most interestingly, eight of 17 patients had a fix superior to 2 u / dl 1 week after dosing . No severe adverse event was observed and once - weekly prophylaxis was well tolerated.77 the studies of valentino75 and kavakli76 showed efficacy of once - weekly prophylaxis with no unexpected safety issues, no thrombotic event, no occurrence of inhibitors, and significant reduction in bleeding episodes . The population size of the studies was small, although this is quite frequent in hemophilia b studies . Forty - one patients were evaluated in the twice - weekly prophylaxis group of the valantino study and 25 patients in the kavakli study . Besides, the study conducted by valantino was of short duration . This is relatively a too short period to confirm safety and efficacy of a product . The kavakli study was conducted over a 52-week period and supports safety and efficacy of this regimen in a better way . Otherwise, the two studies were conducted in previously treated patients and 92% of the patients included in the kavakli study had tertiary prophylaxis . The benefit of such regimen in untreated patient or in primary prophylaxis or secondary prophylaxis remains to be demonstrated . Effect of switching from twice - weekly to once - weekly prophylaxis is not yet known . The current unmet challenges with regard to management of hemophilia b are financial sustainability, inhibitor occurrence, and poor uptake of prophylaxis . Prophylactic regimen must be more convenient to patients and their caregivers, while being effective and safe to improve adherence to treatment . Further research is needed to identify ideal prophylaxis regimens for patients with varying severity of hemophilia b. once - weekly prophylaxis with rix may be a viable treatment option for patients with hemophilia b. prophylaxis regimen at 100 iu / kg may be a safe and effective alternative to twice - weekly prophylaxis at 50 iu / kg . However, it remains unknown how patients with a less severe bleeding phenotype or with better baseline joint status might respond to once - weekly dosing with rix . In many regions of the world, fix doses less than the standard 50 iu / kg twice - weekly dose are followed . To minimize the cost of treatment and to tailor individual treatment to clinical response, it would be of interest to investigate the effect of once - weekly administration of lower doses of fix.
Protease inhibitors have been purified from an array of leguminous and nonleguminous species encompassing torresea cearensis, erythrina caffra, dolichos lablab, crotalaria paulina, medicago scutellata [5, 6], canavalia gladiata, pisum sativum, dimorphandra mollis, swartzia pickellii, psophocarpus tetragonolobus, delonix regina, poecilanthe parviflora, adenanthera pavonina, cajanus cajan, dolichos biflorus, phaseolus acutifolius, arachis hypogaea, leucaena leucocephala, bauhinia bauhinioides, bauhinia variegata, bauhinia ungulata, vigna unguiculata, lens culinaris, glycine max, peltophorum dubium, pithecellobium dulce, glycine soja, and barley . Protease inhibitors impair the activity of insect midgut proteases and thus adversely affect protein digestion and health in insects . They represent one of the multitude of entomotoxic proteins [30, 31]. In addition to insecticidal activity [3235], protease inhibitors demonstrate antiproliferative and antitumor activities [3645]. The objective of the present study was to isolate and characterize proteins with protease inhibitory activity from white cloud beans . An aqueous extract of the beans (250 g) was produced by blending in distilled water (3 ml / g) followed by centrifugation (14000 g for 25 minutes at 4c). The resulting supernatant was applied to a 5 20 cm column of deae - cellulose (sigma) in 10 mm tris - hcl buffer (ph 7.4). After elution of unadsorbed proteins (fraction d1), the column was eluted successively with 0.2 m nacl and 1 m nacl in the tris - hcl buffer . Fraction d2 eluted with 0.2 m nacl was dialyzed to remove nacl and then subjected to affinity chromatography on a 5 15 cm of affi - gel blue gel (bio - rad) in 10 mm tris hcl buffer (ph 7.4). The unadsorbed fraction (b1) was dialyzed against 10 mm nh4oac buffer (ph 5) and then applied to a 2.5 20 cm column of sp - sepharose (ge healthcare). After elution of unadsorbed proteins (fraction s1), the column was eluted with a 0 - 1 m nacl concentration gradient in the nh4oac buffer . The first and second adsorbed fractions (sp2 and sp3) were then further purified by gel filtration on a superdex 75 hr 10/30 column (ge healthcare) in 0.2 m nh4hco3 buffer (ph 8.5) using an akta purifier (ge healthcare). The molecular mass of the isolated proteins was determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page) following the procedure of laemmli and favre . Gel filtration on an fplc - superdex 75 column, previously calibrated with molecular mass marker proteins (ge healthcare), was employed to determine the molecular mass of the protein . The n - terminal sequence of the protein was analyzed by using a hewlett - packard hp g1000a edman degradation unit and an hp 1000 hplc system . The test sample (20 l) was added to 160 l of a 1% casein solution in 0.1 m tris - hcl buffer (ph 7.4). Trypsin (20 l of a 0.5 mg / ml solution) was then added and the mixture was incubated at 37c for 15 minutes followed by addition of trichloroacetic acid (0.4 ml, 5%) that was added to bring the reaction to an end . After centrifugation the absorbance of the resulting supernatant, which indicates the amount of casein fragments produced by trypsin, was read at 280 nm . The% inhibition of trypsin activity is equal to the% decrease in absorbance of the supernatant . The isolated trypsin inhibitor (2.5 m) was treated with dithiothreitol (dtt) at the final concentration 2.5, 10 and 40 mm for 5, 20, and 80 minutes at 37c . Soybean trypsin inhibitor from sigma (2.5 m) was similarly treated and used as a positive control . The reaction was terminated by adding iodoacetamide at twice the amount of thiol functions at each dtt concentration . Residual trypsin inhibitor activity was measured at ph 7.4 as described above in assay for trypsin inhibitory activity . The highest iodoacetamide concentration used in the test was devoid of any effect on trypsin activity and the trypsin inhibitory activity of isolated trypsin inhibitor and soybean trypsin inhibitor . The cell lines l1210 (human leukemia) and mbl2 (murine lymphoma) were obtained from american type culture collection . The cell line was maintained in dulbecco modified eagles' medium (dmem) supplemented with 10% fetal bovine serum (fbs), 100 mg / l streptomycin, and 100 iu / ml penicillin, at 37c in a humidified atmosphere of 5% co2 . Cells (1 10) in their exponential growth phase were seeded into each well of a 96-well culture plate (nunc, denmark) and incubated for 3 hours prior to addition of the trypsin inhibitor . Incubation was performed for an additional 48 hours . Radioactive precursor, 1 ci ([h - methyl]-thymidine, from ge healthcare), was then introduced to each well and the incubation continued for 6 hours . The assay for hiv reverse transcriptase inhibitory activity was carried out in view of the report that trypsin inhibitors manifest this activity [50, 51]. It was conducted according to instructions supplied with the assay kit from boehringer mannheim (germany). The assay makes following use of the ability of reverse transcriptase to synthesize dna, commencing from the template / primer hybrid poly (a) oligo (dt) 15 . The digoxigenin- and biotin - labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule, which is freshly synthesized by the reverse transcriptase (rt). The detection and quantification of synthesized dna as a parameter for rt activity are based on a sandwich elisa protocol . Biotin - labeled dna binds to the surface of microtiter plate modules that have been precoated with strepatavidin . In the following step, an antibody to digoxigenin, conjugated to peroxidase, binds to the digoxigenin - labeled dna . In the last step the peroxidase enzyme affects the cleavage of the substrate, yielding a colored reaction product . The absorbance of the sample at 405 nm which is directly correlated to the level of rt activity can be measured using a microtiter plate (elisa) reader . A fixed amount (46 ng) of recombinant hiv-1 reverse transcriptase was used . The inhibitory activity of the trypsin inhibitor was calculated as percent inhibition compared to a control without the trypsin inhibitor . The plasmid that expressed his - tagged wild - type hiv-1 integrase, pt7 - 7-his (y \| tx)-hiv-1-in, was a generous gift from professor s.a . A 1-liter culture of e. coli bl21 (de3) cells containing the expressing plasmid was grown at 37c until it reached od600 0.7 - 0.8 . Cells were induced by addition of 0.8 mm iptg (isopropyl--d - thiogalactopyranoside) and harvested, after 4 hours of incubation, by centrifugation at 6000 g for 10 minutes at 4c . Cells were suspended at a concentration of 10 ml / g wet cell paste in 20 mm tris - hcl buffer (ph 8.0) containing 0.1 mm edta, 2 mm -mercaptoethanol, 0.5 m nacl, and 5 mm imidazole . After incubation at 4c for 1 hour, the lysate was sonicated and centrifuged at 40 000 g at 4c for 20 minutes . The pellet was homogenized in 50 ml buffer a (20 mm tris - hcl, ph 8.0, 2 m nacl, 2 mm -mercaptoethanol) which contained 5 mm imidazole . The suspension was rotated at 4c for 1 hour and cleared by centrifugation at 40 000 g at 4c for 20 minutes . The supernatant was applied to a 1 ml chelating sepharose (ge healthcare) column charged with 50 mm imidazole . The column was eluted with five column volumes of buffer a containing 5 mm imidazole, and the protein was eluted with three column volumes of buffer a containing 200 and 400 mm imidazole, respectively . Protein containing fractions were pooled, and edta was added to a final concentration of 5 mm, followed by dialysis against buffer b (20 mm hepes, ph 7.5, 1 mm edta, 1 m nacl, 20% glycerol) containing 2 mm -mercaptoethanol and then against buffer b containing 1 mm dithiothreitol . A nonradioactive elisa - based hiv-1 integrase assay was carried out according to the dna - coated plate method . In this study, 1 g of smal - linearized pbluescript sk was coated onto each well in the presence of 2 m nacl as target dna . The donor dna was prepared by annealing vu5br (5-biotin - gtgtggaaaatctcta- gcagt-3) and vu5 (5-actgctagagattttccacac-3) in 10 mm tris - hc1, ph 8.0, 1 mm edta, and 0.1 m nacl at 80c, followed by 30 minutes at room temperature . Integrase reaction was conducted in 20 mm hepes (ph 7.5) containing 10 mm mncl2, 30 mm nacl, 10 mm dithiothreitol, and 0.05% nonidet - p40 (sigma). After the reaction, biotinylated dna immobilized on the wells was incubated with streptavidin - conjugated alkaline phosphatase (boehringer - mannheim, mannheim, germany), followed by colorimetric detection with 1 mg / ml p - nitrophenyl phosphate in 10% diethanolamine buffer (ph 9.8) containing 0.5 mm mgcl2 . The activity of sars coronavirus (cov) protease was reflected by a cleavage of designed substrate which is composed of two proteins linked by a cleavage site for sars cov protease . The reaction was carried out in a mixture containing 5 m sars cov protease, 5 m sample, and 20 m substrate and buffer [20 mm tris - hcl (ph 7.5), 20 mm nacl and 10 mm beta - mercaptoethanol] for 40 minutes at 37c . The reaction was then terminated by heating at 100c for 2 minutes . Then the reaction mixture was analysed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page). If sars cov protease is inhibited by the test sample, there is only one band, which is the intact substrate, shown in sds - page . The assay for antifungal activity toward botrytis cinerea and fuserium oxysporum was executed in 100 mm 15 mm petri plates containing 10 ml of potato dextrose agar . After the mycelial colony had grown to a sufficiently large size, sterile blank paper disks (0.625 cm in diameter) were laid at a distance of 0.5 cm away from the edge of the mycelial colony . An aliquot (15 l) of the trypsin inhibitor was added to a disk . The plates were exposed at 23c for 72 hours until mycelial growth had enveloped the disks containing the control and had produced crescents of inhibition around disks containing samples with antifungal activity . Anion exchange chromatography of the bean extract on deae - cellulose resolved it into three fractions, an unadsorbed fraction d1 together with two adsorbed fractions d2 and d3 . After affinity chromatography of fraction d2 on affi - gel blue gel, the activity appeared in the unadsorbed fraction b1 (data not shown). Ion exchange chromatography of fraction b1 on sp - sepharose resolved it into a small unadsorbed fraction sp1 and three large adsorbed fractions (sp2, sp3, and sp4) of about the same size (figure 1). Final purification of sp2 on superdex 75 produced two fractions, sp2su1 and sp2su2 (figure 2(a)). Gel filtration of sp3 on superdex 75 yielded two fractions sp3su1 and sp3su2 (figure 2(b)). Fractions sp2su2 and sp3su2, both with a molecular mass of 16 kda as determined by gel filtration on superdex 75, were the only fractions with trypsin inhibitory activity . Both sp2su2 and sp3su2 displayed a molecular mass of 16 kda in sds - page (figure 3) and gel filtration (figure 2). These two white cloud bean trypsin inhibitors inhibited trypsin with an ic50 of about 0.6 m (figure 4). Dtt treatment curtailed the trypsin inhibiting activity in a dose- and time - dependent manner (table 3). The two trypsin inhibitors did not inhibit hiv-1 reverse transcriptase when tested at various concentrations up to 100 m (table 4). They lacked antifungal activity when tested up to 24 g / disk (100 m, 15 l) (data not shown). The ic50 values of the inhibitory effects of the trypsin inhibitors on l1210 cells were, respectively, 21.5 m and 28.8 m (table 4). However, there was no activity toward mbl2 cells when tested up to 100 m . The present study disclosed the production of two trypsin inhibitors with closely related n - terminal sequences chromatographic behavior and bioactivities by the white cloud bean variety of phaseolus vulgaris . The trypsin inhibitors demonstrate the same molecular mass; both are adsorbed on deae - cellulose and unadsorbed on affi - gel blue gel but can be separated by ion exchange chromatography on sp - sepharose . They have approximately the same trypsin inhibitory potency, and neither of them inhibits hiv-1 reverse transcriptase inhibitory activity, hiv-1 integrase inhibitory activity, sars coronavirus proteinase inhibitory activity, or fungal growth . Both inhibitors exhibit an antiproliferative activity toward l1210 cells albeit with a small difference in potency, while there is little activity toward mbl2 cells . The difference in yields of the two trypsin inhibitors from the white cloud beans is only slight . The two trypsin inhibitors exhibit n - terminal sequence homology with those of other leguminous trypsin inhibitors such as inhibitors mungbean (vigna mungo), cowpea (vigna unguiculata), and lima bean (phaseolus lunatus). Whereas leguminous antifungal proteins [48, 49], like nonleguminous antifungal proteins, are unadsorbed on deae - cellulose and adsorbed on affi - gel blue gel, white cloud bean trypsin inhibitors are adsorbed on deae - cellulose and unadsorbed on affi - gel blue gel . Thus the purification procedure adopted in the present investigation can be conveniently used to separate trypsin inhibitors from antifungal proteins . The antiproliferative activity of white cloud bean trypsin inhibitors is consistent with similar observations on field bean trypsin inhibitor [39, 40, 42, 43, 54]. It is noteworthy that white cloud bean trypsin inhibitors do not exert a similar action on lymphoma mbl2 cells . In contrast to broad bean trypsin inhibitor [48, 56], those from white cloud bean do not inhibit hiv-1 reverse transcriptase . This is reminiscent of the finding that lentil and vigna mungo inhibitors have little or no hiv-1 reverse transcriptase inhibitory and antifungal activities [57, 58]. In summary, the isolation of two trypsin inhibitors with very similar biochemical and biological characteristics from white cloud beans was achieved in the present investigation . White cloud bean trypsin inhibitors demonstrate antiproliferative activity against tumor cells but do no inhibit mycelial growth or hiv-1 reverse transcriptase . Previously isolated p. vulgaris trypsin inhibitors have not been so tested [6064]. They reportedly have a molecular mass of about 9 kda [63, 65] or 13 kda, smaller than the value of 16 kda obtained for white cloud bean trypsin inhibitors.
Although about two - thirds of patients with epilepsy (pwe) treated with antiepileptic drugs (aeds) reach seizure - freedom, about one - third remains drug - resistant to the current therapies.1 despite the introduction of new aeds with a better pharmacokinetic and safety profile compared to old generation aeds, today, one of the major causes of failure of antiepileptic treatment is poor adherence often due to occurrence of adverse drug reactions (adrs), leading up to 25% of patients to discontinue treatment before the achievement of effective doses and with a consequent increase of health care costs.2,3 during the last 25 years, many efforts have been directed to the development of new aeds with different mechanisms of action able to reduce brain hyperexcitability; recently considerable interest has been focused on synaptic vesicle protein 2a (sv2a) and its role as a target for aeds.4 the first drug of this new class approved for epilepsy is levetiracetam (lev), and from this lead compound, several racetam analogs have been synthesized.5 based on target - drug program, brivaracetam (brv) (2s)-2-[(4r)-2-oxo-4-propylpyrro - lidinyl]butanamide, an n - propyl analog of lev has been identified and has entered clinical trials for pwe;6 it is an sv2a ligand with high selectivity and a 1030-fold higher potency, depending on the experimental conditions, when compared to lev.7,8 the purpose of this review is to present updated data available on the pharmacology, efficacy, and tolerability of brv . We have conducted a systematic search in the pubmed and cochrane library databases up to august 14, 2015 summarizing all relevant data for efficacy, safety, and tolerability of brv in the treatment of partial - onset seizure . Although the correlation between sv2a binding and anti - convulsant potency in animal models has been previously demonstrated, the role of sv2a in neurotransmission still remains unclear.9 in fact, even if it has been established that sv2a protein is involved in normal synaptic vesicle function, the exact mechanism of synaptic vesicle cycling regulation and neurotransmitter release remains unknown.5,9 several preclinical studies have demonstrated that brv is more potent and efficacious than lev in animal models of both focal and generalized epilepsy.10 in particular, in fully amygdala - kindled rats, brv induced a significant suppression in motor - seizure severity from a dose of 21.2 mg / kg, whereas lev caused a similar effect from a dose of 170 mg / kg . Brv also significantly reduced the after - discharge duration at the highest dose tested (212.3 mg / kg), whereas lev was inactive on this parameter up to 1,700 mg / kg.10 moreover, brv protected significantly against both partial and generalized seizures in fully amygdala - kindled mice resistant to phenytoin (effective dose 50 [ed50]: 68.3 mg / kg, intraperitoneal [ip]).10 recently, it has been demonstrated that brv has a higher brain permeability than lev, with consequently more rapid onset of action after acute dosing in audiogenic mice; this fast onset of action might also have a potential clinical relevance for the treatment of status epilepticus or cluster seizures.11 another study confirmed the protective activity of brv against kindled seizures with focal seizure threshold and significantly severity significantly modified.12 furthermore, a recent study, using a rapid kindling model in p14, p21, p28, and p60 rats, has evaluated two doses of brv 10 and 100 mg / kg, demonstrating that brv 100 mg / kg significantly increased the after - discharge threshold at all ages, whereas brv 10 mg / kg increased after - discharge threshold in p60, p28, and p21 rats . Brv also reduced the after - discharge duration, achieving statistical significance with 10 and 100 mg / kg at p60 and with 100 mg / kg, at p21 . At p60, brv increases the number of stimulations required to reach stage 45 seizures in a dose - dependent manner . At p28 and p21, brv increased the number of stimulations required to develop stage 45 seizures in a dose - dependent manner, with almost complete elimination of stage 45 seizures.13 moreover, brv showed a marked synergism with diazepam to reduce seizure duration in self - sustained status epilepticus induced by stimulation of the perforant path.14 a recent study has evaluated the antiseizure and antimyoclonic activities of brv in comparison to lev in an animal model of posthypoxic myoclonus, showing higher efficacy of brv (0.3 mg / kg) than lev (3 mg / kg) against posthypoxic seizures.15 furthermore, recent experiments conducted in transgenic mice with alzheimer s disease supported an adjunctive and peculiar role of brv that not only revealed an efficacy against spike - wave discharges similar to ethosuximide, but it showed the ability to reverse memory impairment, thus extending the potential spectrum of action of this new aed.16 in addition to sv2a block, brv also exhibits inhibitory activity on neuronal voltage - gated sodium channels (vgsc) playing a role as a partial antagonist, as has been reported for other aeds.17,18 in particular, experimental studies on primary cortical cultures have demonstrated that brv is able to prolong the sodium channel time recovery from fast inactivation, and this effect could reduce the availability of sodium channel during high - frequency repetitive firing.17,19 even if this data has been refuted by another recent experimental study that has showed that this vgsc inhibition does not impair sustained repetitive firing in neurons of neuroblastoma cells,20 this is an important aspect, which deserves to be better clarified, since the lack of effect of brv to reduce neuronal excitability by blocking high repetitive firing in neurons might exclude that the modulation of vgsc contributes to antiseizure effects of brv . Brv presents a favorable pharmacokinetic profile, linear and predictable, with low intersubject variability and almost 100% bioavailability.2123 the pharmacokinetic properties of brv have been studied in healthy adult volunteers, in the elderly, in patients with pwe, and in those with hepatic or renal impairment.2225 the pharmacokinetic differences in elderly subjects compared to healthy volunteers are not so important as to require any dose adjustment.25 absorption of brv is unaffected by the presence of food, including high fat meals; after oral administration, brv is rapidly absorbed by the gastrointestinal tract, and displays linear and dose - proportional profile over the dose range tested.22,26 its distribution volume is close to total body water (vz=0.5 l / kg), and it binds weakly to plasma proteins (17.5%). Its terminal half - life is ~9 hours.21 saliva and plasma brv levels are highly correlated, brv crosses the mucosa by passive diffusion, therefore, the saliva concentration of brv is correlated with plasma concentration . It is possible to speculate that saliva might be a suitable sample for monitoring brv levels when blood sampling could be a limiting factor.23 brv is extensively metabolized through several metabolic pathways and is fully excreted by urine (only 8%11% remains unchanged). Brv is eliminated primarily by metabolism, with the major metabolic pathway involving hydrolysis of the acetamide group resulting in formation of an acid metabolite (brv - ac; 34.2% of a radiolabeled dose in urine).21 a secondary pathway, mainly mediated by cytochrome p450 (cyp) 2c19,27 forms a hydroxy metabolite (brv - oh; 15.9% of dose in urine).21 a combination of these two pathways leads to the formation of a hydroxyacid metabolite (15.2% of dose in urine);21,28 only 8.6% of the dose is recovered as the unchanged compound in urine.21 all three metabolites of brv are pharmacologically inactive (unpublished results) in an open - label study conducted in patients with liver disease, the plasma half - life of brv was prolonged up to 17.4 hours in correlation with the severity of hepatic impairment; however, the exposure to brv is increased by 50%60% in patients with hepatic impairment, irrespective of severity classified by child pugh score.25,26 these data suggest that the maximum daily dose of brv might be reduced by one - third in patients with hepatic impairment.25 in severe renal impairment, the exposure to a single oral dose of 200 mg brv not requiring dialysis (creatinine clearance <15 ml / min), and renal clearance of three metabolites (acid, hydroxy, and hydroxyacid), was decreased 10-fold in patients with severe renal impairment.24 nevertheless, there are data showing a toxicological coverage for metabolites, without the evidence of any safety issues (ucb data file). Based on these observations, a dose adjustment for brv should not be required at any stage of renal dysfunction . The efficacy of brv as add - on therapy in patients with uncontrolled partial seizures has been assessed in six randomized placebo - controlled clinical trials (table 1).29 in the first two studies, brv as adjunctive therapy in adult patients with partial epilepsy and poor control with 12 concomitant aeds, at different doses (5, 20, 50, and 50150 mg / d) has been evaluated.30,31 french et al30 reported a statistically significant reduction of seizure frequency achieved at the 50 mg / d dose, with high tolerability and infrequent adrs . In particular, the percentage reduction over placebo in focal seizure frequency / week was directly correlated to brv dose, respectively 9.8% at 5 mg / d, 14.9% at 20 mg / d, and 22.1% at 50 mg / d, with a median percent reduction from baseline in seizure frequency / week of 21.7% (placebo), 29.9% (brv5), 42.6% (brv20), and 53.1% (brv50); 50% responder rates were 16.7% (placebo), 32.0% (brv5), 44.2% (brv20), and 55.8% (brv50); seizure freedom rates during the 7-week treatment period were 1.9% (placebo), 8.0% (brv5), 7.7% (brv20), and 7.7% (brv50). On the other hand, higher doses of brv would not seem to be more effective, in fact, van paesschen et al31 did not find significant differences in seizure frequency reduction between brv 50 and 150 mg / d during the 7-week maintenance period . In particular, the median seizure frequency / week was 1.00, 1.96, and 1.86 in the group treated with brv 50 mg / d, brv 150 mg / d, and placebo, respectively.31 the reduction in baseline - adjusted seizure frequency / week over placebo during the maintenance period was 14.7% in the brv 50 mg / d group and was 13.6% in the brv 150 mg / d group; however, a significant difference over placebo was observed on several secondary efficacy outcomes (10 weeks combined up - titration and maintenance period).31 in fact, after the 10-week treatment period, the median seizure frequency / week was 1.10, 2.05, and 1.95 in the brv 50 mg / d, brv 150 mg / d, and placebo groups, respectively . In the maintenance period, 50% responder rates were 23.1% for placebo compared with 39.6% for brv50 and 33.3% for brv150 . During the treatment period, 50% responder rates were 17.3% for placebo compared with 35.8% for brv50 and 30.8% for brv150 . Nine patients were seizure - free during the 10-week treatment period in the brv50 group (three in the brv150 group, only one in the placebo group). In the study by ryvlin et al,32 the efficacy and safety / tolerability of brv (at doses of 20, 50, and 100 mg / d) in patients with uncontrolled partial seizures with / without secondary generalization, despite treatment with one or two concomitant aeds, was investigated . The percent reduction over placebo in baseline - adjusted seizure frequency / week was 6.8%, 6.5%, and 11.7% in the brv 20, 50, and 100 mg / d groups, respectively . The percent reduction over placebo in baseline - adjusted seizure frequency/28 days was 9.2% and 20.5% in the brv 50 and 100 mg / d groups, respectively . Median percent reductions from baseline were 30.0% for brv 20 mg / d, 26.8% for brv 50 mg / d, and 32.5% for brv 100 mg / d in comparison to 17.0% for placebo . Responder rates (50%) were 27.3%, 27.3%, and 36.0% for brv 20, 50, and 100 mg / d, respectively, in comparison to 20.0% for placebo . Based on these results, only brv 100 mg / d was able to significantly reduce seizure frequency / week over placebo . Indeed, in their randomized placebo - controlled trial, adjunctive brv at a daily dose of 50 mg significantly decreased seizure frequency, while lower dosages (5 and 20 mg / d) did not achieve significant differences.33 in more detail, the percent reduction in partial - onset seizure frequency / week in comparison to placebo was 0.9% (p=0.885) for brv 5 mg / d, 4.1% (p=0.492) for brv 20 mg / d, and 12.8% (p=0.025) for brv 50 mg / d; in the brv 50 mg / d group, statistical significance was also observed for the 50% responder rate (brv 32.7% vs placebo 16.7%) and median percent reduction from baseline in focal seizure frequency / week (brv 30.5% vs placebo 17.8%). Recently a randomized, multicenter, double - blind phase iii trial was conducted by klein et al34 to evaluate the efficacy and the safety profile of brv at fixed doses of 100200 mg / d in adult patients with refractory partial onset seizures . Responder rate was 21.6% for placebo group, 38.9% for brv 100 mg / d, and 37.8% for brv 200 mg / d; the percent reduction of partial onset seizures in 28-day frequency was 22.8% for brv 100 mg and 23.2% for brv 200 mg . Kwan et al35 conducted a double - blind, randomized, placebo - controlled trial investigating the safety and tolerability profile of adjunctive brv (at individualized tailored doses ranging from 20 to 150 mg / d) in patients with partial or generalized refractory epilepsies . The percent of reduction of focal seizure frequency / week in the brv group in comparison to placebo was 7.3%, while only in the generalized seizures group, the number of seizure days / week decreased from 1.42 at baseline to 0.63 during the treatment period in brv - treated patients (n=36), and from 1.47 at baseline to 1.26 during the treatment period in the placebo group (n=13).35 the median percent reduction in baseline - adjusted seizure frequency / week was 26.9% brv vs 18.9% placebo, and the 50% responder rate was 30.3% brv vs 16.7% placebo . The median percent reduction from baseline in generalized seizure days / week was 42.6% vs 20.7%, and the 50% responder rate was 44.4% vs 15.4% in brv - treated and placebo - treated patients, respectively . Similar to lev, brv might become a useful aed for the treatment of myoclonic seizures occurring in the setting of idiopathic generalized epilepsies (eg, juvenile myoclonic epilepsy)36 or of progressive myoclonic epilepsies.37 two randomized, placebo - controlled trials evaluating efficacy and safety of adjunctive brv (5150 mg / d) in unverricht lundborg disease, the most common and less severe form of progressive myoclonic epilepsies,38 failed to show a significant improvement of myoclonus in these patients.39 however, sample size was small (106 patients randomized in the two trials), and the patients were allowed to receive lev.39 moreover, it is well known that myoclonus may present high inter- and intrapatients variability (with patients experiencing good days and bad days) in unverricht currently an open - label, multicenter, follow - up study to evaluate the long - term safety and efficacy of brv is ongoing (brite study - nct01339559). Finally, to date, in all studies performed, brv was evaluated as an oral tablet formulation, and no data are available about intravenous infusion since the study nct02088957, aiming at a comparison of the efficacy and safety of intravenous brv vs phenytoin in adult subjects with nonconvulsive electrographic seizures, was terminated for low enrollment . The most commonly reported adverse effects with brv in adults were primarily related to the central nervous system and included somnolence, fatigue, and dizziness.22 these adverse effects were mild to moderate, and the tolerability profile is so excellent that it did not impair therapeutic compliance . In fact, the daily dose of brv (20150 mg) was well tolerated and associated with 6.1% of discontinuation rates due to adrs compared to 5.0% of the placebo group.35 furthermore, adrs induced by brv seem to be time - related, disappearing during the course of treatment . The entity of sedative effects of brv measured by psychometric tests is dose - related in healthy men and appeared clearly from 600 mg upwards as a decrease in attention, motor control, and alertness.22 moreover, the type and the severity of adrs are not influenced by food.22 as demonstrated in healthy males, a twice - daily dosing regimen could be a good clinical practice to reduce blood fluctuations and peak, which might influence the appearance of adverse events.22 no effects on cardiac function were reported even at very high daily dosages (up to 800 mg / d).40 no data about fertility and/or potential teratogenic effect of brv in humans are currently available; however, no adverse effects were detected up to the highest tested oral dose of 400 mg / kg / d on fertility, and no effects on pregnancy or fetal development at 600 mg / d were observed in animals.26 seizure aggravation or the appearance of new generalized seizures was rare: three studies reported this adverse event, occurring in similar proportions between placebo and treated group (4.3% vs 5.2%, p=0.67).32,33,35 in the above - reported five randomized clinical trials, 1,639 subjects were included in an intention - to - treat analysis (1,214 treated with brv and 425 with placebo).3035 no differences were observed in the proportion of subjects experiencing at least one adverse event (65.5% with brv vs 60.5% with placebo, p=0.10). Most events were mild to moderate; actually, comprehensive withdrawal rate due to adverse events was quite low and similar in brv and placebo arms (5.4% with brv vs 4.2% with placebo, p=0.37). Serious adverse events were quite rare and equally distributed (2.9% with brv vs 4.4% with placebo, p=0.16). Adverse events that were observed in at least 5% of subjects in either group are listed in table 2 . The proportion of patients reporting fatigue and somnolence was significantly higher in brv group compared to placebo (table 2). Irritability was reported in three studies only, and it was present in a small proportion of subjects (3% receiving brv, 1% receiving placebo, p=0.36). Because of its advantageous pharmacokinetic profile, brv treatment does not appear to influence plasma concentrations of other aeds such as carbamazepine, lamotrigine, lev, oxcarbazepine, topiramate, or valproic acid.41 however, carbamazepine plasma levels are slightly reduced by coadministration of brv (400 mg / d), while levels of carbamazepine - epoxide are increased in a dose - dependent manner.26,42 this increase of plasma concentration of carbamazepine - epoxide is the result of inhibition by brv of epoxide hydrolase that metabolizes carbamazepine - epoxide into carbamazepine - diol.42 high doses of brv (400 mg / d) cause a moderate decrease of ethinylestradiol and levonorgestrel plasma levels (components of oral contraceptives) but this posological range has no impact on ovulation . No effect on contraceptive disposition is reported at therapeutic doses of brv 100 mg / d.39 interestingly, there is a possible negative interaction between brv and lev, in fact, the concomitant use of both drugs may reduce brv efficacy; however, this evidence is not robust because the number of patients with concomitant lev was very small, other studies may be useful to assess this apparent pharmacodynamic interaction.33 brv is a novel aed whose efficacy in partial epilepsies has been studied and established in five randomized controlled trials;3035 furthermore, two recent meta - analyses have confirmed significant effects for brv in patients with refractory partial seizures.29,43 considering that brv shares part of its mechanism of action with lev and that its ability to inhibit vgsc (still debated) is in common with several other aeds, it will be very intriguing to see how this drug will behave in real - life clinical practice . Accordingly, it could be hypothesized that brv might possess at least the same effectiveness as lev . Based on this hypothesis, brv may be reasonably considered as a valuable add - on aed in patients with partial seizures, also considering its suggested good tolerability . However, specific studies are needed to confirm its efficacy in specific epileptic syndromes, for example, a decreased expression of sv2a in the hippocampus of patients with temporal lobe epilepsy with hippocampal sclerosis has been documented and might represent a pharmacoresistance mechanism in some cases; however, lev has been reported to be effective.44,45 in addition, because of its good safety and pharmacokinetic profile, brv might be ideal for use in monotherapy, as previously demonstrated for lev.46 finally, few studies have been performed, and more randomized double - blind studies are needed to confirm these considerations and to demonstrate if brv might really confirm its promises and become a new tool for epileptologists.
Acute intussusception is one of the most common causes of acute abdomen in infants . Management includes both surgical and non - surgical methods, with gradually increasing importance recently being accorded to the latter . Conservative measures, including hydrostatic reduction, are the methods of choice for initial management . Though widely accepted, the expertise is still not widely available, especially in developing countries . A six - month - old child presented with complaints of repeated episodes of vomiting that contained only ingested milk . This was associated with three episodes of loose stools, initially watery and later with fresh red blood per rectum with jelly - like consistency . There was no history of fever, decreased urine output, lethargy, abnormal body movements, or difficulty in breathing . Her immunizations were fully up to date with no history of rota virus immunization . On admission abdominal examination was remarkable for an ill - defined mass that could be felt in the right hypochondrium . She was initially kept nil per oral and started on conservative measures, including antibiotics . An urgent abdominal ultrasonography was done, which showed a round mass - like lesion giving a target appearance in the right hypochondrium (suggestive of ileocolic intussusception). She was taken for immediate intervention, and as per surgical opinion, a hydrostatic reduction under radiological guidance was started . The child was first stabilized using intravenous fluids to treat dehydration, and a pre - procedure x - ray abdomen was performed to rule out intestinal perforation . The procedure was done in the ultrasonography room in the presence of a treating physician and a pediatric surgeon . Without using any sedation, a 16 f foley catheter was inserted into the rectum in the supine position and the balloon was gently inflated while maintaining a tight anal seal . The patient s vitals, which included abdominal girth and distension, were monitored throughout the procedure . Lactate solution, warmed to body temperature, was then slowly introduced into the catheter through a bag hanging at a height of approximately 100 cm . During reduction, the intussusceptum was observed under continuous ultrasound guidance as it proceeded to the cecum and then reduced across the ileocecal valve (figures 1 - 4). Ileal loop (star) within ascending colon (arrow), longitudinal view . This was associated with the patient s improved condition, which included less crying and irritability . The parents were told about the possibility of recurrence and asked to pay close attention for the onset of symptoms . Acute intussusception is defined as the telescoping of the proximal bowel (intussusceptum) into the distal bowel (intussuscipiens). The usual locations include the ileocolic region and ileocecal junction, which comprise the majority of cases . Intussusception mostly presents between four months and two years, while the peak incidence is found around middle infancy . Diagnosis usually needs a high suspicion on the part of the treating physician, as presentation can be quite varied, including irritability, vomiting, and poor feeding . Characteristic manifestations include crying episodes, fresh red blood in stools (hematochezia), and a mass in the abdomen . Differential diagnoses include acute gastroenteritis, dysentery, sepsis syndrome, and volvulus, amongst others . The diagnosis is usually confirmed sonographically, which is highly accurate and has a reported sensitivity that exceeds 90% . Various signs have been described in the literature, depicting intussusception as an abdominal mass with a target sign on the transverse section, a pseudokidney (or sandwich sign) on the longitudinal section, and a crescent - in - doughnut sign on axial images . The management of intussusception has seen a paradigm shift in recent years . In the past, the preferred non - surgical method involved use of a barium enema for reduction, followed by the air insufflation method . Hydrostatic reduction using ultrasound - guided saline reduction has recently gained acceptance as the procedure of choice for initial nonsurgical management of intussusceptions in children . This popularity can be attributed to the avoidance of radiation exposure to the child and the treating team, as well as to the high success rates that have been achieved . There is still some controversy regarding the exact method of the procedure, which can be attributed to the poor training of physicians and to the general belief that intussusception is entirely a surgical problem, especially in developing countries . This procedure should be undertaken with a close liaison between pediatrics, pediatric surgery, and radiology teams to obtain the best results . The surgeon and physician should always be present at the time of reduction for clinical monitoring . After adequate optimization, including temperature controlled settings, the procedure should ideally be undertaken without any sedation . This is helpful for assessing a successful reduction using clinical criteria, as described later . We used ringer s lactate due to its near physiological constitution, avoiding the risk of chemical peritonitis due to a barium enema or tension pneumoperitoneum due to an air enema, as seen in cases of intestinal perforation . The risk of intestinal perforation following such a procedure is rarely encountered due to less hydrostatic pressure exerted and an even amount of pressure exerted on the bowel walls as compared to air insufflations . Even if intestinal perforation occurs we chose 100 cm h2o pressure to lessen the risk of perforation, though different studies have used a wide range of pressures (75 to 125 cm h2o).6, 9 the main ultrasonographical criteria of successful reduction is the flow of the fluid from the cecum into the terminal ileum . General improvement in the clinical profile of the patient including relief from crying, stable vitals, increasing abdominal distension during the procedure (which indicates filling of the small bowel), and the disappearance of the abdominal mass, should also be given due consideration when deciding to terminate the procedure . A repeat ultrasound should always be done after the procedure as the chances of recurrence is high, especially in the immediate post - procedure period ., this method should be undertaken as teamwork, and not considered the work of a single specialty, which is the best way of treating the patients according to the standards of today.
Aging, a multifactorial process of enormous complexity, is characterized by impairment of physiochemical and biological aspects of cellular functions (harman 1992). Oxidative stress, an unavoidable consequence in the metabolism of oxygen in aerobic cells, is a major factor in the aging process and, in the course of many chronic diseases, associated with aging (mattson 2002). Many predisposing conditions which increase in prevalence during aging, such as obesity, insulin resistance, inflammation, changes in the activity of the hypothalamus hypophysis suprarenal axis, stress, and hypertension, contribute to increase prevalence of cardiovascular diseases (veronica and esther 2012). Lipid infiltration in the myocardium is the foremost disorder encountered in the development of the aging process (johannsen and ravussin 2010). Aging is frequently accompanied by several pathological conditions and some associated phenomena such as increased lipid peroxidation, generation of free radicals, and increased peroxidation of nitric oxide (no) to its toxic species, resulting from oxidative stress which significantly alters the incidence of cardiovascular diseases (guarner et al . Alteration in glutathione - dependent antioxidant system is expected to exert a significant impact on physiological and metabolic functions of cellular membranes . Enhanced lipid peroxidation and deterioration of membrane structure has been well established during the aging process (yu 2005). Preventing and treating cardiovascular diseases would be useful in promoting normal aging . The identification of natural molecule with antioxidant, antilipidemic, and membrane - stabilizing properties is, therefore, one strategy to facilitate healthy aging . Chitosan is one of the most abundant naturally occurring polysaccharides present in shellfish, clams, krill, oysters, squid, fungi, and insects (cardenas et al . It is a polymer of -(1 - 4)-d - glucosamine (fig . 1), and it is chemically similar to that of the plant fiber, cellulose . It has been reported to possess antilipidemic (santhosh et al . Previously, anandan et al . (2004) observed the antiulcerogenic potential of chitosan against hcl the free radical quenching property of this marine polysaccharide has also been studied in detail (xing et al . It has profound applications in the fields of clarification and purification, chromatography, paper and textiles, photography, food and nutrition, agriculture, pharmaceutical and medical, cosmetics, biodegradable membranes, and biotechnology (santhosh et al . Though the beneficial effects of chitosan have been extensively studied, the antiaging effect of chitosan has not yet been explored.fig . 1structure of chitosan structure of chitosan in the present study, an attempt has been made to assess the salubrious effect of dietary chitosan intake on myocardial lipid peroxidation and glutathione - dependent antioxidant status in young and aged rats by virtue of its antioxidant and hypolipidemic properties . Reduced glutathione, tetraethoxypropane, and 2-thiobarbituric acid were procured from m / s sigma chemical company, st . Chitosan (mw, 750,000 da; viscosity, 8 cp; deacetylation rate, 8587%; purity, 98.6%) used in the experiment was a kind gift from dr . T. k. thankkappan, principal scientist, central institute of fisheries technology, cochin, india . Male wistar strain albino rats, weighing 120150 g [18 young rats of 23 months old (mean age, 79.2 6.53 days)] and 350400 g [18 aged rats of 2025 months old (mean age, 712 51.6 days)], were selected for the study . The animals were housed individually in polypropylene cages under hygienic and standard environmental conditions (28 2 c; humidity, 6070%; 12 h light / dark cycle). The animals were allowed a standard diet (m / s sai foods, bangalore, india; table 1) and water ad libitum . The experiment was carried out according to guidelines of the committee for the purpose of control and supervision of experiments on animals, new delhi, india, and approved by the institutional animal ethics committee of the central institute of fisheries technology, cochin, india.table 1composition of standard diets . No.ingredientscomposition (g/100 g diet)1carbohydrate (nitrogen free)56.22crude protein22.03ash7.54crude oil4.25crude fiber3.06glucose2.57vitamins1.88sand silica1.49calcium0.810phosphorus0.6 composition of standard diet seven days after acclimatization, the animals were divided into two major groups: group i consisted of 18 normal young rats and group ii consisted of 18 normal aged rats . Each group was further subdivided into three groups (six rats each): one control group (group ia and group iia) and two experimental groups based on the duration of supplementation of chitosan at 2% level along with feed 30 days (group ib and group iib) and 60 days (group ic and group iic). At the end of the experimental period, the animals were sacrificed, and the blood was collected in a heparinized tube for the separation of plasma . The heart tissue was excised immediately and homogenized in ice - cold 0.1-m tris hcl buffer in a potter the homogenate was used for the estimation of lipid peroxides (lpo; ohkawa et al . 1979), reduced glutathione (gsh; ellman 1959), glutathione reductase (gr; stall et al . High - density lipoprotein - cholesterol (hdl - cholesterol) in plasma was determined by the method of izzo et al . (1981), and low - density lipoprotein - cholesterol (ldl - cholesterol) was estimated according to the calculation of friedwald et al . (1974) after extracting total lipids according to the method of folch et al . Multiple comparisons of the significant anova were performed by duncan's multiple range comparison test . A p value <0.05 was considered as statistically significant . All data were analyzed with the aid of statistical package program spss 12.0 for windows . Reduced glutathione, tetraethoxypropane, and 2-thiobarbituric acid were procured from m / s sigma chemical company, st . Chitosan (mw, 750,000 da; viscosity, 8 cp; deacetylation rate, 8587%; purity, 98.6%) used in the experiment was a kind gift from dr . T. k. thankkappan, principal scientist, central institute of fisheries technology, cochin, india . Male wistar strain albino rats, weighing 120150 g [18 young rats of 23 months old (mean age, 79.2 6.53 days)] and 350400 g [18 aged rats of 2025 months old (mean age, 712 51.6 days)], were selected for the study . The animals were housed individually in polypropylene cages under hygienic and standard environmental conditions (28 2 c; humidity, 6070%; 12 h light / dark cycle). The animals were allowed a standard diet (m / s sai foods, bangalore, india; table 1) and water ad libitum . The experiment was carried out according to guidelines of the committee for the purpose of control and supervision of experiments on animals, new delhi, india, and approved by the institutional animal ethics committee of the central institute of fisheries technology, cochin, india.table 1composition of standard diets . No.ingredientscomposition (g/100 g diet)1carbohydrate (nitrogen free)56.22crude protein22.03ash7.54crude oil4.25crude fiber3.06glucose2.57vitamins1.88sand silica1.49calcium0.810phosphorus0.6 composition of standard diet seven days after acclimatization, the animals were divided into two major groups: group i consisted of 18 normal young rats and group ii consisted of 18 normal aged rats . Each group was further subdivided into three groups (six rats each): one control group (group ia and group iia) and two experimental groups based on the duration of supplementation of chitosan at 2% level along with feed 30 days (group ib and group iib) and 60 days (group ic and group iic). At the end of the experimental period, the animals were sacrificed, and the blood was collected in a heparinized tube for the separation of plasma . The heart tissue was excised immediately and homogenized in ice - cold 0.1-m tris hcl buffer in a potter the homogenate was used for the estimation of lipid peroxides (lpo; ohkawa et al . 1979), reduced glutathione (gsh; ellman 1959), glutathione reductase (gr; stall et al . 1969), and glutathione peroxidase (gpx; paglia and valentine 1967). High - density lipoprotein - cholesterol (hdl - cholesterol) in plasma was determined by the method of izzo et al . (1981), and low - density lipoprotein - cholesterol (ldl - cholesterol) was estimated according to the calculation of friedwald et al . (1974) after extracting total lipids according to the method of folch et al . Multiple comparisons of the significant anova were performed by duncan's multiple range comparison test . A p value <0.05 was considered as statistically significant . All data were analyzed with the aid of statistical package program spss 12.0 for windows . Significant (p <0.05) variation was observed in the body weight (grams) of young (initial, 129 9.05; final, 249 18.3) and aged (initial, 382 27.2; final, 336 23.1) chitosan - supplemented groups of rats . The significant (p <0.05) loss observed in the body weight of chitosan - supplemented aged animals might be related to the fibrous nature of chitosan . Interestingly, the total food intake (grams per 60 days) of aged chitosan - supplemented rats (904 78.4) was comparable to that of young rats (745 59.5). Level of total cholesterol was significantly (p <0.05) higher in plasma and heart tissue of group iia aged rats as compared to group ia young control rats, indicating the development of mild age - associated hypercholesterolemic condition (tables 2 and 3). The level of ldl - cholesterol was slightly (p <0.05) higher in group iia aged rats, whereas hdl - cholesterol levels were significantly lower compared to group ia young animals (table 2). This aspect might be due to the augmented mobilization of ldl - cholesterol from the blood into the cell membranes, resulting in abnormal cholesterol deposition in the myocardium . In the present study, the dietary supplementation with chitosan significantly reduced the total cholesterol level in plasma and myocardial tissue of group iic aged rats as compared to group iia rats . It also kept the levels of ldl - cholesterol and hdl - cholesterol in plasma comparable to that of group ic rats . 2009), which showed that the positively charged amino groups of chitosan possess the ability to bind negatively charged molecules such as lipids and bile acids, inducing a greater fractional excretion in the feces . Also (2007) suggested that chitosan improve lipid metabolism by regulating total cholesterol and ldl - cholesterol by upregulation of hepatic ldl receptor mrna expression, increasing the excretion of fecal bile acids . Previous studies (yao and chiang 2002) pointed out that chitosan supplementation was capable of lowering the levels of plasma total cholesterol and ldl - cholesterol in experimental animals . In the present study, a slight decline in the level of total cholesterol and ldl - cholesterol were also noted in group ic chitosan - fed young rats, ascertaining the anticholesterolemic property of chitosan (ylitalo et al . 2002).table 2effect of dietary chitosan supplementation on total cholesterol, hdl - cholesterol, and ldl - cholesterol in plasma of young and aged ratsparametersyoung ratsaged ratsgroup ia (control)group ib (30 days)group ic (60 days)group iia (control)group iib (30 days)group iic (60 days)total cholesterol79.9 5.36 a, b76.2 4.98 a, b73.1 5.07 a98.4 9.12 c91.7 8.56 c87.3 8.21 b, chdl - cholesterol42.2 3.11 a, b, c44.9 3.48 b, c47.5 3.29 c33.2 2.68 d36.4 2.56 a39.7 3.74 a, bldl - cholesterol24.3 1.42 a22.7 1.31 a, b20.3 1.18 b48.4 2.72 c43.1 2.14 d38.3 2.27 eresults are mean sd for six rats . Values expressed: total cholesterol, hdl - cholesterol, and ldl - cholesterol, milligrams per deciliter . Values that have a different letter (a, b, c, d, e, f) differ significantly with each other (p <0.05; duncan's multiple range test)table 3effect of dietary chitosan supplementation on the levels of total cholesterol, lipid peroxides, and reduced glutathione (gsh) and the activities of glutathione - dependent antioxidant enzymes [glutathione peroxidase (gpx) and glutathione reductase (gr)] in the heart tissue of young and aged ratsparametersyoung ratsaged ratsgroup ia (control)group ib (30 days)group ic (60 days)group iia (control)group iib (30 days)group iic (60 days)total cholesterol2.72 0.17 a, b2.56 0.14 a2.43 0.16 a3.68 0.32 c3.29 0.21 d3.04 0.25 b, dlipid peroxides1.08 0.07 a0.98 0.06 a0.97 0.07 a2.58 0.14 b1.96 0.09 c1.54 0.11 dgsh10.9 0.84 a12.3 1.02 a, b14.5 1.17 c7.54 0.61 d8.76 0.69 d, e9.52 0.85 a, egpx5.23 0.35 a5.51 0.41 a, b5.98 0.37 b3.12 0.18 c4.05 0.26 d4.56 0.34 dgr0.43 0.02 a0.52 0.04 b0.58 0.03 c0.22 0.01 d0.29 0.03 e0.36 0.02 fresults are mean sd for six animals . Values expressed: total cholesterol, milligrams per gram wet tissue; lipid peroxides, nanomoles mda released per milligram protein; gsh, micrograms per milligram protein; gpx, micrograms gsh oxidized per minute per milligram protein; gr, nanomoles nadph oxidized per minute per milligram protein . Values that have a different letter (a, b, c, d, e, f) differ significantly with each other (p <0.05; duncan's multiple range test) effect of dietary chitosan supplementation on total cholesterol, hdl - cholesterol, and ldl - cholesterol in plasma of young and aged rats results are mean sd for six rats . Values expressed: total cholesterol, hdl - cholesterol, and ldl - cholesterol, milligrams per deciliter . Values that have a different letter (a, b, c, d, e, f) differ significantly with each other (p <0.05; duncan's multiple range test) effect of dietary chitosan supplementation on the levels of total cholesterol, lipid peroxides, and reduced glutathione (gsh) and the activities of glutathione - dependent antioxidant enzymes [glutathione peroxidase (gpx) and glutathione reductase (gr)] in the heart tissue of young and aged rats results are mean sd for six animals . Values expressed: total cholesterol, milligrams per gram wet tissue; lipid peroxides, nanomoles mda released per milligram protein; gsh, micrograms per milligram protein; gpx, micrograms gsh oxidized per minute per milligram protein; gr, nanomoles nadph oxidized per minute per milligram protein . Values that have a different letter (a, b, c, d, e, f) differ significantly with each other (p <0.05; duncan's multiple range test) sumiyoshi and kimura (2006) suggested that the lipid - lowering effects of chitosan might be mediated by increases in fecal fat and/or bile acid excretion resulting from the binding of bile acids, and by a decrease in the absorption of dietary cholesterol from the small intestine . (2009) have shown that normal intestinal microbiota might also positively influence immune responses and protect against the development of inflammatory diseases systemically through the formation of short - chain fatty acids by fermentation of dietary fiber in the intestine . Chitosan, a biopolymer of glucosamine derived from chitin that is chemically similar to that of cellulose, acts as a dietary fiber in gastrointestinal tract (gallaher et al . It is possible that chitosan may function through the generation of gut metabolites, such as short - chain fatty acids / short - chain oligosaccharides, in attenuating the development of inflammatory processes related to aging . In the present study, there was a significant (p <0.05) increase in the level of lipid peroxidation observed with a concomitant reduction in the level of nonenzymatic (gsh) and enzymatic (gpx and gr) antioxidants in the heart tissue of group iia aged rats as compared to group ia young control animals (table 3). This concurs with the earlier findings (subramanian and james 2010), which indicated that the higher vulnerability of aged myocardium to peroxidative damage was mainly due to a decline in the level of free radical scavengers . Depletion of gsh results in enhanced lipid peroxidation, and excessive lipid peroxidation can cause increased gsh consumption during aging (denniss et al . The reduction in the activity of gpx and gr may be due to the reduced availability of gsh . Gpx offers protection to the cellular and subcellular membranes from the peroxidative damage by eliminating hydrogen peroxide and lipid peroxide (li et al . Inhibition of gsh - dependent antioxidant enzymes makes myocardial cell membranes more susceptible to oxidative damage in aging . Gsh and gsh - dependent enzyme systems may be directly related to the pathogenic mechanisms related to age - associated disorders (goncharova et al . 2007). In our study, the dietary chitosan intake significantly attenuated the age - associated oxidative stress and maintained the level of the glutathione - dependent antioxidant status in the heart tissue at near normal . (2003) have shown that chitosan has strong antioxidative effects, which decrease free radical production and increase antioxidant enzyme activities during ccl4-induced lipid peroxidation in rats . (2004) have suggested that chitosan may eliminate various free radicals by the action of nitrogen on the c-2 position of the chitosan . (2001) reported that the scavenging mechanism of chitosan is related to the fact that the free radicals can react with the hydrogen ion from the ammonium ions to form a stable molecule . The normal young rats receiving chitosan (group ic) did not show any significant change when compared with normal (group ia) rats, indicating that it does not per se have any adverse effects . In conclusion, the overall antiaging effect of dietary chitosan intake is probably related to its ability to inhibit the increased accumulation of lipids both in the systemic circulation and in the myocardium by its antilipidemic property, or to normal maintenance of the activities of glutathione - dependent antioxidant enzymes and the level of gsh, which protect myocardial membrane against oxidative stress by decreasing lipid peroxidation reactions.
Cleft lip and palate (clp) patients are going through an extensive treatment with orthodontic and surgical correction of the position of teeth and hard and soft tissues . The aim of the primary surgery is to close the clefts of the lip and the soft palate . The aim of the secondary surgery is to close the cleft of the alveolus by bone grafting . Surgery of the clefts induces formation of scar tissue, which affects the soft tissue matrix that guides the development of the maxillary complex . The scar tissue as well as a reduced growth of the mid face often causes a maxillary retrognatism, that adversely affects the chewing function, the ability to speak, the facial aesthetics, social life as well as psychological and general wellbeing of the clp - patients . For that reason, between 25 to 60% of the clp - patients need a surgical treatment of the maxillary retrognatism . This surgical - orthodontic treatment is the last part of the rehabilitation of the clp - patient, which has the objective to create a good dental occlusion and function, more harmonic facial aesthetics and increase the quality of life . Since the 1970's the standard treatment of clp - patients with a maxillary hypoplasia has been a le fort i osteotomy with a bone graft . Treating extensive sagittal discrepancies maxillary surgery has to be combined with mandibular set - back . Maxillary surgery is difficult in clp - patients because of the scar tissue, a tendency to relapse, poor bone quality and quantity and a reduced blood supply in the affected area . These conditions are responsible for a typical relapse of 20 - 25% . To compensate for this an overcorrection is often made during the surgical procedure, which can lead to an unpredictable final result . Polley and figueroa described the use of distraction osteogenesis (do) as an alternative treatment of maxillary hypoplasia using an external bone distraction device . The principle of this treatment was to induce formation of immature bone in the gap after a le fort i osteotomy by gradual tensile strength separating the two segments . Studies of the treatment have shown a significantly reduced tendency of relapse, favourable changes of the soft tissue and changes of the velopharyngeal closure similar to that of conventional advancement . The duration of the course of treatment is up to 16 weeks longer when choosing do . In this period the appliance penetrates the buccal mucosa in the sulcus and the patient must take care of the daily activation and keep it clean . The aim of this retrospective pilot study was to examine and compare the cleft lip and palate patients' satisfaction after treatment with either maxillary distraction or traditional advancement of the maxilla after a le fort i osteotomy . In the period 1996 - 2007 forty - two clp - patients with need for advancement of the maxilla were surgically treated at the department of oral and maxillofacial surgery, aarhus university hospital by either traditional advancement of the maxilla or distraction osteogenesis . Until 2000 the standard regime of treatment was a traditional advancement of the maxillary complex after le fort i osteotomy . Since 2000 maxillary distraction has provided an alternative in treatment of clp - patients requiring a maxillary advancement of 10 mm or more . Consequently, the patients in this study consisted of two groups of clp - patients with the need for surgical - orthodontic correction of maxillary hypoplasia . A criterion for both groups was that the postsurgical orthodontics had to be finished if the patient was to be included in the study . Conventional group (conv) included patients treated with traditional advancement of the maxilla after a le fort i osteotomy in the period 1996 - 2007 . The group do included patients treated with maxillary distraction table 1 . Patients' characteristics according to gender, type of cleft, type of surgery performed, type of distractors used and age at surgery unsegmented le fort i osteotomy and internal distraction . Unsegmented le fort i osteotomy and external distraction . M = male; f = female; uclp = unilateral cleft lip and palate; bclp = bilateral cleft lip and palate; cl . Fifteen patients met the inclusion criteria in the do group and ten patients in the conv group . The composition of the two groups according to gender, type of cleft, type of distraction, type of distractors used, age at surgery and type of surgery performed is presented in table 1 . Group do consisted of 10 males and 5 females with a mean age at the time of surgery of 17.5 (sd 2.3) years while group conv consisted of 5 males and 5 females with a mean age at the time of surgery of 17.8 (sd 2.6) years . A quantitative method, a questionnaire, existing literature was reviewed and a list of relevant subjects was made and operationalized in a questionnaire containing 13 questions about the patients' perception of aesthetics and function . The patients were asked to answer by making a mark on a vas - scale scaled 0 - 100 . The score 0 indicating the highest level of satisfaction with functional parameters or facial aesthetics and the score 100 indicating the lowest level of satisfaction . The end points of the scales were unequivocal and easy to understand, for example no pain / intolerable pain; very satisfied / very unsatisfied; no discomfort / worst imaginable discomfort . The accompanying letter contained information about the aim of the study and how to answer . The patients were asked to return the questionnaire by pre - paid mail after two weeks . The study was approved by the central denmark region committees on biomedical research ethics . All patients had presurgical orthodontic treatment and were treated surgically with a high le fort i osteotomy . In the conv group the treatment was a conventional combined orthodontic - surgical treatment with model surgery and splint fabrication for the maxillary position and completed with postsurgical orthodontics . The patients in the conv group had an average advancement of 6.98 mm (range 5 - 11 mm). Patients from the do group had the distraction device placed in pre - planned position calculated from lateral cephalograms and adjusted on a three - dimensional print of the skull based on a computed tomography (ct) or a cone beam ct (cbct). The appliances used for distraction were synthes internal distractor (synthes, west chester, pa, usa), kls martin internal distractor (kls martin group, tuttlingen, germany) and rigid external device (red) kls martin (kls martin group, tuttlingen, germany) for the external distraction procedures (table 1). All the patients treated with distraction were registered for model surgery using the same procedure as for the conventional treated patients using face bow registration, wax bite and articulator mounting . The patients had surgical splints fabricated and in the cases of do the splint was used as a guide for using intermaxillary elastics after the active distraction . The maxilla was thereby manipulated into the final planned position . In five of the do patients the palatal defect caused an unstable intersegmental position . The use of the splint was refrained from and intermaxillary elastics were used to guide the segments into occlusion . During the surgical procedure the internal distraction devices were adapted to the maxilla in the planned position . The screw holes were marked and the devices were removed in order to complete the le fort i osteotomy . After ensuring mobility of the maxilla the devices were fixed to the maxilla and tested for interference free activation . The mean latency period after device placement was 4.9 days (range 4 - 7 days). During the active phase of distraction the appliances were activated on a daily basis either by the patient or an assistant, usually a family member . Activation was done twice a day with a rate of 0.5 mm, corresponding to 1.0 mm daily . Activation went on until the planned position of the maxilla was achieved and intermaxillary elastics were then used to adjust the final position . The mean periodof active distraction was 17.7 days (range 13 - 28 days) and the mean advancement of the maxilla was 12 mm (range 6 - 16 mm). After an average consolidation period of 77.3 days (range 35 - 213 days), the patients were readmitted and the devices removed in general anaesthesia . The treatment was a conventional combined orthodontic - surgical treatment with model surgery and splint fabrication for the maxillary position and completed with postsurgical orthodontics . Unpaired t - tests were performed to analyse intergroup differences regarding duration of orthodontic treatment and vas - scores in spss 18.0 (ibm, usa). Statistical significance was defined as p <0.05 . The duration of the orthodontic treatment was in average 10 months longer in the do group than the conv group and this difference was significant (p <0.05). Neither the age distribution nor the period of time between surgery and completion of the questionnaire differed significantly (p <0.05) table 2 . Distribution of the patients' groups according to gender, age at surgery and period between surgery and completion of the questionnaire after the primary surgery with insertion of the distractors . After the surgical procedure . Unpaired t - test, states p <0.05 between patients of do and conv groups . Nineteen out of 25 patients returned the questionnaires and the total response ratio was 76% . Results of the patients' satisfaction on a continuous visual analog - scale (vas) on a vas - scale scaled 0 - 100 with the score 0 indicating the highest level of satisfaction with functional parameters or facial aesthetics and the score 100 indicating the lowest level of satisfaction . Both groups felt a great deal of satisfaction with their appearance both according to themselves and the perceptions of relatives and other people's reaction in general . Both groups were satisfied with their general well being and felt few restrictions or discomfort during social activities . According to functional parameters the groups were alike . Both groups were minimally affected by pain and sensory disturbances and reported a great deal of satisfaction with speech and breathing . The discomfort during eating and drinking and sleep were low as well in both groups . The greatest difference occurred in the parameter satisfaction with the duration of the treatment course . The do group scored higher on the vas - scale according to less satisfaction with the duration than the conv group . Statistical analyses revealed no significant differences (p> 0.05) among the groups which may be explained by the numbers of patients included in the study . Distraction osteogenesis has become a widely used treatment of maxillary hypoplasia in clp - patients because of the reports of better stability and the possibility for larger advancements . However, it is a more complicated treatment because of the period with active distraction, the obligate need for good cooperation and the long consolidation phase with the patient still wearing the appliance . The present study is a pilot study and evaluates retrospectively patients' satisfaction in two groups of clp - patients treated for maxillary hypoplasia with either maxillary distraction or conventional le fort i advancement . The number of patients was limited and especially the conv group was small and the number of patients responding was lower than in the do group . The results indicated a high level of satisfaction with the facial aesthetic at the end of treatment in both the do and the conv group . The parameters pain and sensory disturbances did not differ a lot between the groups in this study . It could be hypothesized that more extensive osteotomies and preoperative advancements in conv could induced more sensory disturbances and pain compared with a gradual movement and distraction histogenesis of soft tissues when using do . The groups had comparable scores according to the functional parameters eating and drinking, sleep, speech and breathing . Do was significantly less satisfied with the duration of the treatment than conv probably according to a longer duration of the orthodontic treatment . The influence of the period with the distractors mounted on the level of satisfaction was not measured but it can be hypothesized that this period is troublesome . Further studies of this period between mounting and removal is needed as the prolonged total treatment time is a disadvantage and perhaps the most important difference when choosing do instead of conventional treatment . A greater understanding of the cellular processes during the period of distraction and the period of consolidation and possibility to accelerate the genesis and maturation of bone by the use of pharmacological agents could reduce the duration of these periods . By a reduction of the duration of these aforementioned periods the probable inconveniences could be reduced and possibly of a greater patients' satisfaction and lesser discomfort could be achieved . Another way to reduce the strain on the do patients would be the continuous development of the distractors, e.g. Reduction in size, partly removable appliances, continuous activation etc . . Patients' satisfaction in association with distraction is a subject sparsely examined in the existing literature . No studies describes patients' satisfaction during internal distraction while only a single retrospective survey with a quite small population has studied patients' satisfaction during external distraction . Concluded that most of the patients receiving red treatment had an increased satisfaction with facial aesthetics after the treatment than before the treatment in spite of a great deal of dissatisfaction with the facial aesthetics during the course of treatment . A long term study of patients' perception of function and satisfaction showed that orthognathic surgery resulted in a subjective estimation of function, appearance, health, and interpersonal relationships that was higher than that among pretreatment and no - treatment control groups . A newly prospective study of patients receiving orthognathic surgery showed that perception of function and general satisfaction significantly increased after orthognathic surgery and that the parameters were positive correlated . This study also showed a general high level of satisfaction with appearance and functional parameters in clp - patients treated with either do or conventional treatment . The retrospective design of this survey and the two years between the operation and the completion of the questionnaire diminished the reliability of the patients' answers . The use of a questionnaires cause problems with the internal validity, because the questionnaire is designed with a limited amount of topics, that might not all be the most valid according to every single patient but representing a selection bias . Further use of this questionnaire necessitates a validation . Instead a validated questionnaire, for instance the oral health impact profile or orthognathic quality of life questionnaire can be used in further studies . Prospective surveys should be based on a validated questionnaire and include the period between mounting and removal of the distractors . A comparative study of patients' satisfaction during three different courses of treatment, traditional advancement, external distraction and internal distraction, could probably lead to a greater understanding of this important subject . 1 . Cleft lip and palate patients experience a high level of satisfaction with functional parameters and aesthetics after surgical - orthodontic treatment of maxillary hypoplasia . Patients treated with distraction osteogenesis were less satisfied with the duration of their treatment than the conventional group . No external funding, apart from the support of the authors' institution, was available for this study.
A rising trend in the incidence and prevalence of inflammatory bowel disease (ibd) in asia has been recognized for the past two decades . The thiopurine drugs azathioprine (aza) and mercaptopurine are widely used for the treatment of ibd and have proven to be effective in both inducing and maintaining remission of crohn's disease and ulcerative colitis; however adverse effects can occur in 928% of patients, and these adverse effects often necessitate a dose reduction or discontinuation . Nevertheless, some reports suggested that neutropenia during aza therapy reduced the relapse rates in ibd patients [1, 2], and that the eradication of sensitized leukocytes, which was done by leukapheresis or bone marrow transplantation, improved ibd in some cases [3, 4]. There have also been some cases of ulcerative colitis with prolonged remission following aza - induced pancytopenia [5, 6]. A 22-year - old man with a known history of crohn's colitis who had undergone fistulectomy for complex perianal fistula 2 years previously was admitted with a complaint of severe weight loss (25 kg during 2 years), diarrhea, abdominal pain and recurrent perianal tenderness . Physical exam revealed fistular openings and subcutaneous abscess pockets with severe tenderness on the anus and buttock . He was not able to maintain a sitting or supine position, so his social activity was limited . Contrast - enhanced computed tomography scan of the abdomen showed multifocal inflammatory wall thickening and thick - walled abscess pockets with the beaded appearance of subcutaneous small abscesses in the precoccygeal and perianal area (fig . Abscess drainage and a seton operation were performed, then aza was administered at a starting dose of 25 mg; this was increased up to 50 mg after 2 weeks . He then revisited the hospital and presented with high fever (39.2c) and myalgia . On general peripheral blood test, leukocytes were 180/l (segmented cells 6%), hemoglobin was 6.6 g / dl and platelets were 48,000/l . After 2 weeks of administering human recombinant granulocyte colony - stimulating factor and broad - spectrum antibiotic therapy, his fever subsided and the cell count fully recovered . Escherichia coli was found on blood culture, but it was sensitive to the antibiotics . After recovering from cytopenia, the frequency of bowel movements decreased and the perianal pain and oozing improved gradually . Follow - up colonoscopy was performed 3 months later and demonstrated diffuse fibrotic scar instead of multiple ulcerations (fig . He gained 10 kg within 6 months and is now on regular follow - up without clinical recurrence (cdai score = 114). Therefore, a conventional immunosuppressive dose of less than 2.5 mg / kg / day causes predictable mild leukopenia . Rarely however, sudden, severe and unexpected myelosuppression, which is possibly idiosyncratic, has also been reported when low to moderate doses (<2 mg / kg / day) of the drug were used . In these cases, the cytopenic patients are vulnerable to the development of sepsis that necessitates hospital admission for intensive care . However, the preferential suppression of neutrophils develops when leukopenia occurs, which suggests that aza's antiinflammatory effects are mediated through its effects on neutrophils as well . In fact, clinical relapse of ibd is characterized by increased neutrophil migration into the intestine . . Moreover, some reports have described cases of ulcerative colitis with prolonged remission following pancytopenia . Burke et al . Reported prolonged remission in excess of 4 years following induced pancytopenia, where 3 of the 4 patients who developed severe neutropenia remained in remission for up to 21 months following that episode . First, given that altered host immunity plays an important role in ibd activity, it is possible that this patient's host immunity was reset following the pancytopenia caused by aza . According to one report that analyzed the course of patients with crohn's disease and ulcerative colitis and who underwent allogeneic stem cell transplantation for myeloid leukemia and myelodysplastic syndrome, 10 of 11 patients showed no ibd activity after stem cell transplantation, and the colonoscopy after complete discontinuation of prophylactic posttransplant immunosuppression revealed no pathologic findings . Second, the possible benefit of granulocyte macrophage colony - stimulating factor (gm - csf) can be hypothesized . Dieckgraefe and korzenik investigated the safety and possible benefit of gm - csf for the treatment of 15 patients with moderate to severe crohn's disease . Third, the original bowel flora is temporarily altered after total gut decontamination and long - term antibacterial treatment . Therefore, the most likely theory seems to be the modification of a genetically determined immune abnormality that is responsible for the chronic intestinal inflammation by the reset of the immune system or modification of an altered microbial environment . However, it is unclear whether this mechanism can affect the long - term clinical course . Some studies have reported that tpmt genotype or activity does not predict the development of aza - induced myelotoxicity . On the other hand, some reports have suggested a substantial correlation between low enzyme activity and the development of myelotoxicity [13, 14]. Reported that assessment of the tpmt genotype or activity can reduce the risk of myelotoxicity in approximately one - third of patients . In our case, the tpmt was the wild type and this result raised doubts whether tpmt can be a reasonable screening test to identify patients with a high risk of severe myelotoxicity due to an inherited deficiency of tpmt activity . In conclusion, we report here a patient who had crohn's colitis and complicated perianal fistulas that required immunosuppression, and who achieved endoscopically determined remission that showed accelerated mucosal healing as well as clinical remission after aza - induced pancytopenia.
Diabetic macular edema (dme) is a common complication of diabetic retinopathy (dr) and a leading cause of visual loss in this population [1, 2]. Major components of dme are retinal microvascular dysfunction and blood - retinal barrier (brb) breakdown with consequent increase in vascular permeability that allows plasma compounds to leak into the retina [35]. There is evidence that upregulation of angiogenic and inflammatory factors, including vascular endothelial growth factor (vegf), and downregulation of antiangiogenic factors as well as redox shift contribute to the breakdown of the brb in dr [510]. Oxidative stress and inflammation also play an important role in the pathogenesis of dr and dme . Vegf causes conformational changes in the tight junctions of the retinal vascular endothelial cells and plays a major role in the increased vascular permeability and brb breakdown in diabetic eyes [5, 7, 11, 12]. Vitreous vegf levels correlate significantly with the severity of dr, but dme can occur in nonproliferative dr (npdr) as well as proliferative dr (pdr). Interventional studies on ranibizumab, a monoclonal antibody against vegf, have shown that intraocular injections of ranibizumab significantly reduce foveal thickness and improve visual acuity in patients with dme [14, 15]. However, the conclusions of the studies were not based on the comparison of the real intravitreal concentration of vegf with the foveal thickness in oct; they only asses the retinal thickness before and after therapy . In our earlier study, we found that also the intravitreal uric acid (ua) concentrations correlated significantly with degree of dr . We suspect that ua may play a role in the pathogenesis of dr and dme: studies of ua strongly suggest that its redox potential affects endothelial function and might contribute to the brb breakdown . The correlation of intravitreal ua with vegf in npdr and dme has not been studied yet . Optical coherence tomography (oct) has enabled clinicians to noninvasively evaluate the effect of dr on retinal thickness in a standard clinical setting [18, 19]. However, there are very limited data on how oct parameters in dme correlate with vitreous levels of vegf and other biochemical parameters . The aim of our study was to analyse the vitreous and serum of diabetic patients with dme and severe npdr and compare them to nondiabetic controls . The analysis focused on vegf and ua as two possible pathogenetic factors in the development of dme . We compared blood and vitreous levels of vegf, ua, and protein between the two study groups and describe their correlation with the changes seen in oct . First group involved 16 subjects with type 2 diabetes mellitus (dm) with npdr and cystoid dme . In this group, the mean duration of dm was 18.6 8.3 years and 15 patients (93.75%) were treated with insulin and 1 patient (6.25%) was treated with peroral antidiabetics . A group of 13 nondiabetic subjects with idiopathic epiretinal membrane and diffuse retinal thickening served as control . Dm duration was defined as the duration from the first diagnosis of dm to the time of vitreous sampling . All patients underwent a standard ophthalmologic examination including measurement of best corrected visual acuity, slit - lamp biomicroscopy, indirect ophthalmoscopy, and oct . The retinopathy was graded according to the early treatment diabetic retinopathy study research group and patients enrolled in the study had moderate to severe nonproliferative dr (npdr). The center involving dme was defined clinically and confirmed by retinal thickening in cross - sectional spectral domain (sd) oct scans . The indications for vitrectomy in this study were macular edema and preoperative best corrected visual acuity (bcva) more than 0.3 logmar (logarithm of the minimum angle of resolution) and in the diabetic group no or poor response to previous therapy with photocoagulation or intravitreal injection . Exclusion criteria were as follows: (a) history of intraocular haemorrhage, (b) prior vitreoretinal surgery, (c) other ocular surgeries or laser coagulation less than 6 months prior to the operation, (d) history of ocular inflammation, (e) proliferative dr or other retinal conditions causing neovascularisation, (f) ophthalmic disorders associated with macular edema, and (g) treatment with intravitreal anti - vegf or steroid injections (e.g., triamcinolone, dexamethasone, bevacizumab, ranibizumab, and aflibercept) less than 6 months prior to the operation . At the time of the study, all patients were in a stable clinical condition without clinical or laboratory signs of acute inflammation . The research was approved by the local institutional ethics committee, faculty of medicine and dentistry, palacky university olomouc, czech republic . Data and sample collection was independent of all treatment decisions . It did not affect a patient's access to treatment and fully complied with all ethical and legal requirements for noninterventional data collection in the czech republic . All patients gave written informed consent to the treatment, as well as data collection . The reported investigations were in accordance with the principles of the current version of the declaration of helsinki . Oct examinations were performed one day before vitrectomy with spectral domain oct (cirrus hd - oct, carl zeiss meditec ag, jena, germany) using macular cube acquisition according to the manufacturer's protocol . The macular cube 512 128 scan consists of 128 raster scans with 512 a - scans, within a 6 6 mm macular area . The mean central retinal thickness (crt, i.e., central subfield thickness) from the internal limiting membrane to the retinal pigment epithelium at the fovea was defined as the mean retinal thickness in a 1 mm diameter circular zone concentred on the fovea . Also cube volume (cv) and cube average thickness (cat) of the scanned area were calculated by cirrus hd - oct software and checked for accuracy . The cv is calculated from the 1 mm diameter zone and cat from the central 6 mm diameter zone concentred on the fovea . Based on previous studies that evaluated morphological changes in dme [22, 23], the central scan through the fovea was assessed for the presence of intraretinal cysts and serous retinal detachment (srd) by an independent examiner . Vitrectomy was performed to improve visual acuity and to decrease retinal thickness in the macula . Each patient underwent standard three - port therapeutic pars plana vitrectomy using current surgical techniques (the alcon constellation vision system). Before opening the infusion port at the start of the vitrectomy, undiluted vitreous samples were obtained and collected in sterile tubes (cca . 0.3 ml). Overnight fasting blood samples were drawn from the antecubital vein at the time of vitrectomy and used for biochemical assay . The concentration of ua was estimated using enzymatic methods (uricase - peroxidase) with photometric detection (modular, roche, germany). Hba1c was measured by high performance liquid chromatography and calibration was traced to the reference method of the international federation of clinical chemistry (variant ii, bio - rad; http://www.bio-rad.com/). The concentration of vegf was quantified by enzyme linked immunosorbent assay (elisa) using a commercial human vegf kit (r and d systems, minneapolis, mn, usa) according to the manufacturer's protocol . The limits of quantification for vegf were min = 31.2 pg / ml and max = 1000 pg / ml, respectively . We calculated the median with 1st and 3rd quartile (iqr, interquartile range). In 16 subjects, the intravitreal vegf and in 3 subjects the intravitreal ua concentration were under the detection limit; these subjects were included in the statistical analysis to avoid selection bias . Hence, we used the nonparametric analysis for ordinal variables, and the concentrations under the detection limit were assigned the comparison between dme group and control group was done by mann - whitney u test and fisher's exact test . Biochemical analysis of the vitreous showed significant differences between dm and control group in the concentration of vegf, ua, and total protein but not albumin as shown in table 2 and figures 13 . In all nondiabetic control subjects, the concentration of vegf in vitreous was under the detection limit of 31.2 pg / ml . In the diabetic group, ua concentration in vitreous correlated significantly with vitreous vegf concentration (= 0.559, p = 0.03). However, in dme vitreous vegf and ua did not correlate with the total vitreous protein . Further, in the control group, no significant correlation between the biochemical analytes in vitreous was found . Figure 4 shows the relationship between vitreous vegf and vitreous ua of dme and control group . Median of serum concentration of ua in diabetic patients was significantly elevated compared with the control group (337.0 mol / l, iqr: 324.0407.0 mol / l in dm group versus 259.5 mol / l, iqr: 220.0334.8 mol / l in control group; p = 0.025). Also median concentration of vegf in serum of diabetic patients (414.3 pg / ml, iqr: 293.1512.0 pg / ml) was higher than in controls (332.7 pg / ml, iqr: 149.4551.8 pg / ml), but the difference was not significant . There was a significant correlation between ua concentrations in serum and vitreous (= 0.652, p = 0.016) in the control group but not in dme . Further, no significant correlation between concentrations of vegf in serum and vitreous was found in both groups . The median crt, cat, and cv did not differ significantly between both groups and are listed in table 3 . Significant difference was found in presence of srd between the groups as shown in table 3 . In the diabetic group, there was a significant correlation between crt and cat (= 0.589, p = 0.016). The crt of dm subjects also correlated significantly with the cv (= 0.581, p = 0.018). However, the strongest correlation in the dm group was between cat and cv (= 0.999, p <0.001). The srd was found in the oct scans of 6 diabetic eyes, but its presence did not correlate with any of the other oct parameters . Further, among all oct parameters, only cv correlated significantly with the concentration of vitreous vegf in the dm group (= 0.515, p = 0.041). The crt, cat, cv, and srd show in dm and control subjects no significant correlation to vitreous concentrations of ua, albumin, or total protein . The correlation of logmar bcva with changes in oct parameters and vitreous content was also evaluated and we found it to be nonsignificant in both groups . There was also no correlation between oct parameters and serous concentrations of ua or vegf . The results demonstrate that biochemical analysis of the vitreous showed significant higher concentrations of vegf, ua, and total protein in dm and control group . Moreover, in patients with dme intravitreal levels of ua correlate significantly with intravitreal levels of vegf . Furthermore, we found that the cv measured with cirrus hd - oct correlate significantly with the concentration of vegf in the vitreous of patients with npdr and dme . In our earlier study, we showed that the levels of intravitreal ua correlated significantly with the degree of dr and recently also serum ua concentration has been found to be associated with increase in severity of dr . Finding significant higher ua concentration in vitreous of dm compared to controls and a correlation between ua and vegf in the vitreous of npdr patients supports our assumption that ua too may be one contributing causal factor in the pathogenesis of dr . Ua is a degradation product of metabolism and under normal conditions ua acts as an antioxidant . In diabetics, hyperglycaemia induces redox stress, which leads to consumption of the naturally occurring local antioxidants protecting capillary endothelium . This results in urate redox shuttle, meaning that ua paradoxically becomes prooxidant and contributes to endothelial dysfunction through oxidative - redox stress . Johnson et al . Showed that local ischemia results, via enzymatic activation, in increased ua production as well as oxidant formation . Decreased total antioxidant status was shown to contribute to the progression of pdr via induction of vegf . On the other side, high ua concentration in the vitreous of diabetic patients the vegf - induced production of reactive oxygen species was attenuated by urate; however, it did not modify the vegf - induced changes in permeability of monolayers . This could explain the correlation of ua and vegf in the vitreous of diabetic group found in the present study . It has to be elucidated whether ua is originating from leakage of retinal vessels, which is increased in dr, or from local production . Although total vitreous protein was significantly higher in the diabetic group compared to controls, its level did not correlate with both ua and vegf . Furthermore, in the diabetic subjects we found no correlation between serum and vitreous level of ua . However, to be able to distinguish the origin of increased ua in the vitreous, further analyses, for example, with tagged ua, should be done . Since there was a correlation between vitreous ua and vegf but no correlation between vitreous ua and oct parameters, we conclude that ua has a probable relation with diabetic microangiopathy and accordingly dr but not directly with the development of dme . Recent studies have shown that vegf causes conformational changes in the tight junctions of retinal vascular endothelial cells and plays a major role in the elevated vascular permeability in diabetic eyes with dme . It is well known that the vitreous vegf levels correlate significantly with the severity of dr [1113, 28]. Few authors have also found a significant correlation between retinal thickness at the fovea measured on oct and vegf concentrations in the vitreous [29, 30] and aqueous . The association of vitreous vegf levels and dme morphology was studied by sonoda et al . And these authors showed no significant differences in vegf concentrations in cystoid versus diffuse aspect of dme . In the present study, there was no significant correlation between vitreous vegf levels and crt of diabetic patients . However, the results show that in dme increase in cv correlated with increased concentration of vegf in the vitreous . The retinal thickness at the central fovea in funatsu et al . Was calculated as average foveal thickness from 4 manual measurements per patient . Used the average thickness of the central area with 1000 m in diameter calculated by humphrey oct . Defined the central macular thickness as the average thickness of the central 500 m in diameter . The mean crt in dme is widely accepted as the new surrogate marker for evaluating treatment efficacy . This is also because the changes in the fovea are deciding for the visual acuity . In our study, the crt, cat, and cv in both dm and control group did not correlate with the logmar bcva . There are also studies evaluating the effect of the anti - vegf therapy using both the mean foveal thickness and cv . On the other hand, since dme usually affects the macular area and not only the foveal region, assessment of vegf concentration in clinical practice using the cv is comprehensible . Like us, sonoda et al . Showed that there was no significant correlation between intravitreal vegf levels and the amount of subretinal fluid in dme . Other studies have reported that eyes with serous retinal detachment often have a poor prognosis after treatment [22, 36]. The strength of the study described here is that it determined the relationship between the levels of intravitreal biochemical parameters and retinal morphology at the same time . The limitation was the small sample size (29 eyes). This was caused by decreasing use of vitrectomy for dme and this curtailed collection of vitreous samples . Although our vitrectomy for dme might be considered overtreatment, it was a comparatively effective method as it stabilized the intraocular condition of dme and the efficacy was maintained for a long period . In interpreting or generalizing our results, it should be remembered that the findings demonstrate association of vegf levels in the vitreous with the cube volume, but they do not prove cause and effect . Further, the study was focused on vegf and ua; however, the pathogenesis of the dme is complex and still not fully understood . Vitreous concentrations of uric acid and vegf were significantly higher in dm subjects than in controls . Moreover, vitreous ua concentration correlated significantly with the vitreous vegf concentrations in patients with npdr and cystoid dme . Increased vegf concentrations are known to be involved in the pathogenesis of dme . Our results suggest that, apart from vegf, the role of ua in the pathogenesis and progression of dr should also be considered . Comparing oct parameters to the vitreous levels of ua and vegf, we found that increased concentration of intravitreal vegf in patients with npdr and cystoid dme correlated with increase of cube volume calculated by cirrus hd - oct . Since dme usually affects the macular area and not only the foveal region, the assessment of the vegf concentration in clinical practice using the cube volume is comprehensible . This oct parameter could be used to assess the efficacy of anti - vegf therapy.
Diabetes is the most frequent cause of end - stage renal disease in industrialised countries [1, 2]. Clinically, diabetic nephropathy is characterized by the development of albuminuria and a subsequent decline in glomerular filtration rate . This severe complication significantly influences the risk of cardiovascular disease as well as mortality and quality of life [3, 4]. At present the most important identified risk factors are diabetes duration, arterial blood pressure, and glycaemic regulation . As evident from an increasing incidence of affected patients, however, there is still a great need for new strategies in the treatment and prevention of diabetic nephropathy . Clear evidence indicates that the pathogenesis of diabetic nephropathy is multifactorial and triggered by a complex series of pathophysiological events . The inflammatory response in diabetes is highly complex involving proinflammatory cytokines and chemokines, for example, il1 the impact of complement activation on the diabetic kidney may well, in part, be mediated through induction of cytokine response and inflammation [911]. Several studies have linked diabetic late - complications to the complement system of the innate immune system [12, 13]. The complement system plays a crucial role in recognition and clearance of infectious microbes and the system forms a link between innate and adaptive immunity . The activation of the complement system results in the release of multiple inflammatory signaling molecules . Ultimately complement activation leads to the formation of pore - forming membrane attack complexes (macs) that are inserted in the cell membranes to mediate lyses of the cell through osmotic stress . However, in mammalian cells, it has been shown that sublytic amounts of mac can increase production of il-8 and monocyte chemoattractant protein 1 dependent on nfb nuclear translocation . Furthermore, macs are shown to have a mitogenic effect and cause release of basic fibroblast growth factor and platelet - derived growth factor from endothelial cells leading to fibrosis in neighboring cells including glomerular mesangial cells [16, 17]. The latter effects of mac may explain the link between complement and diabetic kidney damage . Three activations pathways exist: the classical, the alternative, and the lectin pathway . The present paper focuses on the lectin pathway, in which at least five soluble pattern - recognition molecules are characterized that may activate the complement system, that is, mannan - binding lectin (mbl), h - ficolin, l - ficolin, m - ficolin, and collectin - k1 . The three ficolins utilize a fibrinogen - like domain that binds, for example, n - acetylglucosamine, n - acetylgalactosamine, and n - acetyl - neuraminic acid, whereas mbl and cl - k1 have a carbohydrate recognition domain and through this bind specifically to patterns of monosaccharides . A very recent publication reports a close association between ficolin and diabetic nephropathy in patients with type 1 diabetes . The observational design of the study, however, limits its ability to study a cause - effect relationship . When ficolins bind they all initiate activation of associated serine proteases (mbl associated serine proteases, masps), which subsequently cleave the complement factors, c2 and c4, leading to further complement activation [20, 21]. Eventually, complement activation leads to the formation of macs causing cell lyses or induction of fibrosis . The balance between activation and inhibition of the complement cascade is tightly controlled by regulatory proteins in order to prevent damage of healthy host cells . In diabetes, inappropriate effects of the complement system may be present as glycation - induced dysfunction of the complement inhibitory mechanism and consequently overactivation of the system is indicated [2325]. It is speculated that diabetic patients are exposed to uncontrolled complement attack partly due to altered molecular patterns on the cell surfaces as a consequence of high blood glucose [24, 25]. Most significantly, an association is seen between diabetic nephropathy and the lectin pathway [2628]. We have previously demonstrated direct cause - effect relationship between presence of mbl and worsening of kidney injury in a mouse model of diabetic nephropathy [29, 30]. We speculate that ficolins also exert detrimental effects in diabetes similar to mbl through activation of the lectin pathway as indicated in patients with type 1 diabetes . This study aimed to investigate the impact of ficolin b (the orthologue to human m - ficolin) on the development of diabetic nephropathy in a mouse model of type 1 diabetes . We used 11-week - old, female ficolin b knockout mice and age - matched, female c57bl/6j bomtac wild - type mice (taconic, ry, denmark). The knockout ficolin b model was backcrossed more than 10 generations to a c57bl/6j bomtac genetic background (own breeding). In each cage there were three to eight mice and they had free access to tap water and standard chow (altromin number 1324; lage, germany). The environment was stable with a 12-hour light - dark cycle, temperature at 21 1c, and humidity of 55 5% . The ficolin b knockout mice and the wild - type mice were randomized into a diabetic and nondiabetic group; thus four groups were made: (1) diabetic knockout mice (n = 6), (2) nondiabetic knockout mice (n = 7), (3) diabetic wild - type mice (n = 11), and (4) nondiabetic wild - type mice (n = 11). Diabetes was induced by intraperitoneal injections of streptozotocin (stz) dissolved in a cold 10 mm citrate buffer (doses of 55 mg / kg body weight, sigma aldrich, st louis, mo, usa) on five consecutive days . The 18-week experiment was initiated when the mice were classified as diabetic (blood glucose> 15 mm). Animals with more than 15% sustained weight loss, signs of illness, or persistent ketonuria were excluded from the study . Blood glucose was measured from tail vein by contour (bayer diabetes care, kgs . Lyngby, denmark). With combur test d strip (roche diagnostics gmbh, mannheim, germany) two mice from each diabetic group were excluded because of insufficient increase in blood glucose levels . Furthermore, two mice from the diabetic knockout group were excluded because of weight loss> 15% of body weight . The excluded mice were not included in the number of animals per group indicated above . Spot urine was collected in eppendorf tubes on five consecutive days prior to sacrifice of the animals . The blood samples were drawn from under the tongue at baseline and from the retroorbital venous plexus at study end and collected in potassium edta tubes (sarstedt, nmbrecht, germany). The animals were anesthetized by an intraperitoneal dose of ketamine at 0.5 mg / g body weight and xylazine at 0.2 mg / g body weight (ketaminol 4 vet and narcoxyl vet, resp ., urinary albumin excretion was determined by mouse albumin elisa quantification kit (bethyl laboratories, inc ., urine creatinine was measured by isocratic high - performance liquid chromatography (hplc) on a zorbax scx300 column (agilent, usa) using a slight modification of a method first reported by yuen et al . . In brief, 5 l urine was added to 100 l acetonitrile containing 0.5% acetic acid and vortexed for 15 seconds to extract the creatinine . After 15 min of 20c storage and centrifugation the supernatants were evaporated and then reconstituted with 25 l 5 mm sodium acetate, ph 4.1 . Duplicate samples (10 ul each) were fractionated on a 50 mm 2.1 mm zorbax scx300 column with an in - front scx guard column . Isocratic hplc was performed at a flow rate of 1 ml / min, and uv absorbance was monitored at 225 nm . A standard curve was created by including a 2-fold dilution series of creatinine anhydrous (sigma aldrich). This study was designed with two independent factors; diabetes / nondiabetes and knockout / wild - type and thus analysed by two - way anova for normal distributed variable with equal variance . The main focus of interest was the interaction between the diabetic factor and the knockout factor; that is, does ficolin b modify the effects of diabetes on the effect parameters? If no interaction was found, the independent effects of diabetes and ficolin b on the kidney were estimated . For pairwise comparison, normal distributed data was tested with student's t - test, whereas otherwise the wilcoxon mann - whitney rank sum test was used . Data are given as mean (95% confidence interval (ci)) unless else is stated . At baseline, the knockout mice on average weighed 20.0 g, which was slightly less than the wild type mice, 20.8 g (p = 0.04). No difference was found between the two diabetic groups or between the two nondiabetic groups (table 1). After 18 weeks an expected difference in body weight was observed between the diabetic and the nondiabetic mice independently of knockout status (p <0.001). The nondiabetic mice weighed 3.2 g (ci: 2.0 g4.3 g) more than the diabetic mice . Furthermore the diabetic knockout mice were significantly smaller than the diabetic wild type (p <0.05). As presented in table 1, blood glucose, estimated as area under the curve (auc), did not differ between the two diabetic groups (p = 0.69) or between the two nondiabetic groups (p = 0.13). The kidney weight was equally increased in diabetic wild - type mice, 24% (ci: 13%36%), and in the diabetic knockout mice, 29% (ci: 12%47%), compared to the respective control groups (figure 2(a)). No interaction between knockout and diabetes was found (p = 0.60), indicating that wild - type and knockout mice develop the same degree of diabetes - induced renal hypertrophy . The considerable body weight difference between the two diabetic groups at study end indicated that the kidney weight was to be normalised to the body weight . Ficolin b did not modify the diabetes - induced increase in kidney weight when testing for interaction (p = 0.11). Furthermore, no significant statistical difference was found in kidney weight per body weight between the diabetic wild - type, 1.95 mg / g, and the diabetic knockout, 2.89 mg / g (p = 0.09). The albumin - to - creatinine ration (acr) was higher among the diabetic wild - type mice, 76 mg / g (ci: 50103 mg / g), compared to the nondiabetic wild - type mice, 44 mg / g (ci: 2563 mg / g), p = 0.07 . Similarly, the acr of diabetic knockout mice was 96 mg / g (ci: 71122 mg / g) compared to the nondiabetic knockout group, 34 mg / g (ci: 2344 mg / g), p <0.001 . As depicted in figure 3 no interaction was observed between diabetes and ficolin b knockout, p = 0.21 . In the present study we found no association between diabetes - induced kidney changes and the presence of ficolin b. we conclude that ficolin b is not responsible for, or a crucial contributory factor in, the pathophysiology of diabetic nephropathy . In our study, the kidney weight and to some extent the acr were altered by diabetes as expected . The diabetes - induced increase in kidney weight, measured by comparing the diabetic mice with the nondiabetic mice, was not statistically different between the wild - type and ficolin b knockout mice . The diabetes - induced increase in kidney weight was 24% in the wild - type mice and 29% in the ficolin b knockout mice . Taking the lower body weight of the knockout mice into account, the difference in renal hypertrophy was still insignificant when comparing the wild - type mice and the ficolin b knockout mice . Similarly, the diabetic change seen in acr was not altered in the absence of ficolin b. the experimental setup including four groups matched on age, body weight, and genetical background was a strength to the study, as the diabetes factor and the knockout factor were the only modulators of the outcome . Most importantly both diabetic groups did reach and sustain blood glucose levels of above 15 mm . At study end, the body weight differed among groups, which impeded the analyses of the diabetic kidney damage, because the knockout mice appear to be more vulnerable to type 1 diabetes mellitus . Our study provides important new information on the association between the lectin pathway and diabetic kidney damage . We are the first to investigate the role of ficolin b (which corresponds to ficolin m in human) in the inflammatory response of diabetic nephropathy . In mice with deficiency of mbl, the classical functional and physical renal changes normally seen in this experimental model of type 1 diabetes were modified [29, 30]. The fact that ficolin b does not appear to modulate diabetic effects on the kidney emphasizes the importance of mbl compared with ficolin b. both mbl and ficolin b activate the lectin pathway of the complement system, but only deficiency of mbl has been shown to protect against diabetic kidney damage . This indicates that the role of the lectin pathway in the development of diabetic nephropathy is complex and may depend on the specific carbohydrate - binding properties of mbl as previously described . The function of other complement factors in the first parts of the lectin pathway (e.g., ficolin a and masps) in the pathology of diabetic kidney disease remains unknown and must be explored in further studies . One study indicates that ficolin a and ficolin b exert a cooperatively defensive role in destroying streptococcus pneumoniae, suggesting a synergetic immunological effect . This emphasises the need for further investigations involving both mouse ficolins . In order to fully understand the involvement of the lectin pathway in the development of diabetic nephropathy, an additional parallel experiment with masps is of particular interest given that they represent the limiting downward step in the complement activation in conclusion, this study demonstrates that ficolin b does not modify the kidney weight and acr in a type 1 diabetes mouse model . This indicates that the role of the lectin pathway in the development of diabetic nephropathy is specific and that hyperglycaemia - induced glycations on renal cells may be more prone to bind mbl than ficolin b.
How to choose surgical approach in severe multilevel cervical cord compression case is a hot debate on spine surgery . Some surgeons think anterior surgery should be of the primary choice, because of direct decompression, restoring intervertebral height and reconstruction of stability7,12). Some surgeons prefer posterior approach, owing to easy decompression, low surgical risks and complications14). Whereas other surgeons suggest that combined anterior and posterior approach at one stage achieve more sufficient decompression though with increasing of complications and costs10). Multiple factors should be considered when a surgery is to be performed, especially on patients with multilevel cord compression from both ventral and dorsal parts . Which approach is better to decompress the cord sufficiently and maintain the spinal column simultaneously, anterior or posterior? However, in clinical practice, surgeon's experience and surgical skills are usually of the primary consideration . Although anterior discectomy and fusion (acdf), or corpectomy and fusion (accf) has satisfactory results in treatment of multilevel lesions of cervical spondylosis and ossification of the posterior longitudinal ligament (opll), some patients remain complain of persistence or recurrence of new symptoms . Reports elucidated the causes of unsatisfactory results and the revision techniques have so far been limited . Thus, the purpose of this study was to investigate the causes for failed anterior multilevel acdf or accf, and the outcomes of secondary laminoplasty . Between feb 2003 and may 2011, 23 patients who complained of persistence of primary symptoms or occurrence of new symptoms for more than 3 months after multilevel anterior cervical surgery (acdf or accf) in our spine surgery center were reviewed in the study . Patients demonstrated as neck pain, cervical movement restriction, spasticity, sensory loss, weakness, or sphincter dysfunction to different extent . Therapeutic treatments included traction, collar support, physical therapy, functional exercise and analgesics . Of 23 patients, the study population consisted of 12 men and 5 women, with a mean age of 59.95.6 (range, 49 - 78) years old . Main indications for the second surgery were when patients displayed signs of cord compression at least at 2 levels associated with / without canal stenosis corresponding to signs of myelopathy . Major exclusion criteria were symptomatic treatment less than 3 months, axial neck pain as the solitary symptom, cervical instability, active systemic infection, and other contraindications in routine surgery . Instability documented as angle difference (cobb angle) more than 15 degrees and/or listhesis more than 3 mm on extension and flexion lateral views . The patient underwent a general anesthesia and is positioned prone with the head and neck held in slight flexion on the operating table . A cervical midline incision is performed down to the cervical fascia, and the cervical musculature is reflected off of the involved cervical lamina . The decompression levels extended to one level cranial or / and caudal to compressive lesions are included . A 4-mm laminotomy is created just medial to the level of the pedicles on the clinically more symptomatic side, or on the most narrowed side on images, if there is no dominant symptom side . Then the involved ligaments and flavum is removed until the pulse of dura sac is seen . The external cortex is thin out to a 3- to 4-mm trough on the contralateral lamina, again just medial to the level of the pedicles, and inner cortex is preserved . The laminae are lifted carefully to create a greenstick fracture on the hinge side, expanding the spinal canal diameter . Then cervical titanium mini - plate (medtronic sofamer danek, memphis, usa or depuy synthes, bettlach, switzerland) is placed at each level secured by two 4-mm screws onto the lamina and two at the level of the lateral masses . At last the patient is placed in a hard cervical collar for the first 6 weeks after surgery . Patients' ages, gender, disease, the first surgical technique and levels, intervals between the first surgery and the onset of new symptoms or the persistence of primary symptoms, re - surgical levels, and causes for revision were examined . The outcomes were evaluated preoperatively, at 1 week, 2 months, 6 months, and the final visit . Scores are based on the rating of motor function (fingers, 0 to 4 points; shoulder and elbows, -2 to 0 points; and lower extremity, 0 to 4 points), sensory function (upper extremity, 0 to 2 points; lower extremity, 0 to 2 points; and trunk, 0 to 2 points) and urinary bladder function (0 to 3 points). A normal joa score is 17 points . Recovery rate was calculated as: (scores at follow up - scores preoperatively)/(17-scores preoperatively)100% . Recovery rate more than 75% was classified as excellent, 50 - 75% good, 25 - 50% fair, and less than 25% poor; the excellent to good rate was calculated . Patients were required to rate their neck pain, using the visual analogue scale (vas; 0 mm = no pain, 100 mm = worst pain) at different intervals, supplemented with at 1 day postoperatively . Cervical alignment was created by a line parallel to the inferior aspect of the c2 body and a line parallel to that of the c7 body, according to cobb's method on neutral lateral view (fig . All data were analyzed by sas 8.0 (statistical analysis system, sas institute inc ., between feb 2003 and may 2011, 23 patients who complained of persistence of primary symptoms or occurrence of new symptoms for more than 3 months after multilevel anterior cervical surgery (acdf or accf) in our spine surgery center were reviewed in the study . Patients demonstrated as neck pain, cervical movement restriction, spasticity, sensory loss, weakness, or sphincter dysfunction to different extent . Therapeutic treatments included traction, collar support, physical therapy, functional exercise and analgesics . Of 23 patients, the study population consisted of 12 men and 5 women, with a mean age of 59.95.6 (range, 49 - 78) years old . Main indications for the second surgery were when patients displayed signs of cord compression at least at 2 levels associated with / without canal stenosis corresponding to signs of myelopathy . Major exclusion criteria were symptomatic treatment less than 3 months, axial neck pain as the solitary symptom, cervical instability, active systemic infection, and other contraindications in routine surgery . Instability documented as angle difference (cobb angle) more than 15 degrees and/or listhesis more than 3 mm on extension and flexion lateral views . The patient underwent a general anesthesia and is positioned prone with the head and neck held in slight flexion on the operating table . A cervical midline incision is performed down to the cervical fascia, and the cervical musculature is reflected off of the involved cervical lamina . The decompression levels extended to one level cranial or / and caudal to compressive lesions are included . A 4-mm laminotomy is created just medial to the level of the pedicles on the clinically more symptomatic side, or on the most narrowed side on images, if there is no dominant symptom side . Then the involved ligaments and flavum is removed until the pulse of dura sac is seen . The external cortex is thin out to a 3- to 4-mm trough on the contralateral lamina, again just medial to the level of the pedicles, and inner cortex is preserved . The laminae are lifted carefully to create a greenstick fracture on the hinge side, expanding the spinal canal diameter . Then cervical titanium mini - plate (medtronic sofamer danek, memphis, usa or depuy synthes, bettlach, switzerland) is placed at each level secured by two 4-mm screws onto the lamina and two at the level of the lateral masses . At last, the patient is placed in a hard cervical collar for the first 6 weeks after surgery . Patients' ages, gender, disease, the first surgical technique and levels, intervals between the first surgery and the onset of new symptoms or the persistence of primary symptoms, re - surgical levels, and causes for revision were examined . The outcomes were evaluated preoperatively, at 1 week, 2 months, 6 months, and the final visit . Scores are based on the rating of motor function (fingers, 0 to 4 points; shoulder and elbows, -2 to 0 points; and lower extremity, 0 to 4 points), sensory function (upper extremity, 0 to 2 points; lower extremity, 0 to 2 points; and trunk, 0 to 2 points) and urinary bladder function (0 to 3 points). Recovery rate was calculated as: (scores at follow up - scores preoperatively)/(17-scores preoperatively)100% . Recovery rate more than 75% was classified as excellent, 50 - 75% good, 25 - 50% fair, and less than 25% poor; the excellent to good rate was calculated . Patients were required to rate their neck pain, using the visual analogue scale (vas; 0 mm = no pain, 100 mm = worst pain) at different intervals, supplemented with at 1 day postoperatively . Cervical alignment was created by a line parallel to the inferior aspect of the c2 body and a line parallel to that of the c7 body, according to cobb's method on neutral lateral view (fig . All data were analyzed by sas 8.0 (statistical analysis system, sas institute inc ., cary, nc, usa). Mean duration between the first surgery and the onset of new symptoms or persistence of symptoms was 14.54.2 (range, 1 to 44) months . Causes for revision surgery were inappropriate approach in 3 patients (17.6%), insufficient decompression in 4 patients (23.5%), adjacent degeneration in 2 patients (11.8%), and disease progression in 8 patients (47.1%). There were 8 patients of disease progression, with 4 cases of opll and 4 cases of cervical spondylotic myelopathy (csm). The progression levels increased from 1.250.2 at the first surgery to 2.250.4 at the second surgery on opll patients, and from 1.50.2 to 2.50.4 on csm patients . Mean follow - up duration was 29.712.1 (range, 6 - 84) months . Mean joa score was 10.54.1 preoperatively, 11.84.2 at 1 week, 13.54.4 at 2 months, 14.14.8 at 6 months and 13.94.6 at the final visit (table 2); significant improvement achieved at 2 months (p<0.05) and maintained thereafter (p>0.05). Mean recovery rate was 20.0%, 46.1%, 55.4%, and 52.3% after surgery, respectively; significant improvement was detected at 2 months (p<0.05) and maintained with the time (p>0.05). One case had excellent result, 5 good, 7 fair, and 4 poor, with an excellent to good rate of 35.3% at 1 week; two cases had excellent results, 8 good, 5 fair, and 2 poor, with an excellent to good rate of 58.8% at 2 months; three cases had excellent results, 9 good, 4 fair, and 1 poor, with an excellent to good rate of 70.6% at 6 months; three cases had excellent results, 8 good, 4 fair, and 2 poor, with an excellent to good rate of 64.7% at the final visit . Mean vas score was 29.116.7 preoperatively, 51.127.6 at 1 day, 30.717.2 at 1 week, 9.19.9 at 2 months, 8.57.9 at 6 months, and 9.08.2 at the final visit . Vas score decreased at 1 week (p<0.05) and at 2 months (p<0.05). Mean lordotic angle was 11.710.3 preoperatively, 11.110.2 at 1 week, 11.710.3 at 2 months, 10.99.7 at 6 months and 10.39.1 at the final visit; no significance was observed during the follow up (p>0.05) (fig . One case demonstrated as axial pain and relieved 2 weeks after pain management and physical therapy . Acdf or accf has excellent and good results in treatment of cervical spondylosis and opll . The anterior approach removes pathological compression to the cord directly, stabilizes spine column, and maintains cervical alignment . However, if the disease involved multiple levels or posterior compression, the ideal effects of anterior approach are difficult to achieve3,5). It is not easy in performing sufficient decompression and preserving cervical range of motion on acdf or accf . Besides, corpectomy of multilevel vertebrae in series destroys the integrity of spine column and has a risk of nonunion . Especially for patients with congenital or acquired narrowed canal, the compression to cord remains after initial anterior surgery . In the study, 3 patients demonstrated narrowed canal associated with posterior compression and persistent even after aggravation of symptoms after anterior surgery . However, after the secondary laminoplasty, the canal is enlarged significantly and the cord is given more space to breathe1). If the compression was mainly from the posterior part associated with or without canal stenosis, the posterior approach should be performed . Second cause was adjacent degeneration . In a study by matsumoto et al.11) that acdf patients had significantly higher incidence of progression of disc degeneration than control subjects . Prasarn et al.15) thought biomechanics altered after fusion, with an increased adjacent - segment motion in cervical spine . After fixation and fusion, there is a change in the load of the stiffed fusion which accelerated the degeneration of adjacent disc and endplate, especially on the caudal level . Faldini et al.6) thought proper lordotic alignment could prevent adjacent degeneration . In our opinion, though factors of degeneration were not systematically elucidated, maintaining lordosis and exercising cervical dorsal musculatures could help a lot . It was thought that factors influencing decompression included surgical approach, technique and experience of surgeon, and overall knowledge of pathologic structure . Osteophyte, ossified nucleus pulposus, opll and abnormal curvature venous plexus increased complexity of decompression, especially in patients with multilevel lesions or on narrowed canal . The last but not the least was disease progression . In the study, half of patients displayed as extension of cord compression though after sufficient initial anterior decompression . The symptoms aggravated gradually until the patients seek for the surgeons . After the revised posterior surgery, the enlarged canal was enough to compensate for the spinal cord in a narrowed space for a long time . Thus, in the study, symptoms of patients with disease progression alleviated immediately after revision . Factors of progression are not clear, which may include metabolism, heredity, endocrine secretion and biomechanics . Choi et al.4) also believed that age, severity of disease, irreversible changes of gray matter in cord, duration of symptoms, and diabetes mellitus took important parts . Patients' age, general status, extent and range of compression, and safety should be considered, when planning a revision surgery . Pang et al.13) thought although anterior surgery was more effective in relieving cord compromise, inadequate decompression and disease progression might require secondary laminoplasty . Scar tissue around anterior region after anterior surgery increased risks of neurovascular, trachea or esophagus injury . Be - sides, once the cage and vertebrae above and below are fused, it was difficult to remove the protruded or ossified materials along posterior wall of vertebrae, especially in multilevel segments . It was thought that myelopathy could be treated via anterior or posterior approaches, but risks and complications in acdf increase . Average joa score, recovery rate, and excellent to good rate didn't improve until at 2 months, and attained the maximum at 6 months, with a slight deterioration thereafter . However, it was reported by hyun et al.9) that loss of alignment was time - dependent but no further decreased after 18 months . In the study, the lordosis maintained during the entire follow up . In a study by baba et al.2), excellent to good rate of secondary laminoplasty was 55.6% . They thought that narrowed canal added to the risk of recurrence, and additional laminoplasty could prevent structural compromise occurring adjacent vertebrae . Symptoms and signs had no obvious alleviation, with high - signal intensity remained persistent . Yagi et al.18) also thought that long - term outcome was significantly worse in these patients . The outcome was also worse in patients with expansion of high - signal area, which the causes might be not only from necrosis secondary to ischemia of the anterior spinal artery, but also from the repeated minor traumas inflicted on the cord from an unstable spine . Combining the research and related literatures, we thought that multiple factors affect outcomes of revision surgery, which included age, extent lesion of cord, surgical times, mental disease, and so on . Edwards et al.5) deemed that both corpectomy and laminoplasty arrested progression in multilevel myelopathy, which resulted in significant neurologic recovery and pain reduction . However, the laminoplasty cohort required less pain medication than did the corpectomy cohort . Given the higher prevalence of complications in anterior approach, it was believed that laminoplasty might be the preferred treatment in the absence of preoperative kyphosis . However, although it is a safe and effective technique, complications include paralysis of c5 nerve root, axial pain, and instability of spine, cerebral spinal fluid leakage and close of " opening door"16). Laminoplasty preserving the c7 muscle attachment is very important . In the study, except for the decompression extending to t1, all the other cases preserve muscle insertions into the c7 spinous process . It was also reported that this preservation was associated with a significantly decreased frequency of postoperative axial neck pain8). Future studies will seek to research in a prospective, randomized and controlled way with a longer duration . Despite limitations, laminoplasty should be considered as a primary procedure or as a revision procedure versus anterior approach in multilevel lesions because of effective decompression, preservation cervical alignment and minimum morbidity . Laminoplasty has satisfactory results in failed anterior surgery, with a low incidence of complications.
Population aging is the process by which older individuals become a proportionally larger share of the total population . It was the most distinctive global demographic event that the world witnessed in the past century and is an important event for the twenty - first century too . It was initially experienced by the more developed countries and has recently become apparent in the developing countries . Population aging will be faced globally by all countries in this century, although at varying levels of intensity and time . According to the world health organization (who), world's elderly population i.e., people 60 years of age and older is approximately 650 million at present and by 2050, it is forecast to reach 2 billion . In 2008, five out of the top ten causes of mortality worldwide, other than injuries, were non - communicable diseases (ncds) and expected to go up to seven out of ten by the year 2030 . By then about 76% of the deaths in the world will be due to ncds . Government and the who have already recognized the huge burden of preventable disease, disability, death and distress caused by the ncds . The present international health agenda guided by who focuses on four conditions (cardiovascular disease, diabetes, cancer and chronic respiratory disease) responsible for most of the premature mortality and four lifestyle risk factors (smoking, harmful alcohol use, lack of physical activity and high salt, high fat diets) leading to above conditions . Medications play crucial role in geriatric health care as they treat chronic diseases, alleviate pain and improve quality of life . Age - related changes in drug disposition and pharmacodynamic responses have significant clinical implications, and increased use of a number of medications in elderly raises the risk of medicine - related problems that may occur . As medication use and the incidence of adverse drug outcomes increase with advancing age, it is important to ensure quality use of medicines in older people towards attaining a higher goal of healthy and active aging . But, use of drugs by the elderly and their clinical outcomes especially adverse drug reactions (adrs) have not been prominent research topic and continues to be given low priority in national and international public health arena, at least in a developing country like india . However, we feel that this probably needs more attention in india because we have not completely dealt with the scourge of communicable diseases yet . In addition, 70% of all older people now live in low- or middle - income countries, including india where sustainable pharmacovigilance systems has not developed as yet . The relationship between increased use of drugs and elderly is well established . Consequently, increased use of medications in elderly increases the risk of adrs . Studies from around the world have shown a definite correlation between increasing age and adr rate, at least for some medical conditions . Although very few such studies are available in indian settings, harugeri et al . In a hospital setting found that the prevalence of adr - related hospital admissions was 5.9%, while in another such study in india, it was observed to be 6.7% . For india, harugeri et al . Predicted that 18000 bed days in a given time would be due to adrs in elderly and total cost of hospital stay due to adrs is estimated to be us $4350 (inr 200100), that is us $80.5 per patient (108.7% of per capita per year expenditure on health). There has been much debate on whether advancing age by itself is a cause of increased risk of adrs but merely a marker for comorbidity, altered pharmacokinetics, and polypharmacy . Gurwitz and avorn concluded that patient - specific physiological and functional characteristics are probably more important than any chronological measure in predicting both adverse and beneficial outcomes associated with specific drug therapies, while studies around the world have clearly shown that the risks of adrs (including interactions) is related to the number of medicines taken and sometimes due to inappropriate use of medicines . (1991) observed an exponential rather than the linear relation between the risks of adr and the number of medicines taken in 9000 elderly italian patients receiving 10 medicines . Review of several studies by steward rb et al . Found that the patients aged above 65 years use an average of two to six prescribed medications, and 1 - 3.4 non - prescribed medications . Out of all the factors that are most consistently associated with adverse drug reactions, polypharmacy is considered to be the most important . Thus, studies have correlated the integral association between old age and increased rate of adrs arising out of confounding association between age and polypharmacy contributed by age - related changes in pharmacodynamics and pharmacokinetics at least for some medical conditions . The classical pharmacological classification of adrs by rawlins and thompson divides adr into two major subtypes: type a reactions, which are dose - dependent and predictable, and unpredictable type b (bizarre or idiosyncratic) reactions . Majority of adrs (80%) causing admission or occurring in hospital setting are type a reactions . They are predictable and potentially avoidable in nature as they are related to accentuation of known pharmacological effects of the drug . Drugs associated with type a reactions are generally with low therapeutic index and commonly used among elderly . The lists of medicines most likely to be used in the elderly include antibiotics, anticoagulants, digoxin, diuretics, hypoglycemic agents, antineoplastic agents and non - steroidal anti - inflammatory drugs (nsaids) and these are responsible for 60% of adrs leading to hospital admission and 70% of adrs occurring in hospital . Type b adrs are usually uncommon, but rarely may sometimes cause serious toxicities . Therefore, adrs in elderly are largely contributed by prescribing error e.g., large doses of drugs without taking into account, the effect of age and frailty on drug disposition, especially renal and hepatic clearance . The other contributing factor may be because of not considering the increased pharmacodynamic sensitivity of the elderly to several commonly used drugs, e.g., central nervous system and cardiovascular drugs . Since only around 3000 subjects receive a medicine prior to marketing, it is not surprising that less frequent (particularly type b) adrs are often recognized only after marketing . The capacity of premarketing studies to recognize adrs is further reduced by the limited numbers of patients in the age group of 65 or older in trials and that even smaller numbers of the oldest old . In addition, long latency diseases like cancer are difficult to detect on account of the short duration of study . The latter has been defined in the literature in relative terms (for example, the administration of an excessive number of drugs) and in absolute terms, ranging from two to more than six simultaneous medications . In contrast to general population, the incidence of combination therapy is found to be greatest in the elderly . A drug combination may sometimes cause synergistic toxicity, which is greater than the sum of the risks of toxicity of either agent used alone . For example, the combination of corticosteroids and nsaids: the risks of development of nsaid - induced peptic ulcer in older patients may increases by 10% among elderly . However, concurrent use of corticosteroids and nsaids had shown a risk of peptic ulcer disease that was 15 times greater than that of non - users of either drug . Similarly, the relative risk of hospitalization for hemorrhagic peptic ulcer disease in elderly patients (above 65 years) has been observed to be increased by manifolds on concurrent use of oral anticoagulants and nsaids, while the risks were lower when used alone . This suggests that there is certainly a need to recognize and consider the aspects of synergism of toxicity while prescribing medications in elderly . However, polypharmacy at times can effectively control certain disease condition e.g., hypertension and epilepsy . Respecting the individual needs of every individual patient is the preferred approach of prescribing in elderly . Some of the steps that may be helpful in minimizing adrs in elderly may be: maintaining accurate record of all medications in use: may include asking patients to bring all medications to clinic including the use of over - the - counter and complementary medicines.number of medications (prescribed and non - prescribed): monitoring to balance the need and avoid polypharmacy while minimizing under - use of vital drugs.individual doses: reducing the doses wherever appropriate and titrating them carefully from a low starting dose, if pharmacodynamic sensitivity is likely to be the problem.simple regimens of medication: choose the preparation suitable for the patient and minimize the dose whenever needed, but avoid advising the patient to break a single tablet into two or three equal pieces.ensuring safe management of medications by patients: involving patients in decisions on their therapy by educating the patient about important side effects and what to do if they occur . Minimize hoarding of previously used and expired drugs.therapeutic drug monitoring is encouraged, but should not replace clinical observation . Considering an adr as a possible cause for any new problem.utilizing available strategies and inter - disciplinary collaboration to enhance the quality use of medicines . Maintaining accurate record of all medications in use: may include asking patients to bring all medications to clinic including the use of over - the - counter and complementary medicines . Number of medications (prescribed and non - prescribed): monitoring to balance the need and avoid polypharmacy while minimizing under - use of vital drugs . Individual doses: reducing the doses wherever appropriate and titrating them carefully from a low starting dose, if pharmacodynamic sensitivity is likely to be the problem . Simple regimens of medication: choose the preparation suitable for the patient and minimize the dose whenever needed, but avoid advising the patient to break a single tablet into two or three equal pieces . Ensuring safe management of medications by patients: involving patients in decisions on their therapy by educating the patient about important side effects and what to do if they occur . Considering an adr as a possible cause for any new problem . Utilizing available strategies and inter - disciplinary collaboration to enhance the quality use of medicines . Drugs are double - edged weapons and increased use of drugs by elderly increases the risk of adverse drug reactions causing increased morbidity and mortality . In addition, economic consequences of adrs constitute a problem of considerable magnitude . Developing countries have rapidly aging population and the governments are seeking guidance in promoting healthy and active aging . However, strategies to increase opportunities for identifying adrs and related problems have not been emphasized in current policy responses in india to meet the increase in elderly population and chronic conditions . In addition, there is definite paucity of good quality research and data on adrs in india . Further, growing pharmacotherapy with increase in the number of diseases in the elderly will require more timely and accurate drug safety data . Pharmacoepidemiological studies that encompass large numbers of elderly drug users are needed to obtain this information as increased knowledge of the frequency and cost of adverse drug reactions is important in enabling more rational therapeutic decisions by individual clinicians and more optimal social policy . Given quantitative information on medication risks, clinicians will be able to change the use of the drug (prescribe for fewer patients, use safer alternatives when available, and use in lower doses for shorter duration) or can take measures to minimize side effects (prescribe prophylactic medications, increase monitoring for side effects, and intensify patient education). Policy changes that can result from better data on drug toxicities include withdrawal of drug from the market, change in drug labeling, educational programs to physician and changes in academic curriculum of pharmacology . This will result in promoting and ensuring a good health of older people, which can be of great benefit to their families and communities . In the long run, more precise estimates of the true costs associated with adrs could stimulate development of prophylaxis and of alternative therapies.
Day care or ambulatory anaesthesia is as old as the history of anaesthesia itself . In the last few decades, it has gained significant popularity mainly because of the enormous cost savings and has therefore been accepted by the hospitals, patients and the insurance companies alike . However, the quality and excellence of care needs to be defined and defining good care is a challenge . Unanticipated admission, whatever the cause, is an indicator of the quality of care because it negates the basic goal of preventing hospitalisation and same day discharge . While day care surgery in india is limited to a few hospitals, there is a dearth of data on hospitalisation following such intervention . This study aimed to examine the incidence and factors associated with unanticipated admission following day care surgery at a tertiary level hospital in south india . Patients belonging to american society of anesthesiologists (asa) physical status classes 1 and 2, undergoing surgeries under general or regional anaesthetic techniques along with appropriate nerve blocks for post - operative pain relief were included . It was planned to use the median value of controls for continuous exposure variables (such as time of anaesthesia and time of starting last case) to define exposure status . We required 70 cases and 70 controls to detect an odds ratio (or) of 2.5 with a power of 80% and error of 0.05% . To permit adjustment for confounders, two controls per case were chosen, which required the number of subjects to be 53 cases and 106 controls . Demographic characteristics, patients weight, pre - operative medical illness, personal habits, asa status, the diagnosis and surgical procedures, time since last meal, duration of anaesthesia and surgery, experience of the surgeon and anaesthesiologist, and intraoperative management (techniques, drugs, monitoring, etc .) Were documented . The number of hours fasted was divided into 68 h, 911 h and> 11 h. the outcomes measured were the admission of patients following day care surgery, and the causes of unanticipated admissions . Discharge of the patient was decided after both the surgeon and the anaesthesiologist cleared the patient . The admission of the patient to the hospital was also decided by the surgeon or the anaesthetist depending on the complication the patient had . Those who need to stay overnight from the day care centre were considered as unanticipated admissions (the centre functions from 7 am to 9 pm with the morning session from 7:30 am to 11:30 am and the afternoon session from 12:30 pm to 4:30 pm . The reasons for unanticipated admission were classified into four main groups: (i) anaesthetic reasons included nausea vomiting, giddiness, prolonged general anaesthesia and others, (ii) surgical reasons included pain, bleeding, complicated surgery requiring wound dressing, wound drainage or antibiotics, (iii) medical reasons such as fever, drug allergies and other complications, (iv) social reasons included patients or surgeons request . A nested case descriptive statistics in the form of frequencies, median and means, standard deviations and percentages were calculated . The chi - square test and or were calculated to determine which factors were associated with unanticipated admission . The or, their 95% confidence intervals (cis) and corresponding p values were calculated for all variables . For the statistically significant variables, the mantel haenszel or was calculated to identify confounding, if any . For analysing the different narcotics used, each narcotic was given a score using morphine as a reference; 10 mg of morphine = 100 g of fentanyl = 75 mg of pethidine = 100 mg of tramadol . Hence, a narcotic score of one equals 1 mg of morphine = 10 g of fentanyl = 7.5 mg of pethidine = 10 mg of tramadol . For example, if a patient received 100 g of fentanyl and 6 mg of morphine during the surgery, the patient would have a narcotic score of 16 . The equivalent narcotic dose was totalled and the narcotic score for each patient was then calculated . A narcotic score of 10 was used as the cut - off and the tests of significance done . Data were analysed using the statistical package for social sciences (spss) version 13.0 (spss inc . During the 3-month study, from the cohort of 776 patients, 63 patients with unanticipated admission formed the case group and were compared to twice the number of unhospitalised patients, which formed the control group . At the end of the day, after all the patients had been discharged or admitted from the day care centre, lots were drawn from the discharged group, which randomly selected controls . Of these, anaesthetic complications accounted for 33.33%, surgical complications were 15.87%, medical were 6.35% and social reasons were 44.44% . Social reasons included admission on request of the surgeon and admission on request of the patient or parents of a child . The reasons for anaesthetic admission were post - operative nausea and vomiting (ponv), sweating and giddiness in the recovery room, prolonged general anaesthetic (> 2 h), probable aspiration, urinary retention, a failed anaesthetic, which required switching to another technique and post - operative pain . The surgical reasons were bleeding; complicated surgery needing wound drainage, dressings or antibiotics . Medical causes were persistent high blood pressure needing treatment, fever, seizures and drug reactions . Reasons for unanticipated admissions the orthopaedic surgery speciality had the maximum admissions (34.9%), followed by abdominal surgeries, 27% . Ear nose and throat (ent) had 11.1% admissions; vascular surgeries accounted for 3.17% whereas urology had none . The type of surgery and technique of anaesthesia among unanticipated admissions are shown in tables 2 and 3 . Type of surgery requiring readmission technique of anaesthesia in those requiring readmission individual risk factors for admissions and or's were calculated using univariate logistic regression [table 4]. The possibility of confounding was investigated using stratified analysis and calculation of the mantel haenszel or . Univariate analysis showing factors associated with unanticipated admissions the factors significantly associated with unanticipated admissions were the duration of anaesthesia, duration of surgery, experience of the surgeon, time of starting last case, hours of fasting and a high narcotics score . Anaesthesia lasting more than 50 min and surgery lasting more than 35 min were at a higher risk of admission . Patients whose surgery started after 3 pm were also at a significantly high risk of admission . The study found that patients operated on after 3 pm are 8 times at risk of being admitted than if their surgery started before 3 pm . It was found that those fasting shorter hours (68 h) had 2.2 times greater risk of admission . Intraoperative use of narcotics was analysed in both groups, and this was not statistically significant . (p = 0.06 with an or of 0.84 and a 95% ci of 0.411.71) the dose of narcotic used during anaesthesia was analysed using the narcotic score and patients with a score of> 10 were 2.86 times at a greater risk of admission with a 95% ci of 1.177.09 . It was also found that patients operated on by consultant surgeons were 3.2 times more likely to be admitted but this was not analysed further as consultant surgeons operated upon almost 90% of patients . The above statistically significant variables were entered into a bivariate logistic regression model [table 5]. Anaesthesia time 50 min and surgery time 35 min were highly correlated and hence only anaesthesia time 50 min was entered into the model . The anaesthesia time 50 min (p = 0.002; or: 3.18; 95% ci: 1.506.72) and starting of last case after 3 pm (p = 0.002; or: 10.10; 95% ci: 2.4242.15) remained statistically significant for unanticipated admissions . The day care centre is an architecturally and functionally integrated independent unit with areas for admission, surgery, recovery and discharge . It consists of an admission area with a lounge for relatives, two operating theatres, and a four - bedded recovery room with adequate monitoring facilities, oxygen and suction apparatus and in addition a six bedded day surgery ward . The surgical disciplines using the day care centre include ent, orthopaedics, general surgery, vascular surgery and urology . Unanticipated admissions following day care surgery are a quality marker of efficiency of ambulatory surgery services . The incidence of unanticipated admission in this study was 4.51% that compares favourably with other published reports, which document rates between 1.04% and 5.4% . Patients were admitted for an average of 4.5 days however 70% of patients were admitted only for 1 day . The factors associated with unanticipated admissions identified by the study were long duration of anaesthesia and surgery, starting of last case after 3 pm, shorter hours of fasting and greater dose of narcotics used . . Anaesthesia lasting longer than 50 min and surgery lasting longer than 35 min were significantly associated with an increased risk of admissions . The study has found that longer anaesthesia is associated with a 3.85 times greater and longer surgery has four times increased risk of admissions . This study documented that surgeries started after 3 pm had a higher risk of unanticipated admissions and is consistent with those reported in literature . Fortier et al . Have reported that completion of surgery after 3 pm was a significant factor . This may be because patients having surgery later in the day had less time to recover and be fit for discharge as compared to patients whose surgery was started earlier in the day . The use of intraoperative narcotics was statistically significant on the univariate analysis but after adjusting for confounders using bivariate analysis, it was not significant . The study showed that the incidence of ponv was one of the major anaesthetic risk factors for admission, which accounted for 51.14% of anaesthetic admissions . Therefore, a comparison between the narcotic score and ponv was made, and a significant association was found . Patients with a narcotic score of> 10 were nine times more likely to have ponv requiring admission . The ability to provide adequate pain relief without exacerbating ponv remains one of the major challenges for providers of outpatient anaesthesia and surgery . Balanced analgesia techniques involving the use of opioids and other groups of analgesic drugs such as local anaesthetics and non - steroidal anti - inflammatory drugs are now being commonly used . Post - operative pain is the most commonly reported complication of day care anaesthesia with up to 50% of patients experiencing wound pain . However, this study did not show that instead it demonstrated that the use of opiates can lead to ponv requiring admission . It was found that shorter fasting time of <8 h induced a greater risk of unanticipated admission . The univariate analysis showed that shorter fasting time was at 2.2 times the risk of admission . The bivariate analysis showed that shorter fasting times had 1.9 times the risk of admission though not statistically significant . This group of patients may be the patients who are on the afternoon list starting at 12:30 pm, are allowed a light breakfast at 6 am and therefore have a higher risk of ponv . The duration of fasting and time of starting last case were stratified, and the mantel haenszel or calculated . Since both the crude or and the mantel haenszel odd ratio were almost equal it was concluded that there was no confounding factor involved . The uncorrected incidence of unanticipated admission in our study was 8.11%, and this is because our day care facility was relatively new and the threshold level for admission was very low . Surgeons request accounted for 36.5% of all cases when no real discernable reason could be made out . It may be that the surgeon suspects that he might have done a little more extensive procedure on the patient than anticipated, but does not fall under the category of complications . A small number (7.9%) also included request for admission from the patient or parents of a child . The idea that patients can go home on the same day as the surgery is a novel one and one which patients find difficult to reconcile to and therefore request for admission . Therefore, if both these groups are removed, the overall incidence works out to the value of 4.51% . Our setup is part of a large tertiary hospital and in case of any apprehension, admission from the day care centre to the ward is relatively easy and more readily facilitated . The variables that were not significant were weight of the patient, distance travelled, pre - existing medical disease, and asa status, type of anaesthetic and experience of the anaesthetist . Type of surgery and gender also had no detectable effect on admission, as evidenced in other studies . Although it was expected that pre - existing medical disease, type of anaesthetic and type of surgery would be significant, it was not so in our study . A large sample size and doing asa 3 and 4 patients in the day care facility the factors associated with unanticipated admissions included longer duration of anaesthesia and surgery, later time of starting last case, shorter hours of fasting and the greater dose of narcotics used . These factors can guide decision making to facilitate improved efficiency of the day care centre . The low rates of medical causes of admissions reflect a good pre - anaesthetic assessment in terms of the suitability of patients.
The relationship between the clotting cascade and the promotion or inhibition of the anti - inflammatory response continues to be defined through basic research . The potential key role of proteins that were originally believed to be exclusively involved in coagulation in the inflammatory response provides an exciting potential mechanism(s) for modification through the application of new therapies . The accompanying review by riewald and ruf outlines the available information on the possible steps involving some of the various proteins that are common to both coagulation and inflammation . The complexity of the multiple actions of the proteins in both inflammation and coagulation should excite the clinician with regard to potential new therapies that may be beneficial via activation of one or both of the concurrent pathways . However, before clinicians can administer new therapies that utilize new knowledge on the actions of coagulation cascade components, and hence improve the outcomes of their patients, several gaps in our current understanding must be addressed . This information is imperative if the clinician is to take basic knowledge' to the bedside', where individual patients can benefit . Administration of intrinsic or synthetic analogues of coagulation cascade components (e.g. Protein c, thrombin, and factor viia or xa), coagulation inhibitors (e.g. Heparin or analogues), or agonists or antagonists of the protease - activated receptors, in an attempt to alter the individual patient's coagulation / inflammation balance, could theoretically produce responses ranging from beneficial to detrimental . Knowledge of the' most beneficial' balance between augmenting or inhibiting the contribution of the clotting cascade components to inflammation would be required for determining the selection and dosing of potential new therapies . A safe assumption is that any new therapies that alter the intrinsic response to coagulation and inflammatory stimulants would also potentially produce detrimental effects on these, and potentially other, physiological responses . A simple example of such a potential for detrimental effects is the administration of protein c resulting in undesirable haemorrhage . This example illustrates that a broader knowledge of the impact of altering the coagulation/ inflammatory response will be required before clinicians can comfortably utilize new therapies in patient care . It is unlikely that any new therapies resulting from the new knowledge of the contribution of the clotting cascade to inflammation can be applied to all patients . It can be anticipated that certain patients would have contraindications to the new therapies or that dosages would need to be adjusted on the basis of pharmacokinetic or pharmacodynamic variables . The clinician will need to know what these contraindications and variables are before the new therapies can be used confidently . Some, such as coagulation disorders, may be known and obvious, but any new strategies that could potentially alter the balance of a complex physiological response may be anticipated to be influenced by many, as yet unknown, factors in the individual patient . An example is the concurrent administration of heparin, which appears to antagonize the anti - inflammatory effect of antithrombin iii in septic patients . A more complete understanding of the impact of concurrent conditions, therapies and the patient's genetic make - up on outcome will be required if we are to obtain the maximum benefit from new therapies . For any new therapy resulting from our expanded knowledge of the clotting cascade in the inflammatory response to be tailored to the needs of the individual patient, a method(s) for rapidly and easily assessing the patient's dynamic inflammatory response will be required . Unless any new therapies have very benign toxicities, the need to assess the need, dosage and response to treatment will mandate sensitive and specific monitoring techniques . Bedside or clinical laboratory techniques that could be applicable to most clinical environments would allow the benefit of any new therapies to be applied to the greatest number of patients . Conversely, any new treatments that require complex, laborious monitoring techniques will have limited applicability . As our knowledge of the contribution of the intrinsic coagulation components increases, the development of many potential new therapies will hopefully emerge for clinical use . These may be totally new therapeutic entities or new strategies for using existing drugs or clotting factors . Such developments will create the new dilemma for the clinician, namely that of trying to select between different treatment options for the individual patient . Clinical trials outlining the potential anticipated benefit(s) and the appropriate target patient population for each new therapeutic strategy will need to be completed . This will allow the clinician to ensure that the most appropriate strategy can be selected for the individual patient . The above - mentioned concerns aside, the clinician should be excited about the expanding knowledge of the role of coagulation cascade components in modifying the inflammatory response . Such knowledge will hopefully result in new strategies and therapies to manage the septic patient.
Myelofibrosis (mf) belongs to the category of myeloproliferative neoplasms (mpns) and may present as a primary disorder (primary myelofibrosis [pmf]) or evolve from polycythaemia vera (pv) or essential thrombocythaemia (et) to post - pv or post - et mf.1 it is characterised by the clonal proliferation of a pluripotent haematopoietic stem cell,2 in which the abnormal stem cell population releases several cytokines and growth factors into the bone marrow microenvironment, thus leading to an increase in bone marrow fibrosis, stromal changes, involvement of extramedullary organs such as the spleen and liver, and consequent clinical manifestations.3 myelofibrosis has an incidence of about 0.58 new cases per 100 000 person - years, but a higher prevalence of 6 per 100 000 person - years because of its chronic and disabling course.4 median age at diagnosis is 67 years, without any significant difference in distribution between the sexes . The diagnosis of mf is currently based on the world health organization 2016 criteria, which include the jak2v617f mutation that is detected in 50% to 60% of all cases.58 mutations in genes other than jak2 such as mpl mutations (frequency: 5%10%)9,10 and somatically acquired mutations in the calr gene (frequency: 15%20%)11,12 have also been described . However, about 10% of patients with mf do not develop any known mutation and are considered to have triple - negative mf.13 in addition to these 3 driver mutations, numerous other somatic mutations involving epigenetic processes (ezh2, tet2, asxl1, and dnmt3a), spliceosome machinery (srsf2, sf3b1, and u2af1), and disease evolution (eg, tp53, idh1/2, and ikzf) have been identified in mf.1416 some of these mutations, such as those in dntm3a17 or tet2,18 have not been shown to correlate with survival outcome . Conversely, mutations in asxl1, srsf2, and ezh2 predicted short survival in a large cohort of patients . More specifically, a report by tefferi et al19 points to the calr/asxl1 + profile as the most detrimental mutation profile in pmf . The symptoms mainly include those associated with splenomegaly (abdominal distension and pain, early satiety, splenic infarction, dyspnoea, and diarrhoea) and constitutional symptoms such as fatigue, cachexia, pruritus, bone pain, weight loss, and fever; these worsen patients role functioning and quality of life (qol). Median survival ranges from approximately 3.5 to 5.5 years,20,21 and the most frequent cause of death in patients with mf is transformation to acute myeloid leukaemia (20%), but most patients die because of other disease - related events, such as progression without transformation, infections, and thrombo - haemorrhagic complications.20 prognosis is currently based on 3 different prognostic scoring systems, which mainly refer to age, constitutional symptoms, anaemia, white blood cell counts, and percentage of peripheral blood blasts: international prognostic scoring system (ipss), which is applicable at diagnosis20; dynamic international prognostic scoring system (dipss)22; and dipss - plus, which can be applied at any time during followup . The last incorporates 3 additional independent risk factors: red blood cell (rbc) transfusion requirement, platelet counts of <100 10/l, and an unfavourable karyotype (table 1).23 until recently, mf has remained orphan of curative treatments: the only treatment that has a clearly demonstrated impact on disease progression is allogeneic haematopoietic stem cell transplantation (allo - hsct), but treatment - related mortality is high and only a minority of patients are eligible for such intensive therapy.24 the previously used treatments were palliative and have only limited benefits in qol and symptom control . However, the discovery of jak2 mutations, which has established that dysregulation of the jak - stat signalling pathway is a major contributor to the pathogenesis of mpns, has also led to the development of small - molecule jak1/2 inhibitors, the first of which (ruxolitinib) has been approved for the treatment of mf in the united states and europe . In this article, we report on old and new therapeutic strategies that proved effective in early preclinical and clinical trials and subsequently in the daily clinical practice for patients with mf, particularly concerning the topics of anaemia, splenomegaly, iron overload (io), and allo - hsct . The management of anaemia can be one of the most challenging aspects of treating patients with mf (table 2). Blood transfusion is the standard therapy for symptomatically anaemic patients, and the transfusion target should be assessed individually . Corticosteroids (eg, prednisone 0.5 mg / kg / day) may be temporarily effective in treating anaemia and constitutional symptoms and are usually used in combination with other therapies.25 erythropoiesis - stimulating agents (esas) are worth trying in mf patients with moderate, nontransfusion - dependent anaemia and a low serum erythropoietin level (<125 iu / l), although rapid spleen enlargement during treatment has occasionally been reported . Response rates vary from 23% to 60% in different studies, with no clear evidence favouring darbepoetinalfa over conventional recombinant erythropoietins . Furthermore, responses are usually short - lived (<1 year), and as no prospective randomised study of the value of esas has yet been published, they are not indicated in anaemic subjects with established transfusion dependency.26 if there are no contraindications, androgen preparations or danazol (a semisynthetic attenuated androgen) can be used . They have been shown to stimulate erythropoiesis in patients with refractory anaemia, leading to increased haemoglobin (hb) levels, reticulocytosis, and a decreased need for rbc transfusions27; however, documentation of their efficacy as single agents is largely restricted to retrospective studies . One of these reported responses in 11 of 30 patients with mf, including 8 with a complete response,28 with a lack of transfusion dependence and higher pretreatment hb levels predicting response . In another retrospective study, responses were observed in 17 of 39 patients with mf taking danazol, including 8 (21%) with an increase in hb of 1.5 g / dl, hb levels of> 10 g / dl, and transfusion independence for 8 weeks.29 however, there were no identifiable patients characteristics (such as transfusion dependency, baseline hb level, or cytogenetic results) that influenced outcome . These findings have been confirmed in a recent series of 50 patients with mf30; the slightly lower rate of anaemia response (30%) should be attributed to the use of more stringent response criteria.31 in terms of predicting response, the only pretreatment variable showing a trend for an association with response to danazol was transfusion dependency, with only 18.5% of the responders in this subgroup of patients against 43.5% in the subgroup not requiring transfusions . The main limitations of using danazol are toxicities, including fluid retention, increased libido, liver function test abnormalities, headache, and virilisation . All patients receiving danazol should therefore be monitored using monthly liver function tests during initial therapy and periodic liver ultrasound examinations to detect any hepatic malignancy . The antiangiogenic and immunomodulatory properties of thalidomide, lenalidomide, and pomalidomide make them potentially effective medical therapies for mf, with some responses in patients with anaemia, thrombocytopenia, and splenomegaly; potential modifications to the bone marrow microenvironment; and a possible reduction in bone marrow fibrosis . The combination of thalidomide and prednisone has been evaluated in 21 patients with mf, 62% of whom showed an anaemia response.32 however, the high incidence of neuropathy associated with thalidomide limits its usefulness . Furthermore, because of the risk of thrombosis, prophylaxis with aspirin is recommended in all patients with a platelet count of> 50 10/l . This combination is therefore not usually selected for the first - line management of anaemia . A phase ii clinical trial (nct00227591) assessed the therapeutic efficacy of lenalidomide combined with prednisone in 42 patients with mf . Clinical improvements in anaemia and splenomegaly were observed in, respectively, 19% and 10% of the subjects . Similar to thalidomide, lenalidomide was burdened by toxicity, including cytopenia (at least 1 grade 34 event in 88% of patients) and nonhaematologic toxicity (at least 1 grade 34 event in 45% of patients).33 a second study of lenalidomide plus prednisone in 40 patients with intermediate - risk or high - risk mf led to an overall response rate based on international working group criteria of 30% for anaemia and 42% for splenomegaly, with a median time to response of 12 weeks . However, grade 3 and 4 adverse events (aes) were reported, mainly cytopenia.34 a recently updated report of this study after a median follow - up of 9 years35 showed that treatment responses improved over time, with 14 patients (35%) responding overall . More specifically, 39% of the patients showed a response in terms of reduction in spleen size, and the overall anaemia response rate was 32% . However, there was no significant difference in baseline characteristics between the patients who responded and those who did not . An analysis combining the results of 3 phase ii trials indicated that lenalidomide - based therapy may be more effective than thalidomide - based therapy, and fewer patients treated with lenalidomide plus prednisone discontinued therapy due to toxicity than those receiving thalidomide - based therapy . In addition, there was no significant difference in the response to lenalidomide alone and lenalidomide plus prednisone; however, response duration was significantly longer in patients who received lenalidomide plus prednisone.36 pomalidomide, a more potent immunomodulatory drug, has been evaluated in a multicentre, double - blind, placebo - controlled phase iii study (nct01178281).37 however, the study failed to meet the primary endpoint as an equal proportion of patients with mf in the pomalidomide (n = 152) and placebo arm (n = 77) achieved an anaemia response (16% vs 16%, p = 1). On the contrary, the platelet response was significantly better in patients who received pomalidomide (22% vs 0%). Cytoreductive agents have been the treatment of choice for most mf patients with symptomatic splenomegaly (table 3). Hydroxyurea (hu), an s - phase cell cycle specific nucleotide - depleting agent that inhibits ribonucleotide reductase,38 is one of the most widely used medical therapies for patients with appreciably symptomatic splenomegaly,39,40 although it induces only modest responses at higher doses (12 g daily) and mainly in subjects with nonmassive splenomegaly (<15 cm).45 although hu is generally well tolerated, the modest improvement in symptoms is temporary, and exacerbated cytopenia frequently limits treatment . In patients who do not respond to hu, it has been shown that the oral alkylating agents, melphalan and busulfan, improve splenomegaly and other disease symptoms, but they may also exacerbate cytopenia and possibly increase the frequency of leukaemic transformation . Furthermore, they are mainly used in older patients as they are relatively manageable insofar as frequent laboratory monitoring is not required, unlike in the case of hu or other cytoreductive agents.41,46 in cases of massive refractory splenomegaly, it has been found that monthly courses of intravenous cladribine (2-chlorodeoxyadenosine) lead to a response in up to 50% of patients, with severe but reversible cytopenia being the main toxicity.42 interferon - alfa (standard and pegylated versions) has proved to have only a minimal clinical effect in reducing splenomegaly, and therefore, its use is not generally recommended.43 hypomethylating agents, such as azacitidine and decitabine, have also been studied in mf, but currently play only a limited role in its treatment.47 more recently, thomas et al48 demonstrated that methotrexate (mtx) may act as an inhibitor of the jak - stat pathway and that this activity is likely to be specific and not related to a general effect on protein phosphorylation: the drug s in vitro activity was observed at a concentration equivalent to that used in patients taking low - dose mtx (525 mg / wk). What is important is that its efficacy in controlling haematologic parameters, systemic symptoms, and splenomegaly has been confirmed in vivo in 2 recent case reports.49 splenectomy is a palliative debulking measure used in patients with mpns . Its indications are mainly symptomatic massive splenomegaly, symptomatic portal hypertension with oesophageal varices and/or bleeding, profound cachexia, transfusion - dependent anaemia, and/or severe hypercatabolic symptoms . Removal of the spleen improves mechanical symptoms (ie, early satiety and pain) in most cases and is often followed by weight gain in cachectic patients, but it is usually not effective against other constitutional symptoms . Improvements in anaemia and thrombocytopenia after splenectomy have been reported in, respectively, 50% and <30% of patients . Progressive hepatomegaly sometimes follows splenectomy, probably due to the migration of haematopoiesis, and a markedly enlarged liver is a contraindication to splenectomy . Current data concerning an increased rate of leukaemic transformation after spleen removal are still discordant.44,50 however, given the high complication rate and limited benefit of splenectomy, appropriate patient selection is crucial.51 splenic radiotherapy, on a fractioned basis, at a daily dose of 0.4 to 1 gy, with weekly evaluation of spleen size and haematologic values until therapeutic effect is achieved or haematologic toxicity develops, can be used to treat mpns with an adequate platelet count (> 50 10/l), as extramedullary haematopoiesis has proved to be considerably sensitive to external beam radiotherapy in patients with mf . However, it leads to only transient benefits and may exacerbate cytopenia, particularly thrombocytopenia.50 it also has to be remembered that radiation can also cause local fibrosis with splenic adhesions to surrounding tissues that make a subsequent splenectomy technically more complicated and increase the morbidity and mortality of the procedure . In general, traditional treatment options are limited and insufficient to address the morbidity and mortality associated with mf . However, as mentioned above, the discovery of mutations leading to constitutive activation of the jak - stat signalling pathway raises hope that mf may be cured by selective jak1/2 inhibitors, as happens in the case of chronic myeloid leukaemia treated with bcr - abl1 tyrosine kinase inhibitors . Ruxolitinib (jakavi; novartis, basel, switzerland) was the first jak1/2 inhibitor to become commercially available for the treatment of mf.52 in preclinical jak2v617f - positive mpn mouse models, it induced a considerable downregulation of jak - dependent proinflammatory cytokines, reduced mouse splenomegaly, and showed antiproliferative and proapoptotic activities . It is the only jak inhibitor approved in the united states for the treatment of splenomegaly in subjects with intermediate / high - risk mf and in europe for the treatment of splenomegaly and/or constitutional symptoms in patients with intermediate-2/high - risk mf.53 these approvals were based on the results of 2 phase iii randomised studies: comfort - i (ruxolitinib vs placebo) and comfort - ii (ruxolitinib vs best available therapy [bat]).54,55 the primary endpoint of both studies was a> 35% reduction in spleen volume after 24 (comfort - i) or 48 weeks of treatment (comfort - ii), which was reached by, respectively, 41.7% and 28.5% of the patients treated with ruxolitinib, as against, respectively, 0.7% and 0% of the patients receiving placebo or bat (p <.0001).54,55 overall, more than 90% of the patients enrolled in both studies experienced some reduction in spleen volume at some time during the follow - up, and the reduction remained stable in most of the patients after a median follow - up of 3 (comfort - i) and 5 years (comfort - ii).56,57 the therapeutic success of ruxolitinib is not limited to reducing spleen volume because, unlike the drugs previously used to treat mf, it is efficacious in relieving constitutional symptoms; reducing abdominal discomfort, appetite loss, itching, fatigue, and night sweats; and improving the qol of most treated patients . As the drug s activity is independent of jak2 mutational status and not specific for the neoplastic clone, the response rate is similar in patients with and without the jak2v617f mutation because of its anti - jak1mediated effect . The uk, open - label, phase ii robust study evaluated its safety and efficacy in patients with mf, including those at intermediate-1 risk . The treatment was successful in 50% of the population as a whole and 57% of the intermediate-1risk patients . Reduction in spleen length and symptoms was observed in all of the risk groups, and improvements in the myelofibrosis symptom assessment form total symptom score were seen in 80% of intermediate-1, 72.7% of intermediate-2, and 72.2% of high - risk patients.58 similarly, the phase iiib expanded - access jak inhibitor ruxolitinib in myelofibrosis patients (jump) trial for patients with mf without access to ruxolitinib outside of a clinical study found that the drug s safety and efficacy profile in intermediate-1risk patients was consistent with that in the study population as a whole and with that previously reported in intermediate-2risk and high - risk patients.59 the toxicity of ruxolitinib treatment is mainly haematologic due to the drug s interference with an essential pathway for haematopoiesis, as demonstrated in the comfort studies; in both trials, thrombocytopenia was the dose - limiting toxicity, and anaemia was the most common haematologic ae . A number of studies have shown that ruxolitinib affects many cytokines and interferes with the immune process necessary for the pathogenesis of mpns, but it also affects the function of various immune cells and may therefore favour an increased incidence of opportunistic and nonopportunistic infections.60,61 for example, ruxolitinib impairs natural killer cell differentiation and function and inhibits dendritic cell activation and migration, and antigen - specific t - cell responses in a dose - dependent manner in vitro and in vivo . However, despite warnings about this increased risk,6264 a recent update of the jump study described a low incidence of infections: the all - grade infections observed in 1% of patients included nasopharyngitis (6.3%), urinary tract infection (6%), pneumonia (5.3%), bronchitis (4.2%), herpes zoster (3.6%), influenza (3%), upper respiratory tract infection (2.9%), cystitis (2.5%), gastroenteritis (1.8%), respiratory tract infection (1.8%), and oral herpes (1.6%). Other infections included tuberculosis in 3 patients (0.3%) and legionella pneumonia in 1 patient (0.1%). No hepatitis b reactivation was reported, and only 6 patients (0.5%) discontinued treatment because of grade 3 pneumonia.59 the patients receiving ruxolitinib in the comfort - ii study experienced higher rates of viral and bacterial infection than those receiving conventional therapy, but most of the infections were grade 1 or 2 and did not lead to any dose reductions or the discontinuation of trial medication . Furthermore, the rates of infection tended to decrease with longer exposure to the drug . However, as patients with mf are already predisposed to infections65 and the long - term risks of ruxolitinib treatment are still unknown, treated patients should be carefully monitored, and prophylaxis for herpes zoster or other infections should be considered on a case - by - case basis, depending on local risk . Given its promising results, a further indication for ruxolitinib treatment is as a therapeutic bridge to allo - hsct . Furthermore, an increasing number of reports appeared in the literature, describing the morphologic changes in the bone marrow occurring in ruxolitinib - treated patients, mostly focusing on modifications in bone marrow fibrosis degree.6670 by the beginning of 2014, a number of other jak2 inhibitors were being tested: fedratinib,71 pacritinib,72 ly2784544,73 and momelotinib.74 however, the clinical trials of fedratinib and pacritinib were soon discontinued because of safety problems: wernicke encephalopathy (fedratinib) and bleeding (pacritinib). Momelotinib (formerly known as cyt387) is a small - molecule, adenosine triphosphate its kinase profiling indicates that it has good selectivity over other jak family kinases (jak3, tyk2) and excellent selectivity over other tyrosine and serine / threonine kinases.75 the preclinical data provide a rationale for the use of momelotinib in bcr - abl1negative mpns, and a multicentre phase i / ii trial involving 166 patients with intermediate / high - risk mf showed that the drug is well tolerated at oral doses of 150 or 300 mg once daily or 250 mg twice daily and led to improvements in splenomegaly, constitutional symptoms, and transfusion requirement.74 of particular interest are the transfusion independence responses, which were observed in more than half of the rbc transfusion dependent subjects with a maximum transfusion - free period exceeding 2 years . In addition, the percentage of all subjects requiring rbc transfusions substantially decreased over the treatment period . More precisely, the overall anaemia response rate was 54% in transfusion - dependent patients with a median time to a confirmed anaemia response of 12 weeks (range: 84293 days). As has been previously reported, treatment with momelotinib led to a rapid and sustained reduction in splenomegaly in approximately 31% of all cases, with a median time to response of 15 days, and the constitutional symptoms of most of the patients disappeared within 6 months . In terms of safety, about 20% of the patients experienced a first - dose effect (dizziness, flushing, and hypotension) that was self - limited . Grade 3/4 haematologic and nonhaematologic aes were infrequent with the exception of thrombocytopenia, which occurred in approximately 17% of patients . Grade 3/4 nonhaematologic laboratory aes included hyperlipasaemia (4%) and increased liver enzymes (grade 3 and 4 increase in aspartate aminotransferase in, respectively, 1% and <1% of the patients; a grade 3 increase in alanine aminotransferase in 2%). Mainly, grade 1 treatment related sensory peripheral neuropathy was reported, but there were no treatment - related deaths . In brief, momelotinib seems to lead to a significant and lasting improvement in anaemia, splenomegaly, and constitutional symptoms at doses of 150 or 300 mg / day or 150 mg twice daily . The efficacy and aes of momelotinib will be further evaluated in 2 currently ongoing phase iii trials: a randomised bat - controlled study of mf patients with anaemia and thrombocytopenia previously treated with ruxolitinib and a randomised study comparing momelotinib and ruxolitinib in patients with mf (nct02101268-nct01969838). More recently, the efficacy and safety of 3 dose levels of a potent and selective oral jak1 inhibitor, incb039110, have been evaluated in an open - label phase ii study, resulting in clinically meaningful symptom relief, modest spleen volume reduction, and limited myelosuppression.76 in particular, only 1 patient discontinued for grade 3 thrombocytopenia, whereas nonhaematologic aes were largely of grade 1 or 2 and most commonly represented by fatigue . Nearly 40% of patients with mf are anaemic at the time of diagnosis, including 25% who are already transfusion dependent,77,78 and more than 60% will develop clinically significant anaemia during the course of follow - up.49 the clinical impact of io and its potential relationship to the heightened inflammatory response of patients with mf warrant consideration not only because potential liver dysfunction, cardiac disease, and other complications of io probably contribute to patient morbidity and mortality but also because the growing evidence of impaired haematopoiesis attributable to bone marrow haemosiderosis suggests a viable therapeutic target.79,80 each unit of rbc contains 200 to 250 mg of iron, and as the reticuloendothelial system can clear approximately 10 to 15 g (corresponding to 50 rbc units), any excess is deposited in tissues and leads to organ damage.81 iron overload is a concern when treating patients with mf, which is why iron chelation therapy (ict) has been used to counteract its potentially negative effects . However, it has to be admitted that there is a lack of prospective, randomised, controlled trials of the use of ict in patients with mf . One small retrospective study of 10 patients with mf demonstrated an erythroid response in 40% of cases receiving oral ict with deferasirox (dfx), thus allowing these patients to reduce their transfusion requirement; it also revealed a trend towards better overall survival in the responding patients.82 other data coming from a number of reported case studies79,80,83,84 also indicate that ict improves anaemia and decreases transfusion dependence in patients with mf . Finally, a recent retrospective, multicentre analysis of 28 patients with mf and io secondary to transfusion dependence found that 11 patients (42.3%) achieved a stable and consistent reduction in ferritin levels (<1000 ng / ml), and 6 of 26 patients (23%) showed a persistent (> 3 months) increase in hb levels to> 1.5 g / dl, with the disappearance of transfusion dependence in 4 cases . However, comparison of the baseline characteristics of the patients who achieved an erythroid response and those who did not achieve did not reveal any significant differences that could be considered predictive.85 deferoxamine (desferal) is a linear ligand that forms 1:1 complexes with iron that maintain a net charge, allow for membrane permeability, and provide access to intracellular iron stores86 that are then excreted primarily in urine.87,88 it is administered in the form of an intravenous or subcutaneous infusion, and because of its short plasma half - life, the efficacy of the treatment correlates with the duration of infusion, and it is only effective if administered at high doses between 5 and 7 times per week.89 when administered as a continuous 24-hour infusion for 6 to 7 days per week in patients with high - risk -thalassemia, it can reverse iron - induced cardiac dysfunction and increase long - term survival.90 however, treatment - related side effects include infusion site discomfort (nearly 100%, with the development of local erythema or induration in some cases),91 visual changes (0%10%), generally transient auditory neurotoxicity (20%25%),92,93 increased serum creatinine levels (22%), vomiting (16%), abdominal discomfort (14%), constipation (14%), arthralgia (14%), nausea (11%), rash (5%), and diarrhoea (5%).94 deferasirox is an oral iron chelator frequently used in clinical practice in the united states and europe that has a long half - life of 8 to 16 hours and can be administered once daily.89 it forms a 2:1 complex with iron,95 which is then excreted largely in the bile and faeces (much less in urine).96,97 unlike other iron chelators, it is thought that dfx also affects haematopoietic stem cell differentiation by means of a reactive oxygen species mediated mechanism, which may underlie the erythropoeitic response seen in some dfx - treated patients.95 the most frequently reported adverse effects are gastrointestinal toxicity (21%64%), diarrhoea (46%), abdominal pain (15%28%), nausea (24%), vomiting (21%), and constipation (10%).94,98101 patients have also been reported to experience renal dysfunction (10%64%, usually nonprogressive at the start of treatment and improving after a dose reduction), skin rash (4%39%), arthralgia (15%),94 and transaminitis (4%70%),81,98101 and there have been rarer reports of auditory neurotoxicity (1%6%) as a potential side effect.100,101 it is not entirely clear whether ict can reverse the ill effects of io, and there are no completed studies that provide prospective evidence of a beneficial impact in terms of the restoration of normal haematopoiesis or outcomes in patients with mf . Consequently, treatment decisions concerning the use of ict in patients with mf continue to be extrapolated from the data of myelodysplastic syndromes . Allogeneic haematopoietic stem cell transplantation is still the only intervention that has been shown to be a potential cure for mf or a means of prolonging the survival of these patients . Data from the most recent studies suggest that the expected 3-year progression - free survival rate is in the range of 40% to 50%.102 the adoption of reduced intensity conditioning regimens has recently made allo - hsct applicable to a larger proportion of patients.103 however, decisions concerning allo - hsct are based on inductive reasoning and require a considerable professional experience . Key questions include patient selection, donor selection, pre- and posttransplant management, conditioning regimen, and prevention and management of post - transplant relapses . International prognostic scoring systems (ie, ipss, dipss, and dipss - plus)20,22,23 are the most comprehensive means of risk stratification currently available to guide therapeutic decision making, although the influence of driver mutations and the acquisition of additional mutations during the natural course of the disease may further refine this process . All patients with mf aged <70 years with ipss, dipss, or dipss - plus intermediate-2risk or high - risk disease and a reasonable performance status, and without any significant competing comorbid conditions, should be considered potential candidates for allo - hsct . Patients aged <65 years with intermediate-1risk disease should only be considered candidates if they present with refractory, transfusion - dependent anaemia or> 2% of peripheral blood blasts, or adverse cytogenetics (as defined by the dipss - plus classification). Finally, patients with low - risk disease should not undergo allo - hsct.104 individual transplant - specific prognostic factors should be considered in every candidate for allo - hsct to be able to make individualised decisions . In this context, the transplant - specific high - risk factors include a spleen extending more than 22 cm below the costal margin, having been transfused with more than 20 rbc units, having received a transplantation from an hla nonidentical donor, a poor performance status (an eastern cooperative oncology group status of> 2), a high comorbidity index (a haematopoietic cell transplantation comorbidity index score of> 3), and the presence of portal hypertension . Completely matched rather than mismatched donors should be selected because, as reported in the european blood and marrow transplantation registry, the cumulative incidence of nonrelapsed mortality after 1 year is, respectively, 12% and 38% and is not different between hla - identical siblings and 10/10 matched unrelated donors (10% vs 13%).105 however, haploidentical related donors are an attractive alternative source of haematopoietic stem cells.106 it is important to note that peripheral blood is considered the most appropriate source of haematopoietic stem cells in the case of hla - matched sibling and unrelated donors . When splenectomy is performed before allo - hsct, it may facilitate disease eradication . Some reports have also shown faster engraftment in splenectomised patients; however, the pretransplant use of splenectomy remains controversial as no study has yet prospectively evaluated the effect of protocol - based splenectomy before transplantation . In the case of older patients and/or those with comorbidities, a less intense conditioning regimen is more appropriate, whereas patients with advanced disease and a good performance status should undergo a more intensified regimen.104 finally, in patients relapsing with constitutional symptoms or splenomegaly, jak1/2 inhibitor treatment is recommended but remains experimental . To address this question, ruxolitinib is being administered to eligible patients with mf for 60 days before definitive allo - hsct in a prospective multicentre phase ii study conducted by the myeloproliferative disorders research consortium (nct01790295). Traditional mf treatments are primarily palliative and have proved to be inadequate to address the considerable morbidity and mortality associated with this disabling disease . More specifically, concerning anaemia, there have been various therapeutic attempts, but rbc transfusions still remain the most frequently used approach, even though io represents an increasingly frequent clinical challenge . Considering instead splenomegaly, besides hu, ruxolitinib, as well as other investigational jak1/2 inhibitors, offers new hope for these patients as they have been shown to lead not only to significant reduction in splenomegaly but also to the palliation of disease - related symptoms . However, allo - hsct is still the only intervention that has evidence indicating it is potentially curative . Obviously, in such a context, participation into a clinical trial should be encouraged whenever possible, with the purpose of making new drugs available.
A 45-year - old iranian woman was referred to the private clinic in the city of hamadan for implant consultation . Her past medical history was not notable, and there was no evidence of systemic disease . She had no history of trauma to the mandible . In the extra - oral examination, intra - oral examination revealed normal oral mucosa, the absence of soft tissue expansion, and teeth of a normal color . All of the teeth were asymptomatic, with no pain or tenderness on percussion or palpation . The involved teeth were vital in an electric stimulation test . For assessment before implant insertion, cbct had been ordered . During evaluation of the implant insertion areas on promax3d cbct (planmeca oy, helsinki, finland), a radiolucent - radiopaque mixed lesion located on the apices of the lower incisors was observed . On the axial, sagittal, and coronal cbct images, the extension of the lesion was observed from the mesial side of the right mandibular lateral incisor to the distal side of the left mandibular lateral incisor . It was a multifocal lesion in which solitary lesions were reached together and made a larger lesion . The total dimension of the lesion was about 16.6 mm in the mesiodistal direction and 6.9 mm in the longest superior - inferior direction . On the panoramic reconstructed cbct image, the lesion associated with the left mandibular lateral incisor was radiolucent, whereas the lesion on the apex of the left central incisor was mixed radiolucent - radiopaque and the lesion associated with the right central incisor was radiopaque with a radiolucent rim around the lesion of this tooth (fig . The state of the lesion relative to the buccal and lingual cortical plates could be assessed, which might not be possible on the conventional radiographs . On the axial image, one of them was located at the mesial side of the right canine and the other between the left central and lateral incisors (fig . Cross - sectional images, the discontinuity of the lingual cortex was found at the area between the two central incisors on several consecutive sectional images (fig . This cortical discontinuity was more obvious on the three - dimensional (3d) cbct images (fig . However, it should be considered that the cortical bone could be seen to have destruction on 3d images, even though it would have been thin without discontinuity on the cross - sectional images . On the digital periapical radiograph that was taken for periodontal ligament space widening was found, especially around the root of the left lateral incisor . Based on the patient clinical and radiographic findings, a diagnosis of multifocal periapical cemento - osseous dysplasia was made . Pcod is a specific lesion within this group of conditions that usually occurs in middle - aged black women.4 a systematic review of the literature on cod indicated an ethnic distribution of 59%, 37%, and 3% for blacks, asians (japanese, chinese, and korean), and caucasians including indian cases, respectively, in case reports.8 zegarelli et al reported the incidence of pcod in the general population to be 2 - 3/1000.9 periapical cemento - osseous dysplasia generally does not cause cortical bone expansion or perforation.9 - 12 alsufyani and lam reviewed the clinical and radiographic characteristics of 118 patients with cod.13 they showed that 71.6% of 118 patients had no cortical expansion, 76% had intact lamina dura, and 93% had a normal periodontal ligament space . Modern advances in imaging help the dentist to use the cbct with the appropriate field of view and spatial resolution; therefore, the internal mineralized structure of the pathologic lesions can be investigated with a low radiation dose.14 axial cbct images clearly demonstrate the location and extension of the lesion . The expansion and perforation of the cortical plates can be evaluated on cbct even if they are slight . This report showed the discontinuity of the lingual cortex on some axial and cross - sectional cbct images . This characteristic of the lesion was an unusual feature among the cases of pcod reported up to now that may be due to its large size and needed to be checked in further regular follow - ups . This feature also could be found in an exaggerated fashion on the 3d cbct images . Although 3d imaging could help the radiologist in diagnosing the lesion for the first look, it should be noted that it is not a reliable viewing modality for assigning the real status of the lesion in relation to its surrounding bone structures . In many cod cases that have been misdiagnosed and/or mismanaged, the lesions were identified in their early stages as a periapical rarefying osteitis such as periapical abscess, granuloma, or cyst, and unnecessary endodontic treatment was performed . Therefore, vitality tests are especially important for differential diagnosis . In the radiolucent - radiopaque mixed stage and the radiopaque stage, the differential diagnosis might include chronic sclerosing osteomyelitis, cementoossifying fibroma, odontoma, cementoblastoma, and osteoblastoma.13 periapical cemento - osseous dysplasia is usually detected on routine radiological examinations . However, the differential diagnosis of our case could be calcifying cystic odontogenic tumor and cemento - ossifying fibroma . By using cbct discrimination of pcod from these lesions that exhibit similar internal calcification on conventional radiography high density mass in pcod which is centered in low density area is different from findings of calcifying cystic odontogenic tumor in which calcification is observed at or near the cyst wall.15 also, cemento - ossifying fibroma has more obvious concentric buccolingual expansion on multiplanar cbct image . Unless pcod is symptomatic, treatment is usually not needed because development and maturation of the lesion is self - limiting . Intervention may cause secondary infection of the cementum - like radiopacities, which may in turn induce osteomyelitis in these lesions.13 however, if pcod demonstrates unusual changes or becomes symptomatic, surgical intervention would then be needed.7 this case was considered as no treatment with only periodic follow - up check.
Controversy exists as to whether endodontic procedures are the primary cause for loss of strength . Panitvisai and messer (1995) reported that cuspal deflection increased with increasing the extension of cavity preparations and was greatest when endodontic access was incorporated into a preparation . It has been reported that endodontically treated teeth and their contralateral vital pairs exhibited similar biomechanical properties, such as punch shear strength, toughness, and load required for fracture . Although the insertion of a post does not strengthen or reinforce endodontically treated teeth, it is basically used to provide sufficient retention of the core material which in turn is used to retain a fixed restoration . For the inserted post to perform its function, several variables must be put into consideration such as post length, diameter, geometric design, and surface configuration . Additionally, special attention should be given to the material the post is made of in order to ensure adequate distribution of the absorbed stresses and to prevent root fracture during . Regarding with consideration to the well - known success of metallic posts there are now many concerns regarding the associated inhomogeneous stress distribution, biological side effects due to microleakage and corrosion, and the influence of their dark color under an all - ceramic restorations . The introduction of non - metallic fiber reinforced composite (frc) posts helped improving stress distribution because their elastic modulus is similar to that of dentin as indicated by several clinical and laboratory studies . Frc post systems showed more frequent favorable failure modes than did metal post systems . Although fiber posts proved effective in withstanding compressive loads in posterior teeth, they behave differently in anterior teeth where non - axial biting forces prevail and where their flexural behavior becomes more effective . Recent studies suggested that glass - fiber posts contributed to the reinforcement and strengthening of endodontically treated teeth under full coverage crowns . A ferrule or encircling band of cast metal around the remaining coronal surface can provide protective reinforcement to endodontically treated teeth by encapsulation of the remaining coronal structure and by resisting functional lever forces during mastication . A minimum 1 to 2 mm of ferrule height is necessary to achieve such protective effect . Recent clinical studies reported that the ferrule structure have a direct influence on the clinical success rate of endodontically treated premolars restored with fiber posts and the failure events were due mainly to post debonding . An in vitro study reported that incomplete crown ferrule was associated with greater variation in load capacity after chewing simulation, while other studies found no effect of different ferrule heights in teeth restored with fiber posts and resin cores . The aim of this study was to evaluate the fracture resistance and failure pattern of endodontically treated mandibular premolars restored with different ferrule heights in combination with fiber posts and all - ceramic crowns . The null hypothesis was that different ferrule heights do not improve the fracture resistance or the failure pattern of the tested restorations . All external debris were removed with an ultrasonic scaler, and examined stereoscopically at 10x magnification to verify the absence of cracks, defects and dental caries . Teeth were stored in a 0.5% chloramine t (prolabo, paris, france) in saline solution . Buccolingual and mesiodistal coronal dimensions plus root length of all selected teeth were measured using a digital caliper (digimatic calipers, mitsutoyo, tokyo, japan) and only teeth with the following mean dimensions were selected: 14.1 mm root length, 7.3 mm buccolingual width, and 4.9 mm mesiodistal width . Standardized root canal preparations were made using the following procedure: initial probing using no . 10 k - files (flexo files, maillefer, ballaigues, switzerland); the root canal length was established through direct observation of the file extruding from the apical foramen . The specimens were then prepared endodontically with a stepback procedure with size 45 (flex r file; union broach, york pa). After intermittent rinsing with 2.5% sodium hypochlorite solution, the endodontic treatment was completed using manual lateral condensation method (ah plus, dentsply, de tray, konstanz, germany). After endodontic treatment, each root was thinly covered with a silicone impression material (aquasil, dentsply) to simulate thickness of periodontal ligament . All specimens were embedded in self - polymerized acrylic resin (orthoresin, dentsply, degudent gmbh, postfach 1364, d-63403 hanau, germany) poured into a mold while maintaining 2 mm below the cervical line exposed . The teeth were embedded along their long axis using a surveyor (ney surveyor; dentsply). A new diamond point (lot - nr 1599, dfs dental and technical products, gmbh, germany) was attached to the milling machine (k9 milling apparatus-990, kavo, germany) for every group . The mrd gauged diamond had a self - limiting tip, which produced a 1-mm - deep chamfer and the margins and the angle of convergence were standardized . After preparation of the finish line, the coronal dentinal extension was modified accordingly (figure 1): group 1 (control): 1 mm circumferential ferrule from the gingival margin without a fiber post; group 2: 1 mm circumferential ferrule with fiber post and resin core; group 3: non - uniform ferrule height (2 mm buccally and 1 mm lingually) with fiber post and resin core; group 4: a non - uniform ferrule height (3 mm buccally and 2 mm lingually) with fiber and resin core post; group 5: received no ferrule preparation with fiber post and resin core . Schematic representation of the different tested groups in the control group, excess gutta - percha was removed to a depth of 2 mm from the coronal surface of the preparation, using a carbide bur (171l-012, brasseler, usa). The coronal walls were etched with 36% phosphoric acid (total etch, ivoclar - vivadent, schaan, liechtenstein) for 15 s, washed with water spray and then gently air - dried . One coat of adhesive resin (excite, ivoclar - vivadent) was applied using a microbrush and light polymerized for 20 s (astralis 10, ivoclar - vivadent). A hybrid composite resin (tetric ceram, ivoclar - vivadent) was applied and light polymerized for 40 s. for the remaining groups, post space was created using no . 1 peeso reamer (union broach co., long island, ny, usa) and corresponding calibrating drill (frc postec plus, #1, ivoclar - vivadent) leaving 4 mm of apical gutta - percha intact . A translucent glass fiber reinforced composite post (frc postec plus #1 ivoclar - vivadent, schaan, liechtenstein) was used . Each post was cut to a suitable length with a diamond bur so that it was covered with at least 2 mm of resin composite occlusally . Post cementation was carried out with self - polymerized resin cement (multilink, ivoclar vivadent) following manufacturer s instructions . Silane coupling agent (monobond - s, ivoclar - vivadent) was applied on the post surface for 60 s and then air - dried . After post cementation, the surrounding dentin surface was etched with 36% phosphoric acid (total etch, ivoclar - vivadent) for 15 s, washed with water spray and gently air - dried . One coat of adhesive resin (excite, ivoclar - vivadent) was applied using a microbrush and light polymerized for 20 s (astralis 10, ivoclar - vivadent). Then core build - up was performed using hybrid composite resin (tetric ceram, ivoclar - vivadent) in 1-mm - thick increments and light polymerized (astralis 10; ivocar - vivadent) for 40 s until the core was restored to predetermined dimensions . The final layer was placed using a transparent matrix to allow for shape consistency between specimens . The dimensions of the prepared cores were confirmed with a measuring microscope with 30x magnification lens with precision of 5 um . A single - phase impression was made using polyvinylsiloxane impression material (virtual, ivoclar - vivadent) and master dies were fabricated with type 4 die stone (jad stone, whip mix, louisville, kentucky, usa). A press ceramic (e max, a3, ivoclar - vivadent) was selected to fabricate all - ceramic crowns of the restored specimens . Crown dimensions were standardized by using a mold for the external shape of each specimen . The fitting surface of the crowns was pretreated with hydrofluoric acid (ips ceramic etching gel, ivoclar - vivadent) for 20 s, rinsed off, air dried, silanized for 60 s and air dried . Dentine primer liquids were mixed and applied on the whole prepared tooth surface for 15 s. resin cement was dispensed from the automix syringe directly into the inner surface of the crowns which were seated and held in position under fixed load of 20 n; excess resin was removed immediately with a micro brush . Exposed margins were covered with glycerin gel and rinsed off after complete polymerization of the resin cement . The specimens were stored in distilled water at 37c for 7 days prior to testing . All specimens were subjected to cyclic loading according to the following regime: sinusoidal load between 50 and 200 n at a rate of 2 hertz . All specimens received 15,000 cycles and surface damage was prevented by insertion of a 0.5 mm silicon sheet between the occlusal surface of the ceramic crown and the loading indenter (3 mm diameter) of the pneumatically activated loading machine . After completion of cyclic loading, a universal testing machine (instron 8500 plus, instron, 100 royal st . Canton, ma, usa) was used to deliver a compressive load to the specimens at a crosshead speed of 1 mm / min at 45 degree angle to the long axis of the teeth (root apex tilted lingually) until failure . Load - time curves were recorded using a universal testing machine s computer software . The failure load of the specimen was determined when the forceversus - time graph showed an abrupt change in load, indicating a sudden decrease in the specimen s resistance to compressive loading . After loading, the failure mode recorded for each specimen and classified as either favorable facture above the cement - enamel junction (repairable) or catastrophic fracture of the root below cement - enamel junction (non repairable). These inspections were made using a stereomicroscope (stereoscopic zoom microscope, smz-1000, nikon, japan) and during inspection, the teeth were trans - illuminated with a fiber optic cable . Complete or partial debonding of the crown or of the post and core were also considered as favorable failure modes . Data were analyzed statistically by one - way anova and tukey s post - hoc test was used for pair - wise comparisons (=0.05). All external debris were removed with an ultrasonic scaler, and examined stereoscopically at 10x magnification to verify the absence of cracks, defects and dental caries . Teeth were stored in a 0.5% chloramine t (prolabo, paris, france) in saline solution . Buccolingual and mesiodistal coronal dimensions plus root length of all selected teeth were measured using a digital caliper (digimatic calipers, mitsutoyo, tokyo, japan) and only teeth with the following mean dimensions were selected: 14.1 mm root length, 7.3 mm buccolingual width, and 4.9 mm mesiodistal width . Standardized root canal preparations were made using the following procedure: initial probing using no . 10 k - files (flexo files, maillefer, ballaigues, switzerland); the root canal length was established through direct observation of the file extruding from the apical foramen . The specimens were then prepared endodontically with a stepback procedure with size 45 (flex r file; union broach, york pa). After intermittent rinsing with 2.5% sodium hypochlorite solution, the endodontic treatment was completed using manual lateral condensation method (ah plus, dentsply, de tray, konstanz, germany). After endodontic treatment, each root was thinly covered with a silicone impression material (aquasil, dentsply) to simulate thickness of periodontal ligament . All specimens were embedded in self - polymerized acrylic resin (orthoresin, dentsply, degudent gmbh, postfach 1364, d-63403 hanau, germany) poured into a mold while maintaining 2 mm below the cervical line exposed . The teeth were embedded along their long axis using a surveyor (ney surveyor; dentsply). A new diamond point (lot - nr 1599, dfs dental and technical products, gmbh, germany) was attached to the milling machine (k9 milling apparatus-990, kavo, germany) for every group . The mrd gauged diamond had a self - limiting tip, which produced a 1-mm - deep chamfer and the margins and the angle of convergence were standardized . After preparation of the finish line, the coronal dentinal extension was modified accordingly (figure 1): group 1 (control): 1 mm circumferential ferrule from the gingival margin without a fiber post; group 2: 1 mm circumferential ferrule with fiber post and resin core; group 3: non - uniform ferrule height (2 mm buccally and 1 mm lingually) with fiber post and resin core; group 4: a non - uniform ferrule height (3 mm buccally and 2 mm lingually) with fiber and resin core post; group 5: received no ferrule preparation with fiber post and resin core . In the control group, excess gutta - percha was removed to a depth of 2 mm from the coronal surface of the preparation, using a carbide bur (171l-012, brasseler, usa). The coronal walls were etched with 36% phosphoric acid (total etch, ivoclar - vivadent, schaan, liechtenstein) for 15 s, washed with water spray and then gently air - dried . One coat of adhesive resin (excite, ivoclar - vivadent) was applied using a microbrush and light polymerized for 20 s (astralis 10, ivoclar - vivadent). A hybrid composite resin (tetric ceram, ivoclar - vivadent) was applied and light polymerized for 40 s. for the remaining groups, post space was created using no . 1 peeso reamer (union broach co., long island, ny, usa) and corresponding calibrating drill (frc postec plus, #1, ivoclar - vivadent) leaving 4 mm of apical gutta - percha intact . A translucent glass fiber reinforced composite post (frc postec plus #1 ivoclar - vivadent, schaan, liechtenstein) was used . Each post was cut to a suitable length with a diamond bur so that it was covered with at least 2 mm of resin composite occlusally . Post cementation was carried out with self - polymerized resin cement (multilink, ivoclar vivadent) following manufacturer s instructions . Silane coupling agent (monobond - s, ivoclar - vivadent) was applied on the post surface for 60 s and then air - dried . After post cementation, the surrounding dentin surface was etched with 36% phosphoric acid (total etch, ivoclar - vivadent) for 15 s, washed with water spray and gently air - dried . One coat of adhesive resin (excite, ivoclar - vivadent) was applied using a microbrush and light polymerized for 20 s (astralis 10, ivoclar - vivadent). Then core build - up was performed using hybrid composite resin (tetric ceram, ivoclar - vivadent) in 1-mm - thick increments and light polymerized (astralis 10; ivocar - vivadent) for 40 s until the core was restored to predetermined dimensions . The final layer was placed using a transparent matrix to allow for shape consistency between specimens . The dimensions of the prepared cores were confirmed with a measuring microscope with 30x magnification lens with precision of 5 um . A single - phase impression was made using polyvinylsiloxane impression material (virtual, ivoclar - vivadent) and master dies were fabricated with type 4 die stone (jad stone, whip mix, louisville, kentucky, usa). A press ceramic (e max, a3, ivoclar - vivadent) was selected to fabricate all - ceramic crowns of the restored specimens . Crown dimensions were standardized by using a mold for the external shape of each specimen . The fitting surface of the crowns was pretreated with hydrofluoric acid (ips ceramic etching gel, ivoclar - vivadent) for 20 s, rinsed off, air dried, silanized for 60 s and air dried . Dentine primer liquids were mixed and applied on the whole prepared tooth surface for 15 s. resin cement was dispensed from the automix syringe directly into the inner surface of the crowns which were seated and held in position under fixed load of 20 n; excess resin was removed immediately with a micro brush . Exposed margins were covered with glycerin gel and rinsed off after complete polymerization of the resin cement . The specimens were stored in distilled water at 37c for 7 days prior to testing . All specimens were subjected to cyclic loading according to the following regime: sinusoidal load between 50 and 200 n at a rate of 2 hertz . All specimens received 15,000 cycles and surface damage was prevented by insertion of a 0.5 mm silicon sheet between the occlusal surface of the ceramic crown and the loading indenter (3 mm diameter) of the pneumatically activated loading machine . After completion of cyclic loading, a universal testing machine (instron 8500 plus, instron, 100 royal st . Canton, ma, usa) was used to deliver a compressive load to the specimens at a crosshead speed of 1 mm / min at 45 degree angle to the long axis of the teeth (root apex tilted lingually) until failure . Load - time curves were recorded using a universal testing machine s computer software . The failure load of the specimen was determined when the forceversus - time graph showed an abrupt change in load, indicating a sudden decrease in the specimen s resistance to compressive loading . After loading, the failure mode recorded for each specimen and classified as either favorable facture above the cement - enamel junction (repairable) or catastrophic fracture of the root below cement - enamel junction (non repairable). These inspections were made using a stereomicroscope (stereoscopic zoom microscope, smz-1000, nikon, japan) and during inspection, the teeth were trans - illuminated with a fiber optic cable . Complete or partial debonding of the crown or of the post and core were also considered as favorable failure modes . Data were analyzed statistically by one - way anova and tukey s post - hoc test was used for pair - wise comparisons (=0.05). Statistical analysis revealed no significant differences between the failure load of the tested groups and the control group restored without fiber post (p<0.780). Although fracture resistance of the control group was comparable to that of the other groups that received fiber post, there was a higher catastrophic failure rate (70%) in the control specimens in the form of vertical root fracture . The groups restored with fiber post, on the other hand, had almost complete favorable fracture in the form of cervical fracture above the cervical line, previous data are summarized in table 1 . Failure load (standard deviation) and failure type of tested groups similar superscript letter indicates no significant difference between different test groups in all specimens, the cemented ceramic crown was fractured without evidence of debonding of either the crown or the cemented post . The internal surface of the fractured ceramic fragments demonstrated evidence of resin cement and part of the core material indicating cohesive fracture of the resin core . Considering the results obtained in this study, different ferrule heights did not improve the fracture resistance or the failure pattern of the tested specimens . On the other hand, the use of fiber posts with a modulus of elasticity close to that of dentin changed the catastrophic failure type of the control group to almost complete favorable fracture for the other four groups . The dynamic cyclic loading program was intended to quickly screen any possible weakness in the cemented restorations . Providing occlusal protection prevented generation of cone cracks in the brittle ceramic crowns which was the reason why all specimens survived without failure . Longer periods of cyclic loading are required to shed light on the long - term performance of these restorations, but investigating this issue was not within the scope of this study . The results of the fracture resistance test showed that the amount of residual coronal structure (ferrule height) did not increase significantly the fracture resistance of endodontically treated teeth . These results are in agreement with those of previous studies; while contrary results were reported by other authors . This could be explained by the fact that fiber posts transmitted the forces and distributed the loading stresses over a bigger surface area of the tooth structure similarly for all tested groups . These results may also be interpreted as the resin bonded fiber posts with resin composite core exerted a reinforcing effect by supporting the remaining tooth structure regardless of the ferrule design . These non - metallic post systems have gained widespread popularity in recent years because of other advantages such as their superior esthetics, ease of retrievability, and simple application technique, which allow the clinician to complete the procedure in a single short appointment . According to the results of the present study, specimens in the control group (no fiber post) revealed higher catastrophic failure rate compared to the other groups, which indicates that insertion of a fiber post may enhance the clinical performance of endodontically treated teeth even if the failure load remains relatively not effected . The presence of a uniform 1-mm - thick coronal structure (group 2) resulted in the highest fracture resistance value, especially when compared to the control group, which is in agreement with the results of previous studies . A point of clinical relevance is that the fracture resistance of specimens with non - uniform coronal structure (group 3 and 4) was lower than the specimens without a ferrule (group 5), with consideration to maintaining as much as possible of sound tooth structure . It could be advised to adjust the coronal structure evenly to provide a flat seat for the core build up material, which could improve the fracture resistance of endodontically, treated teeth . Lithia disilicate - based all - ceramic crowns were used in this study because of its high flexural strength (400 mpa), easily etched using hydrofluoric acid, readily silanized and bonded with any suitable resin cement . It has also been reported that the mean chewing force of adults ranges between 7 to 15 kg, and the maximum biting force could reach up to 90 kg . As the fracture loads in all groups in the present study were found to be greater than the ordinary chewing force, and even greater than the maximum biting force, their mechanical strength could be considered satisfactory from a clinical point of view . In terms of the failure modes, the obtained results are in accordance with those of previous studies, showing that the application of fiber posts resulted in more favorable fracture patternswhile the specimens restored without posts showed a higher incidence of catastrophic failure . It could be postulated that specimens restored with fiber - reinforced post systems offered more homogenous stress distribution due to their modulus of elasticity close to that of dentin resulting in a better stress distribution that occurs at the post - dentin interface . This could explain why all favorable fractures were limited to the cervical portion of the root including the core - dentin interface, since the stresses were concentrated in the cervical area and the outer root surface . Supporting this opinion is the cohesive fracture of the resin core material, which remained attached to the fractured segments of ceramic crowns, thus indicating good bond strength between the core build up material and the cemented ceramic crown . The limited number of specimens and the difficulty related to reproducing the complexity of functional loads in the oral environment may be some of the shortcomings of the present study . Further investigations including finite element analysis and long - term cyclic loading studies are recommended to complement the present study . Within the limitations of this study, it may be concluded that increasing the ferrule length did not improve the fracture resistance of endodontically treated teeth restored with glass ceramic crowns . Insertion of a fiber post could reduce the percentage of catastrophic failure of these restorations under functional loads.
Dichlorvos (dimethyl-2, 2-dichloro vinyl phosphate) is an organophosphorous (op) insecticide with moderate human toxicity, used for insect control in food storage areas, greenhouses, barns and control of insects on livestock . Parenteral dichlorvos administration with suicidal intention is rarely reported in the literature . In one case report, extensive muscle necrosis was reported following intramuscular injection of dichlorvos . In another case, following injection of dichlorvos subcutaneously in both wrists, a young patient developed compartment syndrome requiring fasciotomy . In both cases, i describe the case of a young female patient, who developed atypical signs of op poisoning following subcutaneous injection of dichlorvos . The present case report is about a 37-year - old, previously healthy female, who was admitted to our intensive care unit 18-h after alleged self - injection of 76% dichlorvos (nuvos) 3 ml in the upper left arm (3 times with a 1 ml insulin syringe). Within an hour of the injections, she had normal body temperature with heart rate of 90/min, blood pressure 110/70 mmhg, respiratory rate 14/min and pulse oximeter showing 100% saturation on room air . No crackles were heard on chest auscultation . On the nervous system examination, she was jittery with slurred speech and normal cranial nerves . She had hypertonia, brisk tendon reflexes, bilateral sustained ankle clonus and bilateral extensor plantar reflex . Local examination revealed a blister and a black necrotizing lesion over the anterior aspect of the left arm [figure 1]. She was rapidly atropinized with incremental boluses of atropine (total dose 12 mg) in next 30 min (targeting a dry mouth) followed by infusion of 1.8 mg / h . Pralidoxime infusion was started at 500 mg / h following a loading dose of 1000 mg over 20 min . Intravenous clindamycin (600 mg q 8 h) was administered empirically for presumed local wound infection . Debridement of the wound showed predominant involvement of the subcutaneous tissue plane with minimal necrosis of the superficial fibers of biceps brachii . Initial routine laboratory investigations were unremarkable except elevated total count (16200/mm) and mild respiratory alkalosis . Local skin necrosis with surrounding blisters her clinical course was complicated by respiratory arrest 9-h after hospital admission (27-h after the injection of dichlorovos) leading to hypoxia and bradycardia . Arterial blood gas analysis post resuscitation showed - ph 7.28, po2 - 36 mmhg, pco2 - 54 mmhg, hco3 - 22 mmhg . Serum choline esterase (pseudo cholinesterase) level of the same day showed a very low value of <1 ku / she could be successfully weaned off from the ventilator on day 4 . On day 5, while on atropine infusion she was found to be delirious . She was also noticed to have extrapyramidal manifestations including rigidity and left sided gaze preference . In this case, other than the unusual route of administration, a number of atypical manifestations were found . This includes relatively delayed respiratory failure and central nervous system (cns) manifestations with few muscarinic signs . Two independent mechanisms are thought to be responsible for respiratory failure following op insecticide poisoning cholinergic cns depression in the early stage (associated with other cholinergic features) and late progressive neuromuscular transmission defect . Second mechanism is part of the syndrome described as type ii paralysis by wadia et al . Or intermediate syndrome by senanayake and karalliedde . Our patient developed respiratory arrest 27-h after exposure to dichlorovos . In her clinical course she never had neck or proximal muscle weakness, though the clinical findings were not correlated electrophysiologically . Usually cns signs are manifested early in the course in the presence of peripheral muscarinic signs . In a large series published on neurological manifestations of op compounds, 8 of 200 patients had pyramidal tract signs in the form of only hyper - reflexia manifesting within 24 h of exposure . All these patients were unconscious with florid cholinergic manifestations, including constricted pupils . In their original series of intermediate syndrome senanayake and karalliedde, observed hyperreflexia in one of their patients (with flexor plantar reflex) and even she had typical muscarinic signs . Extrapyramidal signs of op poisoning are often transient and may be missed in a sick patient . In a taiwanese series cns manifestations (both pyramidal tract and extrapyramidal) were delayed and were associated with relatively scanty peripheral muscarinic signs . Possible explanation for these atypical manifestations in our patient could be route specific (in this case subcutaneous) effect of op compound (dichlorovos) causing isolated delayed cns depression, with few peripheral cholinergic signs . Animal experiments have shown route specific (subcutaneous or intravenous) cardiovascular and neuromuscular effect of op compounds in rats.
Transesophageal echocardiography (tee) is considered to be safe and relatively noninvasive diagnostic tool,1) however severe complications have been reported . The incidence of major tee - related complications range from 0.2% to 0.5% and mortality is reported to be <0.01%.2345) among these complications, problems related to insertion of tee probe take a large proportion, including perforation of hypopharynx and esophagus . For our knowledge, there have been reported only few cases of hypopharyngeal perforation, especially not been reported in asian population to date . We describe the clinical presentations and secondary complications associated with oropharyngeal injury with the case of a 74-year - old man who suffered a hypopharyngeal laceration after tee . A 74-year - old man was referred to our cardiovascular center for the evaluation and treatment of mitral regurgitation (mr). He had been complaining of aggravation of dyspnea (new york heart association functional class iv) for the preceding 3 weeks, and atrial fibrillation and severe mr were detected from other clinic . The patient had medical history of hypertension and chronic obstructive pulmonary disease for 15 years, and received endovascular aneurysm repair for abdominal aortic aneurysm 8 years ago . The patient was admitted to the intensive care unit because of uncompensated heart failure and careful control of pulmonary edema with chronic kidney disease . The patient presented tachypnea and orthopnea before the tee procedure, less than minimal dose of sedative agent was administrated to lessen patient's discomfort, 1 mg of lorazepam, 25 mg of fentanyl, intravenously . As we checked for the mental status, the insertion of tee probe was performed with the patient in left lateral decubitus position according to the following standardized technique: the probe was inserted through the midline and gently advanced to pass the first pharyngeal curvature corresponding to the base of the tongue . The probe was then extended, and the patient was asked to swallow, at which point the probe was further advanced to enter the esophageal inlet . When the probe has reached to the root of tongue, the patient suddenly changed his position form left decubitus to supine position and gave force to the neck, and resisted to probe insertion . The probe got lodged at right - side of hypopharyngeal area so that failed to advance . At second attempt, tee demonstrated severe eccentric mr with medial commissural prolapse due to chordae rupture (a3-p3 commissure) with left ventricular dilatation, mild tricuspid regurgitation (fig . The physical examination revealed tenderness and crepitus on right anterolateral area of neck (level iii) 3 hours later after tee . Iatrogenic hypopharyngeal or esophageal injury was suspected, antibiotic treatment with piperacillin / tazobactam was initiated and the patient was not allowed to eat or drink . Computed tomography (ct) scan revealed subcutaneous emphysema without involvement of mediastinum, but the level of injury was not detected (fig . Fiberoptic nasolaryngoscopic examination identified edema of the right posterolateral wall of the hypopharynx and hypopharyngeal bruise but there was no evidence of rupture or perforation . Laboratory analysis revealed an elevation of the white blood cell count (20.36 10/l) and c - reactive protein (crp; 28.54 mg / dl). We tried to find any evidence of esophageal injury because treatment strategy would be different if the esophagus was involved . An ent specialist and a gi specialist agreed with hypopharyngeal injury without esophageal damage after multiple tests . We concluded the lesion was limited to the hypopharynx because the presence of hematoma at the right side of hypopharynx, consistent with the direction and the depth of the probe passage . Further, there was no evidence of esophageal injury on serial follow - up multimodality imaging studies . After 5 days from the injury, the subcutaneous emphysema disappeared and the patient remained afebrile with improvement of the leukocyte count and crp (fig . However painful neck mass around anterolateral area of neck was noticed (level iii). The follow - up ct without contrast showed a right parapharyngeal and retropharyngeal abscess secondary to hypopharyngeal injury (fig . Yellowish fluid in the abscess was analyzed for cytology and revealed as acute inflammatory cells predominantly neutrophils . As the patient's systemic status was stable with decrease of the leukocyte count and crp, the antibiotics (piperacillin / tazobactam) was maintained for 14 days with the drainage of abscess because no organisms were identified from the abscess fluid culture and repeated blood cultures . On 7th day after tee, second swallowing study was performed and no leakage was demonstrated (fig . 4b), infectious parameters were continuously decreased, and follow - up fiberoptic nasolaryngoscopic examination demonstrated no evidence of perforation or rupture in hypopharyngeal cavity . After 14 days of antibiotic therapy, when the infection was controlled completely, the patient had successful mitral valve repair with tricuspid annuloplasty with st . The overall incidence of severe hypopharyngeal and esophageal complications from traditional tee is low.1)3)6) risk factors of hypopharyngeal and upper gastrointestinal complications have been reported in few studies . Patient related risk factors include older age, short stature, presence of cervical spinal disease, chronic steroid use, presence of congestive heart failure.3)7)8) the presence of gastroesophageal pathology such as zenker's diverticulum, esophageal stricture or obstructing mass, fibrosis secondary to prior chest radiation could be potential risk factor . Anatomical variation like massive cardiomegaly1)9) or tracheoesophageal fistula may be correlated to increase the risk . Procedure related risk factors include inexperience of the cardiologist, and resistance to prove insertion with poor cooperation . To avoid direct injury to the hypopharynx and upper esophagus during a blind insertion of probe, appropriate conscious sedation and local anesthetic spray direct visualization of the passage of the probe with direct laryngoscope could be recommended to minimize blind injury of throat . If there are procedural difficulties such as multiple attempts at passage of the tee probe or resistance during insertion, the examination should be stopped . In addition, indirect injury is also result from increased continuous pressure on the probe positioned in the upper esophagus, therefore, the exam should be stopped if the resistance is continuing . Finally, it is very important to prevent sudden position change of the patient during the probe insertion . It needs pre - procedural enough education, adequate sedation, careful insertion of tee probe, and an experienced assistant . In our case, we assumed that the patient's poor cooperation and insufficient sedation might cause the wrong direction of the tee probe and advancing force of tee probe into the throat had led the probe to get lodged into the right pyriform sinus . Treatment of hypopharyngeal perforation depends on the cause, timing of diagnosis, and type of lesion.101112) in a study of hinojar et al.,10) 3 patients out of 7 patients were managed conservatively, whereas other 4 patients got surgical treatment . Mostly in the former cases, the time duration to the diagnosis was less than 1 - 2 days and involvement of infection was limited to pharyngeal space . In current case, our patient complained pain in his throat and we detected subcutaneous emphysema within 3 hours after injury that may prevent aggravation of infection and lead good prognosis . It is important not only to understand possible complications of the procedure and to recognize the risk factors of pharyngeal and esophageal injury, but also to detect the complication as soon as possible if it occurred, to get better prognosis.
It has been reported that the prevalence and incidence of dementia in the united states have been either stable or even declining over the last 2 decades of the 1990s . This question is particularly relevant to the case of japan which is an economically advanced country like the us, but is believed to have a different level of vascular disease risk . Studies conducted in the late 1980s and early 1990s reported that vascular dementia (vad) was more prevalent than alzheimer's disease (ad) in japan, compared with the us and other western countries where ad is more prevalent than vad . In studies among japanese americans conducted in the early 1990s [4, 5], the prevalence ratio of ad to vad was higher than that reported among japanese in japan and more closely resembled that found in the caucasian population . This suggests that there might have been environmental factors that changed the risks of developing subtypes of dementia after japanese immigrated to the us . On the other hand, more recent studies conducted in the late 1990s suggest that the cross - national differences found in the past may have been due to differences in the diagnostic methods used [6, 7]. Standardization of diagnosis is one of the challenges of cross - national comparisons of dementia prevalence . There is an ongoing debate as to whether: (1) the higher proportion of vad found in the past studies in japan could be due to differences in diagnostic criteria used in japan and the us, (2) the similar ad / vad ratio with that of the us found in recent japanese studies could be due to decreased cerebrovascular disease incidence over the past decades in japan, or (3) there is no systematic time trend in ad / vad ratio in japan and the observed variation is due to regional differences within japan . Despite a growing interest in the influence of vascular disease and its risk factors on alzheimer's disease (ad) [812], our aims in the current study are to: (1) summarize epidemiological studies of dementia in japan including relevant details of study protocols and diagnostic criteria, (2) compare age - specific prevalence of all - cause dementia among studies, and (3) assess the trends in ad versus vad over time . Previous studies which concluded an increase in ad / vad over time in japan [1315] did not examine the diagnostic criteria used, nor did they examine age - specific ad / vad ratios . We selected dementia prevalence studies in japan that were designed to be representative of specific communities or prefectures with at least 500 study participants aged 65 and older, and whose age - specific prevalence (for either 5- or 10-year age intervals) for ad and vad was published between 1990 and 2009 in international journals, using medline with the search words japan, dementia, and prevalence . We used the former criterion (n 500) in order to have a large enough sample size to allow meaningful between - cohort comparisons of dementia prevalence . Eight studies met these criteria: the hisayama study [16, 17] conducted at four time points, the okinawa study, the radiation effect research foundation adult health survey (rerf - ahs, a.k.a hiroshima study), the tajiri project, and the ama - cho study . Brief descriptions of each study cohort follow (see also table 1 for further summary). Hisayama is a rural community adjacent to the city of fukuoka, a major city of kyushu island . Cross - sectional dementia prevalence was estimated 4 times, in 1985, 1992, 1998, and 2005 [16, 17]. Random sampling was conducted to recruit study participants in the selected sites between 1991 and 1992 . In 1958, the atomic bomb casualty commission began the adult health study (ahs) to survey the occurrence of illnesses and changes in physiological and biochemical function resulting from exposure to atomicbomb radiation . The original ahs cohort consisted of atomicbomb survivors and their controls, selected from residents in hiroshima and nagasaki . Between 1992 and 1996, those aged 60 and older who were residents of hiroshima and members of the ahs were examined . The tajiri project is a community - based study started in 1988 for the prevention of stroke, dementia, and bed - confinement in tajiri, an agricultural area in northern japan . In 1998, all residents 65 years and older were targeted for the dementia prevalence study [6, 20]. This study was conducted in the municipality of ama - cho, a rural island town in the northwestern part of japan . All residents as of the prevalence day of march 1, 2008 were requested to participate in the dementia prevalence study . Differences in overall dementia prevalence among 8 studies were examined using poisson regression models based on the number of dementia cases (weighted numbers of cases for okinawa studies) and the number of participants by 10 year age groups (except the youngest age group where 5-year ages were used: 6569, 7079, 8089, and 90 +) to provide large enough sample sizes in each group for meaningful statistical comparisons, yet controlling for changing age composition over time . The okinawa 1992 study sample was used as a reference group because it was the largest and was conducted at the mid - point of assessment years among the 8 studies . Table 1 gives a brief description of each study and the criteria used to define dementia and dementia subtypes . All assessments, except in the tajiri study, were based on multistage assessments, during which all participants were screened in phase 1 (screening phase), and selected participants and controls from phase 1 received final diagnoses from physicians in phase 2 (clinical assessment phase). The dementia diagnostic criteria used in japanese studies were either dsm - iii, dsm - iii - r, or dsm - iv . The diagnostic criteria used to define subtypes of dementia varied among the studies in japan . The following criteria were used to define vad: hachinski ischemic scores, ninds - airen, dsm - iv, and the alzheimer's disease diagnostic and treatment centers (addtc) criteria . For ad, dsm - iii - r, dsm - iv, and nincds - adrda were used . The overall prevalence of dementia among those aged 65 and older ranged from 5.6% (hisayama 1992) to 11.3% (ama - cho 2008) (table 1). Poisson regression models for dementia prevalence showed that compared with the okinawa 1992 study, hiroshima 1996 (p = 0.0002), tajiri 1998 (p <0.0001), hisayama 2005 (p <0.0001), and ama - cho 2008 (p = 0.007) had a higher prevalence of all - cause dementia, controlling for sex and age groups (table 2). There were no difference between the okinawa 1992 study and hisayama studies conducted in 1985, 1992, and 1998 . No difference was found between men and women . As a post hoc analysis, we also ran poisson regression models using only the hisayama studies (4 time points). The results showed that compared with the all - cause dementia prevalence in 1985, that of 2005 was higher (coefficient: 0.32, 95% ci: 0.010.64, p = 0.04) controlling for sex and age groups (not shown in table 1). Ad / vad ratios among those aged 65 and older are listed in the last column of table 1 . The okinawa (1992), hiroshima (1996), tajiri (1998), and ama - cho (2008) studies used the same criteria for diagnoses of ad (nincds - adrda) and the ad / vad ratio among those aged 65 and older was 1.85, 1.85, 3.33, and 4.12 for the above studies, respectively . The hisayama studies which conducted 4 cross - sectional studies using the same diagnostic criteria for subtypes of dementia also showed the increasing trend in the ratio of ad / vad, ranging from 0.52 in 1985 to 1.96 in 1998, and 1.92 in 2005 . The tajiri project, which conducted an mri substudy to identify dementia etiologies, showed that the proportion of ad among total dementia cases differed largely depending on the criteria used: 62.5% (ad / vad = 3.3) using ninds - airen criteria, and 40.6% (ad / vad = 1.0) or 56.2% (ad / vad = 2.3) depending on assessors using dsm - iv criteria . Even though we selected studies with relatively large sample sizes (n 500), the number of cases was too small to conduct meaningful comparisons of age - specific ad / vad ratios for the age group 6570 (<5 for each case). Therefore, we list age - specific prevalence of dementia and ad / vad ratios for the age groups 7079, 8089, and 90 and older in table 3 . The youngest age group examined here (age 7079) had a prevalence of vad that was consistently higher than that of ad in japan (i.e., ad / vad <1) except in more recent studies conducted in 2005 (hisayama) and 2008 (ama - cho). Except for these two recent studies (hisayama 2005 and ama - cho 2008), eight major prevalence studies conducted in japan were reviewed in an attempt to identify trends in prevalence of all - cause dementia and subtypes of dementia, paying careful attention to diagnostic protocols . We found that compared with the okinawa 1992 study, studies conducted in later years (1994 (hiroshima), 1998 (tajiri), 2005 (hisayama), and 2008 (ama - cho)) had a higher prevalence of all - cause dementia, after controlling for age groups and sex . Within 4 studies conducted in hisayama (1985, 1992, 1998, and 2005), we also found that the prevalence in 2005 was higher than that in 1985, after controlling for age groups and sex . Thus, the dementia prevalence seems to be increasing in japan, in contrast to the us where decreasing or stable prevalence of all - cause dementia has been reported . Two diseases that could have high impact on the prevalence of dementia at national levels are cerebrovascular disease and type 2 diabetes, as shown by their relatively high population attributable risk% (par%) of dementia in the united states [12, 28]. According to the national nutrition survey in japan, those with hemoglobin a1c values 6.0% (possible type 2 diabetes) were estimated to be 22.8%, 37.4%, and 40.9% among men aged 70 and older in year 1997, 2002 and 2007, respectively, and the comparative figures among women were 27.2%, 28.2%, and 34.6% . On the other hand, the decline in stroke incidence reached plateaus around the late 1990s after a continuous sharp decline beginning in 1960, thus further declines in dementia due to stroke would not be expected after the 1990s . However, it is possible that the prevalence of small vessel cerebrovascular disease with resultant microinfarcts that would not be accounted for in these vascular disease statistics could play a latent or underappreciated role in causing or contributing to dementia . The increase in type 2 diabetes, the metabolic syndrome and its associated vascular complications (risk factors for ad), with a plateau in declining trends in major stroke incidence, could have lead to an increase in dementia prevalence in recent years in japan . It is also possible that increasing public awareness of dementia in recent years is resulting in enhanced recognition of functional and cognitive declines that might previously have been dismissed as longer survival of those who suffered from stroke / tia due to advanced medical treatment could also increase the prevalence of dementia to some extent . Unfortunately, autopsy confirmation in large proportions of the participants in these epidemiologic studies is not available, thus limiting conclusions as to more specific underlying etiologies . We found that okinawa had a lower overall prevalence of dementia compared with other cohorts except hisayama studies in 1985, 1992, and 1998 . These two regions (okinawa and hisayama) have had lower incidence of cerebrovascular diseases in comparison with other regions studied here . For example, the rate of cerebrovascular mortality, as a proxy of cerebrovascular disease incidence, declined sharply between 1975 and 2000 by over 65% in all regions and declined further between 2000 and 2005 by over 10% in all regions (table 4), with the above two regions constantly having lower rates than other regions . Historically, salt consumption has been high in the northern part of japan because vegetables and marine products are cured with salt to preserve them during the longer winter months and consumed with processed white rice . This dietary pattern is believed to be one of the reasons of the higher prevalence of hypertension and large vessel cerebrovascular disease in the northern prefectures in japan . The low dementia prevalence in okinawa and hisayama (except 2008), which is located in the southern part of japan, could not only be due to a lower rate of vad resulting from lower cerebrovascular disease incidence, but also due to lower ad prevalence resulting from reduced vascular injuries [8, 12, 33, 34]. Vascular dementia (vad) was believed to be more prevalent than alzheimer's disease (ad) in japan in the 1980s, in contrast to the us or other western countries, but studies conducted in the late 1990s and after showed patterns that were more similar to the us [13, 15, 35]. Westernization of the dementia prevalence pattern could be partly due to the declining stoke incidence observed during the 1980s as described above . However, it could also be due to changes in diagnostic criteria used in japan . For example, in the tajiri study, patients received a diagnosis of possible ad with cvd by means of the ninds - airen criteria, provided that the vascular effect on dementia was considered to be too ambiguous to diagnose as vad, which lead to relatively high proportion of ad out of all dementia cases (over 62%). The study also demonstrated the difficulty of obtaining consensus on the definition of vad, even for experienced neurologists using the same criteria; two neurologists blinded to each other's diagnosis did not agree when diagnosing vad versus ad under dsm - iv criteria: the proportion of vad out of total dementia cases ranged from 40.6% to 56.2%, depending on assessors . We expect that lack of imaging data may result in underdiagnosis of subcortical vascular brain injury (e.g., white matter hyperintensity, and silent brain infarcts, etc .) And thus lead to underestimation of vad . However, more recent studies including the tajiri, hisayama (1998; 2005), hiroshima and ama - cho studies used imaging data, and these tend to show higher ad / vad ratios (i.e., not higher vad prevalence) compared with earlier studies . Therefore, it is likely that changes in diagnostic criteria at least partly explain the higher proportion of diagnosed ad in recent years . In all studies except more recent studies conducted after 2000, as age increased, the proportion of ad among the total dementia cases increased: the youngest age group examined (age 7079) had a prevalence of vad that was consistently higher than that of ad in japan (i.e., ad / vad <1). On the other hand, among the older age groups, in fact, among the oldest age group (age 90), ad / vad ratios in japan were not necessarily lower than the us figures even among studies conducted in the early 1990s, ranging from 2.5 (hisayama 1985) to 6.0 (hisayama 1992). In the aging, demographics, and memory study (adams) [36, 37], which is the first study in the us to calculate a nationally representative dementia prevalence, the age - specific ad / vad ratios were found to be 2.36, 4.43, and 4.79 for age group 7179, 8089, 90 and older, respectively, using dsm - iii - r and dsm - iv criteria for dementia and nincds - adrda criteria for ad, that is, the age - associated increase in ad prevalence seems to be more evident in japan than in the us . This could be partly due to the fact that the gender gap in life expectancy is larger in japan compared with us and the proportion of women increases more steeply as age increases in japan . For example, in 1990, the life expectancy at age 65 was 18.9 years for women and 15.1 years for men in the us, that is, a gender gap of 3.8 years, while the comparative figure in japan was 20.0 years and 16.2 years, with the gender gap of 4.8 years . Similarly, in 2008, the life expectancy at age 65 was 19.8 years for women and 17.1 years for men in the us, with the gender gap of 2.7 years, while the comparative figure in japan was 23.6 years and 18.6 years, with the gender gap of 5.0 years . Cerebrovascular disease is more common among men than women, and we expect a higher proportion of vad among men than women . A steeper age - associated increase in ad prevalence found in japan, therefore, could be due to a higher proportion of women among the older old age groups in japan . Comparisons of age - specific ad / vad ratios between men and women could clarify this issue, but we were unable to do so due to the small sample size once we stratify prevalence by sex and age groups, especially among those aged 90 and older . However, at least among the younger two age groups (ages 7079; 8089), we see an increasing trend in the proportion of ad cases as age group goes up in both men and women (data not shown). These results also suggest that it is important to consider the age / sex structure of samples when we compare the ad / vad ratio among different cohorts although incidence of dementia and its subtype would give a more accurate picture regarding the trend, there is a paucity of dementia incidence studies in japan . To our knowledge, only three incidence studies have been reported thus far: (1) the hisayama cohort, following their 1985 cohort for 7 years; (2) the tajiri project, following a sub - sample of their 1998 cohort for up to 7 years; (3) the hiroshima study (radiation effects research foundation adult health study), following their 19921996 prevalence cohort until the year 2003 . The incidence of all - cause dementia was 19.3 per 1000 person - years for men and 20.9 for women in the hisayama study, 33.9 for men and 44.0 for women in the tajiri study, and 12.0 for men and 16.6 for women in the hiroshima study . The hisayama and hisorhisma studies reported significant gender differences in subtypes of dementia incidence: hisayama reported that the incidence of vad increased with age and was consistently higher than that of ad for men, while the incidence of ad was higher than that of vad for women age 75 years or older . The incidence of ad markedly increased after the age of 80 in either sex, but overall, vad was more common in the hisayama study . The hiroshima study also reported that probable ad showed the most remarkable increase with age, and probable vad was significantly lower among women . Overall, ad was more common in this study, possibly because the hiroshima study was conducted later than the hisayama study, after a further decline in stroke incidence had occurred . In all 3 incidence studies, nincds - adrda criteria were used for ad and ninds - airen for vad . In japan, ad incidence and prevalence tend to increase with age more than vad and older old women predominantly have ad rather than vad . Therefore, when aggregated, the overall prevalence of ad may become higher as more women survive in the oldest old age group . As we mentioned earlier, it is important to consider the age / sex structure of samples when examining trends in the ad / vad ratio . In conclusion, our systematic review shows that (1) there is an increasing trend in overall dementia in japan . Although we cannot confirm definitively from the current study, the possible explanation of the increase could be a shift in health conditions among the elderly in japan including the increase in diabetes mellitus in more recent years despite the plateau in decline in stroke incidence during the late 1990s; (2) the similar ad / vad ratio found in recent studies in japan with that of the us could be due to a combination of at least 3 factors: (a) shifting diagnostic criteria (more in line with us consensus diagnosis), (b) possible shifting in health conditions among the elderly in japan (decline in stroke incidence, but increase in metabolic disease e.g., type 2 diabetes, hyperlipidemia, and atherosclerosis), (c) an increase in the proportion of the oldest old who had an historically higher prevalence of ad (as opposed to vad) in japan, and (d) regional variations (i.e., a north - to - south gradient in vad) possibly due to large difference in dietary patterns . The study limitations include relatively small sample sizes, especially for the oldest old group . The okinawa study took into account the potential dementia cases among those who were screened at phase 1 (screening phase), but did not participate in phase 2 (clinical assessment phase), using weights generated from phase 1 . However, as with most other epidemiological studies of dementia, there was no way of precisely estimating the frequency of dementia among those who did not participate in the screening phase . Four japanese prevalence studies [15, 4143] were not included in this study because the age - specific prevalence of ad and vad were not provided in the published articles . Although it would be interesting to deconstruct the changes in all - cause - dementia prevalence and ad / vad ratios into the potential explanatory factors, different criteria used to define subtypes of dementia would not allow this type of quantitative assessment . One of the strengths of our study is that we could ascertain the detailed screening procedures and diagnostic criteria by contacting the investigators of the original studies . Future studies could aid in monitoring changing prevalence patterns and their causes by including some of the following elements . First, as with hisayama studies, it would be ideal for studies to examine the prevalence of dementia and its subtypes repeatedly at the same locations using the same criteria . Second, it would be helpful if future epidemiology studies would recruit more participants aged 90 and older (e.g., through aggressive age - stratified sampling protocols) to improve estimates of prevalence in this age group . Third, more comprehensive diagnostic criteria should be used for inter - cohort comparisons: as suggested by viswanathan and colleagues, ad and vad exist in a continuum of disease . It might be more meaningful if we could apply, for example, more specific clinicopathological criteria for mixed dementia [4450] than those currently used . Increased use of standardized neuroimaging such as the alzheimer's disease neuroimaging initiative (adni) might aid in the development of more specific and comparable criteria for the diagnosis of vad . Finally, autopsy confirmation of the underlying potential causes of dementia in epidemiological studies would go a long way to help resolve these important uncertainties in the shifting patterns over time of the dementias.
Chronic low back pain (clbp) refers to chronic pain around the lumbar vertebra sustained for over three months without serious pathological characteristics, regardless of the existence of sciatalgia1 . Clbp causes pain and changes in the muscles, as well as decreases in contractile force and muscle activity . Studies of therapeutic approaches to clbp pain reduction, muscular strength improvement, and spinal movement enhancement have investigated the effects of myofacial release, osteopathy, massage, lumbar stabilization exercises, and proprioceptive neuromuscular facilitation (pnf)5,6,7 . Among these therapeutic approaches, joint mobilization using pnf has a positive effect on pain, muscle strength, and range of motion (rom)8, 9 . It is applied as a passive movement by therapists10, 11 . In particular, kaltenborn - evjenth orthopedic manual therapy (keomt) is an effective treatment for improving rom and pain . Keomt is a safe and well - established procedure that slowly utilizes the convex - concave rule (traction or gliding on the treatment side)10, 12 . Hindle et al . Reported pnf is effective at improving rom and muscular strength13 . Kim et al . Reported that lumbar stabilization therapy using pnf for clbp significantly improved the thicknesses of the external oblique and multifidus muscles, and the functional level8 . In addition, pnf has been used to improve the muscular strength of athletes14 . This case study was a single patient with clbp and a lumbar transitional vertebra who has six lumbar vertebrae (not five). A lumbar transitional vertebra is defined as a congenital malformation that appears as a deformation of the fifth lumbar (l5) or first sacrum (s1) vertebra15 . Vergauwen reported that lumbar transitional vertebrae patients more commonly experience disc protrusion, degeneration, and spinal stenosis than patients without lumbar transitional vertebrae16 . Therefore, the purpose of this study was to identify the effects of joint mobilization using keomt and pnf on a patient with clbp and a lumbar transitional vertebra . The subject was selected from among the patients of sarang hospital (yongin, south korea). The subject agreed to participate in the study after receiving explanations regarding the purpose and procedures of the experiment, and signed an informed consent statement before participation . The protocol for this study was approved by the local ethics committee of the namseoul university of cheonan . Computer tomography (ct) showed six lumbar vertebrae, which is one more lumbar vertebra than a normal person (fig . The subject was aged 29, and had a weight of 67 kg, a height of 158 cm, and a bmi of 26.90 . The initial symptom developed about two years earlier, and at the time of the study the subject was not receiving any medical or physical therapy . The major complaint of the subject was a numb feeling, especially in the hind part of the left leg . She also complained of a stinging pain during daily activities, such as lifting, lumbar twist, and flexion . The program consisted of a 40-min session, 3 days a week for 4 weeks (october 131, 2014), during which joint mobilization was performed using keomt and pnf techniques . Joint mobilization using keomt lasted 20 min, and it included lumbar segmental traction and lumbar segmental mobilization (flexion, extension) in a side - lying position . It was performed by a therapist who had completed a keomt spine advance course . The pnf exercise included shoulder flexion, abduction, and external rotation in a supine position, and dynamic reversal of the antagonist was performed17, which indirectly exercised the abdominal muscle through irradiation of the shoulder muscle . It was performed by a therapist who had completed the pnf level i, ii courses . The subject was assessed for spinal motion, low back pain, and thickness of the multifidus . Spinal motion was assessed using a spinal mouse (idiag, swiss), which measures the spinal curvature, flexion, and extension (thoracic and lumbar vertebrae). Low back pain was assessed using a visual analogue scale (vas) and the oswestry disability index (odi). The vas assesses subjective pain through a which has a possible range of 0 to 10 . The odi assesses low back pain, and has a possible range of 0 to 100% . It is composed of ten items, including pain severity, self - management, lifting, sitting, standing, sleeping, etc . The thickness of the multifidus was measured using ct (general electric, korea). The multifidus muscles of the fourth lumbar vertebra (l4)20 was scanned by a radiological thechnician at sarang hospital . The spinal curvature, flexion, and extension of thoracic and lumbar vertebrae were measured . In the thoracic vertebra, the rom of flexion increased from 14 to 20, and the rom of extension increased from 1 to 4. in the lumbar vertebra, the angle of spinal curvature increased from 12 to 20. the rom in flexion increased from 33 to 47, and the rom of extension increased from 2 to 7 (table 1table 1.change of thoracic and lumbar movementvariablecurvature ()flexion ()extension ()prepostprepostprepostthoracic2632142014lumbar1220334727). The subject mentioned she was less uncomfortable in daily life, e.g. During face washing, dishwashing, hair washing, etc . The odi percentage score decreased from 48.88 to 24.44 (table 2table 2.change of vas and odivariablevas (score)odi (%) prepostprepost7.5348.8824.44). The thickness of the multifidus was measured at the fourth lumbar vertebra (left and right). The left multifidus increased from 572.09 to 662.09 mm, and the right multifidus increased from 479.84 to 530.90 mm (table 3table 3.change of multifidus thicknessvariableprepostmultifidus (mm)l: 572.09l: 662.09r: 479.84r: 530.90l: left; r: right). 3.multifidus thickness on a ct (post) show the multifidus thickness on a ct . Multifidus thickness on a ct (pre) multifidus thickness on a ct (post) this study was a case study of a single patient with clbp and a lumbar transitional vertebra . The purpose of this study was to identify the effects of joint mobilization using keomt and pnf techniques in a patient with clbp and a lumbar transitional vertebra . The spinal motion, pain, and thickness of the multifidus were evaluated and compared between pre- and post - assessment . Florentino et al . Reported that most nurses possess at least one musculoskeletal disorder, among which low back pain is the most frequent (60.9%)21 . After the intervention, the angle of spinal curvature increased, and the roms of flexion and extension of the thoracic and lumbar vertebrae also increased, indication that joint mobilization using keomt and pnf had a positive effect on spinal motion . Ko et al . Reported that clbp patients in a thoracic joint mobilization group showed greater improvements in spinal motion and pain reduction than a william exercise group22 . Our present results were similar, suggesting that joint mobilization has a positive effect on spinal motion . After the intervention, the vas score decreased from 7.5 to 3, and the odi score decreased from 48.88% to 24.44% . Orthopedic manual therapy has positive effects on the spinal function of clbp patients6, especially items of self - management, such as bathing, dressing, and sleeping . Reported pain reduction after joint mobilization using keomt was performed for osteoarthritic elderly people23, and lpez et al . Reported joint postero - anterior mobilization had an effect on the pain and rom of clbp patients9 . Lee reported a pnf group demonstrated greater pain reduction and increased muscle activity than a ball training group, and jung reported pnf reduced the pain of clbp patients24, 25 . . Therefore, the results of previous studies agree with those of the present study . The spinal muscle contracts prior to motion and controls the movement28 . In particular, the multifidus muscle is important for lumbar stabilization . The superficial multifidus plays a role in spinal direction control, and after the intervention, the left multifidus increased from 572.09 to 662.09 mm, and the right multifidus was increased from 479.84 to 530.90 mm . Kim et al . Reported a pnf group showed greater increases in the thicknesses of the multifidus and external oblique muscles than a general physiotherapy group and a lumbar stabilization group8 . In addition, a resistance exercise using pnf not only reinforced muscular strength causing maximal muscular contraction, but it also stabilized the body position30 . These results indicate pnf has a positive effect on the thickness of the multifidus . According to our present results, joint mobilization using keomt and pnf had a positive effect on the spinal motion, pain, and thickness of the multifidus of a patient with clbp and a lumbar transitional vertebra . This study was a single subject case study, but it is meaningful in that it presented spinal motion, pain, and muscle activity in a patient with a lumbar transitional vertebra . A larger number of patients will need to be studied in the future.
To learn about the normal anatomy of the neural tube on mri to recognise the mr appearance of neural tube defects to understand the value of mri in assessing ntds technological developments in diagnostic imaging have improved the diagnosis and treatment of congenital anomalies immensely, and this progress is especially evident in neural tube defects (ntds). Ultrasonography allows accurate assessment of the neural tube . Fetal magnetic resonance imaging (mri) complements ultrasonography, confirming the suspected anomaly and even detecting malformations that were not detected at ultrasonography . The additional information provided by mri may lead to changes in the management of the pregnancy and/or of the delivery [25]. In this article we describe the radiological, pathological and clinical features of the most common ntds, and finally emphasise an integrated clinical and radiological approach for accurate diagnosis . In our institution, all fetal mri studies are performed after ultrasonography and mri never constitutes the first imaging study of the fetus . None of the women or fetuses receives any special preparation . Although it is important to be extremely careful when undertaking any procedure on a fetus, there is no evidence of associated risk to fetal development with fetal mri [68]. We use a 1.5-tesla scanner with a four - element phased - array body coil . A complete study requires between 12 and 15 sequences, so it takes about 2530 min . The fetal mri images are obtained using ultrafast sequences to minimise artefacts due to fetal movements . It seems that slow, rhythmic maternal respiration reduces fetal movements, so it is not necessary to acquire images with maternal breath - holding . T2-weighted single shot fast spin - echo (ssfse) or half - fourier single - shot turbo spin - echo (haste) images provide most of the information necessary for diagnosis . Gradient echo sequences (echo - planar imaging [epi] and true fast imaging with steady state precession [fisp]) have greater ferromagnetic susceptibility, so they make it possible to identify bony and vascular structures [1, 9]. Both the head and spine must be studied in all three planes . Even when the only indication for fetal mri is a suspected ntd, all the fetal structures should always be studied to rule out other possible anomalies . The first phase, gastrulation, begins in the 2nd to 3rd weeks of gestation; in this phase, the two layers of the embryonic plate divide into three definitive layers (the ectoderm, mesoderm and endoderm). In the second phase, neurulation, which takes place in the 3rd to 4th weeks of gestation, the cells that will become the notochord interact with the overlying ectodermic tissue, causing this tissue to thicken, fold over and fuse, thus forming the neural tube . The fusion begins in the medial dorsal zone of the embryo and progresses in a zipper - like fashion cranially as the rostral neuropore and caudally as the caudal neuropore . The closure of the two ends does not occur simultaneously: the cranial end closes before the caudal end . The final phase, secondary neurulation, takes place in the 5th to 6th weeks of gestation; in this phase a secondary neural tube forms from the cells of hensen s node, which is a mass of totipotent cells located dorsally that intervene in the formation of the notochord and later migrate caudally to the presacral region . This secondary neural tube is initially solid; it undergoes a process of retrogressive differentiation called canalisation in which it cavitates to form the medullary cone and the filum terminale . The interruption of this process at any point, whether in the cranial end or in the caudal end, will result in an ntd [11, 12]. The spinal column and spinal cord are the same length until 1215 weeks gestation, after which the spinal column grows more than the spinal cord until the medullary cone is situated approximately at the level of the second lumbar vertebra, where it will be at birth and will remain without significant changes throughout life [11, 12]. Mri is an excellent method for evaluating the structures of the fetal head and spine . Images can be obtained in any plane, regardless of the position of the fetus in the uterus . The cranial vault (fig . 1a, b) is seen as a hypointense structure in t2-weighted images, even early in gestation . 1a) can also be identified in any plane, but their visualisation improves as gestation advances.fig . A sagittal and b coronal t2-weighted haste images of the fetal head show the osseous structures of the base of the skull (arrows in a) and bones of the cranial vault (arrows in b). The vertebral bodies are hypointense with a band of higher intensity in the centre (white arrows), the intervertebral spaces are hyperintense (black arrows). D coronal t2-weighted haste image shows the vertebral pedicles as hypointense rounded structures on both sides of the spinal canal (arrowheads). The interpedicular distance in the lumbar spine is slightly increased (arrows); this is a normal finding that should not be confused with spina bifida . E axial image at the level of l1l2 shows the medullary cone (white arrow) inside the spinal canal (the asterisks mark the kidneys). F the medullary cone is no longer seen in this axial image of the spinal canal below the kidneys; sometimes the nerve roots can be identified (white arrows) a, b normal anatomy of the neural tube . A sagittal and b coronal t2-weighted haste images of the fetal head show the osseous structures of the base of the skull (arrows in a) and bones of the cranial vault (arrows in b). The vertebral bodies are hypointense with a band of higher intensity in the centre (white arrows), the intervertebral spaces are hyperintense (black arrows). D coronal t2-weighted haste image shows the vertebral pedicles as hypointense rounded structures on both sides of the spinal canal (arrowheads). The interpedicular distance in the lumbar spine is slightly increased (arrows); this is a normal finding that should not be confused with spina bifida . E axial image at the level of l1l2 shows the medullary cone (white arrow) inside the spinal canal (the asterisks mark the kidneys). F the medullary cone is no longer seen in this axial image of the spinal canal below the kidneys; sometimes the nerve roots can be identified (white arrows) the vertebral bodies have low signal intensity on t2-weighted sequences and are clearly seen on sagittal images . The intervertebral spaces are hyperintense and are clearly depicted in the sagittal plane (fig . The interpedicular distance should be constant throughout the spinal column, although it does increase slightly in the lumbar region and this widening should not be confused with an ntd (fig . The posterior elements of the vertebrae are best seen in the axial plane (fig . 1e, f). Caution is warranted in the assessment of possible scoliosis or kyphosis, because the spinal column can be altered by the position of the fetus in the uterus; these pseudoalterations are more common as gestation advances, resulting in proportionately less space within the uterine cavity (fig . The spinal canal is hyperintense in t2-weighted sequences because it is filled with cerebrospinal fluid . Although the spinal cord is well depicted in all three planes, it can probably be evaluated best in the axial plane (fig . The position of the fetus within the uterus results in a certain degree of thoracolumbar kyphosis that displaces the spinal cord anteriorly in the thoracic spine, making it difficult to see because it is adjacent to the vertebral bodies (fig . Precaution should be taken to ensure that the spinal cord disappears below the fetal kidneys (fig . 1e, f); sometimes, small hypointense structures that correspond to nerve roots are seen distal to the medullary cone (fig . It is important to evaluate the paravertebral soft tissues in mri studies, especially in fetuses with spina bifida or destructive processes or tumours that could affect these tissues . The skin and the paravertebral muscular structures are hypointense in t2-weighted sequences . The spinal canal should be closed posteriorly by soft tissues (fig . 1c, e, f). Ntds are a heterogeneous group of anomalies that result from the partial or total failure of the neural tube to close . Ntds can affect the cranial region (anencephaly) or the spinal region (spina bifida); spinal ntds can be divided into open or closed defects, depending on whether they are covered by a layer of skin (fig . 2neural tube defects the overall worldwide incidence of ntds is between one and ten per 1,000 live births [14, 15]. The rate of ntds has varied widely over time and has a clear geographical distribution . The highest incidences have been reported in mexico, ireland, india and northern china [16, 17]. A family history of ntds is absent in 9095% of cases, and ntds are reported in 3% of miscarriages . Some genetic factors like single gene mutations (meckel - gruber and roberts syndromes) and chromosomopathies (trisomies 13 and 18, triploidy) have been identified . Some maternal factors (hyperthermia, diabetes, hyperinsulinaemia, obesity, psychosocial or emotional stress [1823]) and some environmental factors (maternal medication with anticonvulsants like valproic acid and carbamazepine) can increase the incidence of ntds . Prenatal screening for ntds includes the measurement of diverse markers like acetylcholinesterase and alpha - fetoprotein in amniotic fluid and in maternal serum (alpha - fetoprotein is a glycoprotein secreted by the liver and by the fetal yolk sac). A recent meta - analysis confirmed that measuring alpha - fetoprotein in maternal serum during the second trimester is useful in screening for ntds . Nevertheless, this marker is only useful for detecting open ntds; furthermore, alpha - fetoprotein levels are also elevated in a long list of fetal anomalies, including omphalocele, turner s syndrome, and sacrococcygeal teratoma, amongst others . It is the absence of a major portion of the brain, skull and scalp above the orbits . There are two subtypes: anencephaly, in which the cranial vault, cerebral hemispheres and diencephalic structures are replaced by amorphous neurovascular tissue, and exencephaly, in which the cranial vault is absent but relatively normal amounts of brain tissue, albeit abnormally developed, is present . Some authors consider exencephaly an early stage of anencephaly, because the unprotected anomalous brain tissue located outside the cranial vault will eventually take on the appearance of anencephaly after being destroyed by the trauma induced by constant collision against the walls of the uterus . In both exencephaly and anencephaly, the base of the skull, the facial structures, and the orbits are present and are usually normal . Mri is usually not used to evaluate fetuses with these conditions because ultrasonography usually detects this anomaly very early and mri is not required to confirm this diagnosis . Fetal mri studies show the absence of osseous structures above the orbits and the absence of brain tissue or alterations in brain tissue when it is present . It is considered a lethal anomaly, the specific name of the anomaly depends on the structures contained in the herniated sac; for example, a meningocele contains meninges and cerebrospinal fluid, an encephalomeningocele contains meninges and brain tissue and an encephalomeningocystocele or encephalomeningohydrocele contains meninges, brain tissue, and part of the ventricular system (fig . 3). Most encephaloceles occur along the midline in the occipital or frontal region, but they can also involve the anterior cranial fossa (into the nasal sinuses) or even the base of the skull (into the sphenoid or ethmoid sinus).fig . 3encephalocele . A sagittal and b axial t2-weighted haste images show the protrusion of the occipital lobe and occipital horn of the ventricular system (black arrows in a and b) through a defect in the bone and soft tissues (white arrows in a). C ultrasound image also shows the protrusion of the occipital lobe (white arrows). D, e images of the same fetus at 31 weeks gestation show a decrease in the extruded encephalic component (arrow in e) and an increase in the associated meningeal component (arrow in d) encephalocele . A sagittal and b axial t2-weighted haste images show the protrusion of the occipital lobe and occipital horn of the ventricular system (black arrows in a and b) through a defect in the bone and soft tissues (white arrows in a). C ultrasound image also shows the protrusion of the occipital lobe (white arrows). D, e images of the same fetus at 31 weeks gestation show a decrease in the extruded encephalic component (arrow in e) and an increase in the associated meningeal component (arrow in d) encephaloceles are usually diagnosed by ultrasonography . Mri s role is to specify the type of lesion, which is more important in smaller lesions and in those that affect the anterior cranial fossa or base of the skull . It is essential to evaluate the rest of the structures in the head and the neural tube, fundamentally the posterior fossa because it can be difficult to evaluate with ultrasonography . 3d) as well as the presence of brain tissue inside the herniated sac in cases of encephalomeningocele (fig . 3a, b, e). Other anomalies like open spina bifida or ventriculomegaly that are often associated with encephaloceles are easy to evaluate with mri . The differential diagnosis for encephalocele is based on the size, content and location of the lesion . The main differential diagnosis for encephaloceles located in the occipital region is with cystic hygroma, whereas for those located in the frontal region, the differential diagnosis is with teratoma and haemangioma of the scalp . Both teratomas and haemangiomas of the scalp are shown as slightly heterogeneous but well - defined solid masses with variable signal intensity; haemangiomas may have areas of signal voiding due to their intense vascularisation . An encephalocele may form part of a syndrome like meckel - gruber syndrome and it can be associated with other anomalies like ventriculomegaly, spina bifida (715% of all encephaloceles), microcephaly, or cleft lip and/or cleft palate . The prognosis depends on the size, location and extent of the lesion as well as on whether other anomalies are present . Iniencephaly is an ntd affecting the occipital bone combined with rachischisis of the cervicothoracic spine and retroflexion of the head (fig . 4). Fetuses with iniencephaly have a variable defect in the occipital bone with an enlarged foramen magnum, the partial or total absence of cervicothoracic vertebrae with irregular fusion of those that are present and incomplete closure of the vertebral bodies and arches . These alterations result in significant shortening of the spine and of the fetus due to marked lordosis with hyperextension of the head . Iniencephaly can be open (associated with encephalocele) or closed (spinal anomaly without encephalocele).fig . The spine is short, the head is hyperextended, and there is a nuchal fold (arrow). The spine is short, the head is hyperextended, and there is a nuchal fold (arrow). B pathological specimen the differential diagnosis is with klippel - feil syndrome (short neck and fusion of the cervical vertebrae). Some authors believe that klippel - feil syndrome is a milder form of iniencephaly . The prognosis of iniencephaly in the neonatal period is dismal, so whether other anomalies are present is unimportant . Spinal dysraphism is the result of a congenital vertebral defect due to the failure of the caudal neuropore to close that leads to the exposure of the contents of the medullary canal to the exterior . The defect usually involves the posterior vertebral elements, although it can also involve the vertebral body itself . The defect is usually located in the lumbosacral region, but it can occur at any level and it can even involve the entire spine . The absence of skin and muscle in these areas is due to the failure to induct the overlying ectodermal and mesodermal tissues . Other theories postulate the existence of an unbalance in the production and reabsorption of cerebrospinal fluid during the embryonic period that leads to an excessive accumulation in the already closed neural tube (hydromyelia), which in turn causes a secondary separation from the dorsal wall . Spinal dysraphism comprises a wide spectrum of anomalies; these can be divided into open and closed defects . Rachischisis is the most severe form of spina bifida; in this lethal condition, an extensive segment of the rachis is absent, resulting in extensive exposure of neural tissue . Myelomeningocele and myeloschisis (myelocele) are the most common spinal dysraphism, accounting for 85% of all cases [12, 32]. In these lesions, neural contents are exposed to the exterior through midline defects in the bone and skin [12, 33]. When the spinal canal is directly exposed, the anomaly is called myeloschisis or myelocele (fig . 5), and when intramedullary structures lined with meninges protrude or herniate due to the expansion of the underlying subarachnoid space, it is called myelomeningocele (fig . 6). Myelomeningocele is the most common open spinal dysraphism [32, 35]. By definition, there must be a vertebral defect, the most common of which is the absence of the posterior vertebral arches (figs . The main characteristic that allows myelomeningoceles to be differentiated from myeloschisis is the position of the neural placode, a remnant of the unfolded (non - neurulated) neural tube, with respect to the surface of the skin . In myelomeningocele, the neural placode protrudes behind the surface of the skin (fig . 6a, b, f), whereas in myeloschisis it is at the same level (fig . Another, extremely rare type of open dysraphism is the hemimyelomeningocele or hemimyelocele: in this anomaly, diastematomyelia is associated with one of the above - mentioned anomalies in which one of the hemicords does not complete neurulation [32, 36]. This finding is important because if diastematomyelia is not suspected, the surgical repair of the myelomeningocele will not include the repair of the tethered cord that is nearly always present in diastematomyelia [1, 9, 37].fig . A, b axial t2-weighted haste image of the fetal cranium shows the deformity of the frontal bones (black arrows in a) and the characteristic beaklike shape of the ventricular horns (arrows in a). The posterior fossa deformity can also be seen: the cisterna magnum is absent and the foramen magnum is enlarged due to the descent of the structures of the posterior fossa toward the spinal canal (arrows in b). C sagittal t2-weighted haste image shows a cutaneous defect or thinning in the lumbosacral region (arrowhead) with no mass protruding through the defect as well as an associated chiari malformation (black arrow). D axial image at the level of the cutaneous defect shows the abnormally low neural placode within the spinal canal without expansion of the subarachnoid spaces a sagittal t2-weighted haste image shows a small cystic mass protruding at the thoracolumbar level (black arrow). Associated hydrocephalus (asterisk) and a chiari malformation (white arrow) are also seen . B axial t2-weighted haste image at the lumbosacral level shows the neural placode protruding outside the surface of the skin due to expansion of the adjacent subarachnoid space (black arrow). C coronal image at the spinal level shows increased interpedicular distance at the level of the neural defect . D ultrasound image of a different patient shows a cystic mass protruding at lumbar level (white arrows). A, b axial t2-weighted haste image of the fetal cranium shows the deformity of the frontal bones (black arrows in a) and the characteristic beaklike shape of the ventricular horns (arrows in a). The posterior fossa deformity can also be seen: the cisterna magnum is absent and the foramen magnum is enlarged due to the descent of the structures of the posterior fossa toward the spinal canal (arrows in b). C sagittal t2-weighted haste image shows a cutaneous defect or thinning in the lumbosacral region (arrowhead) with no mass protruding through the defect as well as an associated chiari malformation (black arrow). D axial image at the level of the cutaneous defect shows the abnormally low neural placode within the spinal canal without expansion of the subarachnoid spaces . A sagittal t2-weighted haste image shows a small cystic mass protruding at the thoracolumbar level (black arrow). Associated hydrocephalus (asterisk) and a chiari malformation (white arrow) are also seen . B axial t2-weighted haste image at the lumbosacral level shows the neural placode protruding outside the surface of the skin due to expansion of the adjacent subarachnoid space (black arrow). C coronal image at the spinal level shows increased interpedicular distance at the level of the neural defect . D ultrasound image of a different patient shows a cystic mass protruding at lumbar level (white arrows). E pathological specimen . F sagittal diagrams of the anomaly open spina bifida is nearly always found together with chiari type ii malformation, which is an anomaly of the posterior fossa consisting of herniation of part of the cerebellum (vermis and fourth ventricle) and of the brainstem through the foramen magnum (figs . This association is thought to be due to the collapse of the primitive fetal ventricular system (brought about by the loss of cerebrospinal fluid through the spinal defect), which prevents the rhombencephalic vesicle (from which the brainstem, fourth ventricle, and cerebellum are derived) from expanding and results in deficient development of the posterior fossa and herniation . Recent studies have reported worse prognoses for fetuses with greater cerebellar herniation, related to an increased incidence of seizures, vesical dysfunction, and dependency for ambulation . Open spina bifida is accompanied by a collapse of the extra - axial subarachnoid spaces (fig . 6a) secondary to mechanical obstruction from the chiari malformation or to dysfunctional reabsorption of cerebrospinal fluid [38, 40] in 75% of cases . This is why it is essential to evaluate the spine carefully to rule out spinal dysraphism when ventriculomegaly is detected at any gestational age . Likewise, the shape of the ventricles is abnormal owing to the acute angle of the ventricular horns (fig . 5a). As in ultrasonography, in mri both direct and the indirect signs of spina bifida at the level of the fetal cranium (the lemon sign: special shape of the cranium due to deformity of the frontal bones [fig . 5a] and the banana sign: special shape of the posterior fossa secondary to displacement of the cerebellar hemispheres toward the cervical canal, which results in the obliteration of the cisterna magnum [fig . Other cranial anomalies, like the collapse of the subarachnoid space, a beak - shaped mesencephalic tectum, dysgenesis of the corpus callosum and subependymal heterotopias are all better seen on mri . The differential diagnosis will depend fundamentally on the contents and location of the spinal defect . The intracranial anomalies that accompany ntds can help in differentiating between these two entities, because teratomas are not usually accompanied by intracranial anomalies . Distinguishing between open and closed spinal dysraphism is of the utmost importance because the long - term functional and neurological prognoses are notably better in cases of closed dysraphism than in cases of open dysraphism [39, 41]. It is of the utmost importance to obtain an accurate and specific assessment of the anomaly due to the progressive incorporation of in utero treatments . Latest trials in fetal surgery for myelomeningocele have shown a significant reduction of vp shunt placement, an improvement in the overall neuromotor function and a significantly reversed hindbrain herniation in those patients who underwent fetal repair compared with those who underwent postnatal surgery . This trial has also revealed that in utero therapy increases the risk of spontaneous rupture of membranes, oligohydramnios and preterm delivery . Fetal mri provides a better overview for the neurosurgeon who is familiar with mri and not as much with ultrasound . Specific details with respect to the level and magnitude of the defect can help the intra - uterine management of the spinal anomaly . The resolution of mri is less than ultrasound, but the diagnostic value of mri is less dependent on the fetal position . The term closed is more appropriate than occult, because cutaneous signs that raise suspicion of these anomalies are present in up to 50% of cases [34, 43]. The soft tissues that cover the spine dorsally can be identified as a hypointense line on sagittal and axial t2-weighted images (figs ., this line is interrupted at the level of the lesion; this finding is essential for differentiating between open and closed dysraphism . Closed dysraphism is not usually associated to cranial alterations like chiari malformation or ventriculomegaly [1, 9] and does not manifest with elevated alpha - fetoprotein . A sagittal t2-weighted haste image shows an interruption in the posterior vertebral arches in the lumbar region (arrow). A posterior fossa anomaly with an elevated vermis and dilation of the iv ventricle is also seen (asterisk). B axial image at the lumbar level showing the cystic mass protruding through the spinal defect (black arrow). Two nerve roots (white arrow) can be seen arising from the non - neurulated neural placode inside the spinal defect . A sagittal t2-weighted haste image shows an interruption in the posterior vertebral arches in the lumbar region (arrow). A posterior fossa anomaly with an elevated vermis and dilation of the iv ventricle is also seen (asterisk). B axial image at the lumbar level showing the cystic mass protruding through the spinal defect (black arrow). Two nerve roots (white arrow) can be seen arising from the non - neurulated neural placode inside the spinal defect . D sagittal diagram of the anomaly there are three main types of closed spinal dysraphisms with masses . In the first, further subdivided into lipomyelomeningocele and lipomyeloschisis (lipomyelocele) (fig . 8), direct contact between the mesenchymal tissue and neural tube affects the differentiation of fatty tissue . Clinically, these lesions are characterised by the presence of a lipomatous subcutaneous mass above the gluteal cleft . In these cases, it is important to note the point where the lipoma joins the neural placode to differentiate between a lipomyelomeningocele and lipomyeloschisis . The point of union in lipomyelomeningoceles is outside the neural canal due to the expansion of the subarachnoid space, whereas in lipomyeloschisis the lipoma joins the neural placode inside the neural canal after entering through a defect in the bone (fig . A sagittal t2-weighted haste image shows a deformity of the spinal canal completely covered by soft tissues (arrows). B coronal image showing the widening of the spinal canal at the lumbar level (white arrows) and abnormally low termination of the medullary cone (black arrow) below the kidneys . C ct study after birth shows the deformed, widened spinal canal (arrowheads) and soft - tissue calcification behind the bone defect (white arrows). D sagittal t1-weighted mri image obtained after birth shows the abnormally low medullary cone, a lipomatous component within the spine (white arrows), and soft - tissue calcification (black arrow). E axial t1-weighted image showing spinal cord duplication (black arrows) and the lipomatous component within the spine (asterisk). A sagittal t2-weighted haste image shows a deformity of the spinal canal completely covered by soft tissues (arrows). B coronal image showing the widening of the spinal canal at the lumbar level (white arrows) and abnormally low termination of the medullary cone (black arrow) below the kidneys . C ct study after birth shows the deformed, widened spinal canal (arrowheads) and soft - tissue calcification behind the bone defect (white arrows). D sagittal t1-weighted mri image obtained after birth shows the abnormally low medullary cone, a lipomatous component within the spine (white arrows), and soft - tissue calcification (black arrow). E axial t1-weighted image showing spinal cord duplication (black arrows) and the lipomatous component within the spine (asterisk). G sagittal diagrams of the anomaly the second type is a meningocele with cutaneous lining . Anterior meningoceles are much less common and tend to be located in the presacral region . The third type, myelocystocele (fig . 7), consists of the herniation of a segment of the spinal cord with a dilated ependymal canal within a meningocele; these lesions can occur at any point in the spine . Myelocystoceles are similar to myelomeningoceles; the two entities are differentiated by the thickness of the wall of the herniated sac (fig . 7b), the absence of the chiari malformation, and the absence of elevated alpha - fetoprotein levels [1, 9]. Simple lesions include intradural lipomas, filum terminale lipomas, tight filum terminale syndrome, persistent terminal ventricle, and dermal sinus . Intradural lipomas are lipomas located within the spine that are contained within the dural sac . These lesions generally affect the lumbosacral region and are usually associated with the tethered cord syndrome . Filum terminale lipomas or fibrolipomatous thickening of the filum terminale can be considered normal variants unless there is clinical evidence of a tethered cord . Filum terminale lipomas are identified as linear thickening of the filum terminale that is hyperintense on t1-weighted sequences . The tight filum terminale syndrome is characterised by shortening and hypertrophy of the filum terminale and consequent cord tethering syndrome . Persistent terminal ventricle consists of small linear cavities inside the medullary cone immediately above the filum terminale . A dermal sinus is an epithelial fistula that connects the neural tissue or the meninges with the surface of the skin . Dermal sinuses are usually located in the lumbosacral region and are usually found together with an intraspinal dermoid cyst . Clinically, they present with a small midline pit, a cutaneous nevus, an area of hyperpigmentation, or a capillary haemangioma . Detecting all these findings prenatally is extremely difficult; however, the fistulous tract can be identified using high resolution mri techniques . These patients require surgical repair after birth because the fistulous tract that communicates the skin with the neural tissue can predispose to infections of the neural contents . Complex closed spinal dysraphisms can be divided into disorders of midline notochord integration and disorders of notochord malformation . Disorders of midline notochord integration include enteric fistula, which is an abnormal connection between the digestive tract and the surface of the skin that may or may not be accompanied by a neurenteric cyst . Neurenteric cysts are lined with epithelial mucosa similar to that of the gastrointestinal tract and are typically located in the anterior cervicothoracic region . 8), which consists of a longitudinal splitting of the spinal cord into two hemicords . There are two types of diastematomyelia: in type 1, both hemicords are wrapped in independent meningeal linings and are separated by an osseous or cartilaginous septum; whereas in type 2, the two hemicords share a common meningeal lining [32, 48]. The septum in type 1 diastematomyelia can be seen as a hypointense linear structure within the spinal canal on t2-weighted sequences . Caution should be taken not to confuse this septum with the spinal cord, as both structures have the same signal intensity . The soft tissues and the skin that cover these anomalies are usually intact unless they form part of a more complex anomaly . Diastematomyelia is normally accompanied by vertebral anomalies like hemivertebrae or butterfly vertebrae, tethered cord, or open spina bifida (hemimyelomeningocele or hemimyelocele). Disorders of notochord malformation include caudal regression syndrome, which consists of the total or partial agenesis of the lumbar and sacral spine, probably secondary to a disturbance in the formation of the secondary neural tube . Caudal regression syndrome is divided into two types: in type 1, the medullary cone is high and ends abruptly; in type 2, the spinal cord is tethered and the medullary cone is low - lying [32, 46]. Caudal regression syndrome comprises a wide spectrum of anomalies ranging from sacral agenesis or dysgenesis to sirenomelia (fusion of the lower limbs). Some authors believe that type 1 and type 2 caudal regression actually represent two distinct entities . Caudal regression can be found in association with other anomalies like omphalocele, bladder exstrophy, imperforate anus and spinal defects (oeis complex); vertebral anomalies, anal anomalies, cardiac anomalies, tracheoesophageal fistula, limb malformations, hydrocephalus (vacterl - h); and sacral agenesis, anal atresia and presacral teratoma or meningocele (currarino triad). Another disorder of notochord malformation is segmental spinal dysgenesis, which includes agenesis or dysgenesis of the thoracic or lumbar spine, alterations of the spinal cord and nerve roots, congenital paraparesis or paraplegia, and deformities in the lower limbs . Their prenatal diagnosis can predict the viability of the fetus, has implications for family counselling about the pregnancy, and orients the prognosis and management of the newborn . Ultrasonography remains the primary imaging test for the prenatal diagnosis of ntds, but sometimes it does not enable an accurate diagnosis so other diagnostic techniques are necessary . Fetal mri has advanced rapidly in the last 25 years, developing from an experimental technique to become a fundamental tool in normal clinical practice in many centres around the world . Mri s ability to detect complex anomalies that involve different organs has been widely reported . Fetal mri should be restricted to centres with experienced staff and should never be considered a replacement for ultrasonography, which continues to be the technique of choice to evaluate fetal morphology.
Patient acuity has been increasing, patient turnover is higher, and patients are frequently of advanced age with several comorbid disorders . Limited resources and the pressure for higher efficiency can result in the suboptimal delivery of healthcare . During treatment within the healthcare system, a significant percentage of patients (316%) suffer from adverse events through system errors- with 1565% of adverse patient events being attributed to communication failure . Patient care can involve several health care providers with several transfers between providers . In this transition, poor or incomplete relay of patient data may result in errors, near misses and adverse events . Standardized and thorough communication of critical information is an essential component of patient safety, . Although individual us based handover guidelines, and handover tools have been developed, these practices are not widespread . A previous survey study observed that 31% of mds experienced daily clinical problems which they believed avoidable if provided with adequate hand - over processes . Widespread us practice of verbal hand - over has been observed to be highly inefficient . The world health organization listed communication during patient care handovers as one of its high 5 patient safety initiatives . The joint commission international listed their 2nd national patient safety goal as to improve the effectiveness of communication among caregivers . There is a lack of studies on how anaesthesiologists hand over their patients, which is surprising given that anesthesiology is a specialty that has always aimed at placing patient safety as its top priority . The standards of postoperative care issued by the american society of anesthesiology does not state how handover should be performed nor what information it should include, leaving the handover process decision up to the individual clinician . Studies analyzing anesthetic team handover in the recovery room observed that the majority of handovers were remarkably brief and insufficient- . In these studies, critical details such as the preoperative state of the patient, type of operation performed, amount and kinds of analgesics given and problems encountered during the case were either barely mentioned or underplayed . In some instances significant intraoperative events were completely disregarded . Additionally, there were frequent distractions present at the time of the hand - over . There are no reported studies on handover practices performed by anesthesia providers when transition of care occurs intraoperatively . This preliminary pilot study was a qualitative rather than quantitative survey to highlight the scope of the current us intraoperative handover of patient information . This study had institutional approval (wayne state university irb). Due to the anonymity of the suvey content, this study used an online questionnaire format with 8 survey questions created using a computerized online survey program (survey monkey, palo alto, ca, usa,). An e - mail with a link to the questionnaire was sent to anesthesia providers at all anesthesia residency programs nationwide (120 out the 132 us programs encompassing around 4500 residents and their attending academic mdas) and a smaller survey selection of crnas (10 institutions about 300 crnas in the metropolitan area of detroit, mi). All questionnaires were anonymous and were only sent to us anesthesia providers working in an academic hospital setting with an associated residency program in anesthesiology . The only demographics of surveyed anesthesia health care providers obtained were for medical training level (certified registered nurse anesthetist -crna, medical doctor of anesthesiology resident mdar, or medical doctor of anesthesiology mda). From all questionnaires received (n = 216), 87 (40.3%) of respondents were mdas, 71 (32.9%) were mdar, and 58 were crnas (26.9%; table 1). The response rate to this survey was low with a response from approximately 5% of the resident population in us anesthesia programs and approximately 20% of the crnas surveyed in the metropolitan detroit area . Of all respondents, 108 (49.1%) stated that they did not have a handover protocol at the institution where they practiced (table 1). Of the respondents who did have an institutional handover policy furrthermmore, 85.7% respondents reported not having a departmental standardized handover form (table 2). Of all responders, 84.8% (173/204) had an experience where they had not received sufficient information during a patient handover . Only 7.4% (15/203) of respondents had never experienced complications or mismanagement due to poor or incomplete handover . In comparison, 59.6% (121/203) reported rarely having complications, 30.5% (62/203) reported sometimes having complications and 2.5% (5/203) reported frequently having complications (table 3). Respondents were asked to respond on the frequency in which they addressed the specific handover information such as allergies and medications, past medical history, intraoperative events (anesthesia related and surgery related), postoperative plans (extubation / icu, etc . ), special concerns (code status, jehovah's witness, type and screen antibodies, airway information (ie . H / o difficult intubation) and significant diagnostic studies (e.g blood lab, ekg, chest x - ray, stress tests, pulmonary function tests, etc . ). For this specific handover information, the majority of providers frequently or always relayed information regarding patient allergies (89.2%), past medical history (99%), anesthesia related intraoperative events (99%), surgery related intraoperative events (91.7%), airway information (97.5%) and significant diagnostic studies (84.2%; fig . 1). However, information about medications (31.4%), postoperative plan (19.1%), code status (75.4%), information about refusal of blood (21.1%) and type and screen antibodies (49%) were either sometimes, rarely or never included during handover . From the specific list of patient handover information factors, 80.5% (165/204) of responders stated that these factors would be sufficient for an effective, complete handover . On a scale from 15, key - (1 = never, 2 = rarely, 3 = sometimes, 4 = frequently, 5 = always . Mdar - medical doctor of anesthesiology resident, crna- certified registered nurse anesthetist, mda - medical doctor of anesthesiology . Ponv: post - operative nauseaand vommitting; mh: malignant hyperthermia; iv: intravenous . This pilot study was a qualitative rather than quantitative survey to highlight the scope of the current problem of us intraoperative handover of patient information . Poor transition of patient care from the operating room has a high potential for adverse patient outcomes . Our study indicates that the current intraoperatvive handover processes among anesthesia providers do not have standard uniform practices, and there may be a high proportion of insufficient patient handover practices . These findings are surprising given that the anesthesiology specialty places patient safety as one of its top objectives and that all survey respondents practiced in an academic institution . Major limitations of this study were the low response rate of 216 respondents and that due to annonymous nature of this survey the number of individual academic institutitions could not be determined . Additionally, there is the possibility that there was a bias towards an increased proportion of responses from anesthesia providers who had prior poor handover experiences and were unsatisified with current practice . The lack of standardized handover process has resulted in substandard patient care in us healthcare setting, ., we hope that our study will stimulate a discussion of us handover practices in the anesthesia field . Increased awareness in handover practices we believe that patient handover practices should be discussed and an optimal response would be the formation of an expert consortium with the aim to develop and implement standardized protocols.
Slc / ccl21, normally expressed in high endothelial venules and in t cell zones of spleen and lymph nodes, strongly attracts t cells and dendritic cells (dc) [1 - 8]. Slc / ccl21 recruits both nave lymphocytes and antigen stimulated dc into t cell zones of secondary lymphoid organs, co - localizing these early immune response constituents that culminate in cognate t cell activation . The capacity to facilitate the co - localization of both dc and t cells forms a strong rationale for the use of slc / ccl21 in cancer therapy . In previous studies we have shown that the slc / ccl21-mediated anti - tumor responses are accompanied by the enhanced elaboration of ifn, ip-10/cxcl10, mig / cxcl9, gm - csf and il-12, but decreased pge2, vegf, il-10, and tgf- at the tumor site . In addition, there was a marked increase in cd4and cd8cells secreting ifn and gm - csf as well as dc expressing cd11c infiltrating the tumors . Mig / cxcl 9 and ip-10/cxcl10 are glutamine - leucine - arginine (elr)-negative cxc chemokines that are potent chemoattractants for activated t cells [11 - 13]. These chemokines also have in common the ability to signal through a g protein - coupled receptor, cxcr3, expressed on activated t lymphocytes . Ip-10/cxcl10, mig / cxcl9 and ifn are known to have potent anti - tumor activities in vivo [14 - 17]. Both mig / cxcl9 and ip-10/cxcl10 are important for the anti - tumor activity mediated by ifn inducing cytokines such as il-12 . Based on these findings we hypothesized that augmentation in ifn, mig / cxcl9, and ip-10/cxcl10 would play an important role in the slc / ccl21-mediated anti - tumor response . Here we show that the cytokines ifn, ip-10/cxcl10 and mig / cxcl9 serve as effector molecules in slc / ccl21-mediated anti - tumor responses . Host antigen presenting cells (apc) are critical for the cross - presentation of tumor antigens . However tumors have the capacity to limit apc maturation, function and infiltration of the tumor site [21 - 24]. Thus molecules that attract host apc and t cells could serve as potent agents for cancer immunotherapy . A potentially effective pathway to restore ag presentation is the establishment of a chemotactic gradient that favors localization of both activated dc and type 1 cytokine producing lymphocytes at the tumor site . Slc / ccl21, a cc chemokine expressed in high endothelial venules and in t cell zones of spleen and lymph nodes, strongly attracts naive t cells and dcs [1 - 8]. Because dcs are potent apcs that function as principal activators of t cells, the capacity to facilitate the co - localization of both dc and t cells may reverse tumor - mediated immune suppression and orchestrate effective cell - mediated immune responses [9,10,25 - 27]. In addition to its immunotherapeutic potential, slc / ccl21 has been found to have potent angiostatic effects, thus adding further support for its use in cancer therapy . Based on these dual capacities, we as well as others have begun pre - clinical evaluation of slc / ccl21 in several tumor models [9,10,25 - 27]. Utilizing two transplantable murine lung cancer models, we have previously shown that the anti - tumor efficacy of slc / ccl21 is t cell - dependent . In both models recombinant slc / ccl21 administered intratumorally led to complete tumor eradication in 40% of the treated mice . Studies performed in cd4 and cd8 knockout mice also revealed a requirement for both cd4 and cd8 lymphocytes subsets for slc / ccl21 mediated tumor regression . Consistent with these findings we found that slc therapy did not alter tumor growth in scid mice . In immune competent mice, intratumoral injection of slc / ccl21 led to a significant increase in cd4 and cd8 t lymphocytes as well as dc infiltrating the tumor and the draining lymph nodes . The cellular infiltrates were accompanied by an enhanced elaboration of il-12, ifn, ip-10/cxcl10 and mig / cxcl9 at the tumor site . Slc / ccl21 is documented to have direct anti - angiogenic effects; the tumor reductions observed in this model could have been due to participation by t cells secreting ifn leading to inhibition of angiogenesis via mig / cxcl9 and ip-10/cxcl10, as well as t cell - dependent immunity . Both mig / cxcl9 and ip-10/cxcl10 are chemotactic for stimulated cxcr3-expressing activated t lymphocytes that could further amplify ifn at the tumor site . Hence we postulated that in addition to ifn, ip-10/cxcl10 and mig / cxcl9 would also be important contributors to the tumor reduction in the context of slc / ccl21 therapy . To determine the importance of mig / cxcl9, ip-10/cxcl10 and ifn in the slc / ccl21 mediated antitumor response, these cytokines were depleted in slc / ccl21-treated mice . Anti - ip-10/cxcl10, mig / cxcl9 and ifn antibodies each partially but yet significantly inhibited the anti - tumor efficacy of slc / ccl21 (figure 1 * p <0.01 compared to the control antibody group). Neutralization of ifn caused a significant decrease in both mig / cxcl9 and ip-10/cxcl10 indicating that these chemokines are largely ifn dependent . Thus, an increase in ifn at the tumor site of slc / ccl21-treated mice could explain the relative increases in ip-10/cxcl10 and mig / cxcl9 . The converse was also observed; ifn production at the tumor site was found to be mig / cxcl9- and ip-10/cxcl10-dependent as indicated by the fact that neutralization of these cytokines caused a significant decrease in ifn(figure 2). Neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines, thus indicating that ifn, mig / cxcl9 and ip-10/cxcl10 are interdependent in the slc / ccl21-mediated anti - tumor responses . Based on these findings, we speculated that the decrease of ip-10/cxcl10 and mig / cxcl9 led to a decrease in ifn by limiting the influx of t cells producing ifn at the tumor site . Because mig / cxcl9 and ip-10/cxcl10 are chemotactic for stimulated cxcr3 expressing activated t lymphocytes, we determined if neutralizing these chemokines in vivo in the slc / ccl21-treated mice would decrease the frequency of cxcr3 expressing t cells at the tumor sites . Compared to slc / ccl21-treated mice receiving control antibody, flow cytometric evaluation of single - cell suspensions of non - necrotic tumors from slc / ccl21-treated mice receiving neutralizing antibodies to mig / cxcl9, ip-10/cxcl10 and ifn showed 10, 12 and 31% decreases, respectively, in the gated cd3 cxcr3 t cell populations (table 1). The fact that neutralization of ifn was the most efficient at decreasing the total number of cxcr3-activated t cells may be due to the decrease in the ifn-dependent cxcr3 ligands mig / cxcl9 and ip-10/cxcl10 . Individually, neutralizing mig / cxcl9 and/or ip-10/cxcl10 only led to a partial decrease in the total number of cxcr3-activated t cells . Because mig / cxcl9 and ip-10/cxcl10 share the same receptor (cxcr3), one possible explanation of a partial decrease in cxcr3-activated t cells is that neutralization of one ligand may overexpose the receptor to the other ligand . Furthermore, because murine slc / ccl21 binds cxcr3 receptor, slc / ccl21 may be recruiting cxcr3 t cells directly . An alternative explanation is that residual cytokines such as ifn - inducible t cell chemoattractant (itac / cxcl11) present after in vivo neutralization of ifn, mig, ip-10 may have remaining activity and attract cxcr3 t cells . Our results imply that all 3 cytokines, ifn, mig and ip-10 play interrelated roles in the recruitment of cxcr3-activated t cells in slc / ccl21-mediated anti - tumor responses . While the number of cd4 and cd8 cells remained unaltered, compared to slc / ccl21-treated mice receiving control antibody, mice administered neutralizing antibodies to mig / cxcl9, ip-10/cxcl10 and ifn showed respective decreases of 38, 28 and 16% in the number of cd11cdec205dc infiltrating the tumor (table 1). At present it is not clear why there is a decrease in dc following neutralization of mig / cxcl9, ip-10/cxcl10 and ifn. A plausible explanation is that these cytokines may be involved in complex interactions with other molecules that are chemotactic for dc . Further studies are required to delineate the mechanisms responsible for mig / cxcl9, ip-10/cxcl10 and ifn mediated localization of dc at the tumor site . 5 days following tumor implantation, mice were treated intratumorally with recombinant murine slc / ccl21 (0.5 g) 3x / week . One day before slc / ccl21 administration, mice were given the respective cytokine antibody by i.p . Antibodies to: ip-10/cxcl10, mig / cxcl9 and ifn significantly inhibited the antitumor efficacy of slc / ccl21 (* p <0.01 compared to the control antibody treated group). N = 8 mice group slc / ccl21-treated mice had a significant induction in ifn, mig / cxcl9 and ip-10/cxcl10 at the tumor site compared to diluent treated control tumor bearing mice (p <0.001). Assessment of cytokine production at the tumor site of slc / ccl21 treated mice receiving anti - ifn, anti mig / cxcl9 and anti ip-10/cxcl10 showed an interdependence of ifn, mig / cxcl9 and ip-10/cxcl10: neutralization of any one of these cytokines in vivo caused a concomitant decrease in all three cytokines (* p <0.01 compared to the control antibody treated group). N= 8 mice per group neutralization of ip-10, mig and ifn reduces the frequency of cd3cxcr3 t lymphocytes and cd11cdec205dc at the tumor site of slc / ccl21-treated mice single cell suspensions of non - necrotic tumor nodules from slc / ccl21 treated mice receiving neutralizing antibodies to p-10/cxcl10, mig / cxcl9, ifn, control ab and diluent treated tumor bearing mice were prepared . Cell surface staining for t cell markers cd4, cd8, cd3, and the chemokine receptor cxcr3 as well as dc markers cd11c and dec205 were evaluated by flow cytometry . Cells were identified as lymphocytes or dc by gating on the forward and side scatter profiles; 10,000 gated events were collected and analyzed using cell quest software . Within the gated t lymphocyte population, intratumoral injection of slc / ccl21 led to an increase in the frequency of cd4, cd8, cd3cxcr3events compared to the diluent control (* p <0.001). Within the gated dc population, intratumoral injection of slc / ccl21 led to an increase in the frequency of cd11cdec205events compared to the diluent control (* p <0.001). Compared to slc / ccl21-treated mice receiving control antibody, mice receiving neutralizing antibodies to ip-10/cxcl10, mig / cxcl9, and ifn showed decrease in the gated cd3cxcr3 t cell as well as cd11cdec205 dc events (* * p <0.01) (n = 8 mice per group). Our results suggest that the anti - tumor properties of slc / ccl21 may be due to its chemotactic capacity in co - localization of dc and t cells as well as in part to the induction of key cytokines such as ifn, ip-10/cxcl10 and mig / cxcl9 . Abs to murine ifn, recombinant ifn were obtained from pharmingen (san diego, ca). Abs to murine mig / cxcl9, ip-10/cxcl10 and recombinant cytokine standards was purchased from r&d (minneapolis, mn). Polyclonal goat anti - murine mig / cxcl9 and anti - murine ip-10/cxcl10 specific anti - serum were produced and characterized as previously described . Anti - mouse ifn monoclonal (r4 - 462, american type culture collection, rockville, maryland, md) neutralizing antibody was purified by affinity chromatography from scid mice ascites, which was generated 34 weeks after intraperitoneal injection of 10r4 - 462 hybridoma cells per mouse . For flow cytometry, labeled antibodies (fitc, pe, percp) against the t cell surface markers to cd3, cd4, cd8 and against the dc markers, cd11c and dec 205 and the appropriate controls were purchased from pharmingen . A weakly immunogenic lung cancer, lewis lung carcinoma (3ll, h-2) obtained from atcc (crl-1642, llc) was utilized for assessment of cytokines important for slc / ccl21 mediated anti - tumor responses in vivo . The cells were routinely cultured as monolayers in 25 cmtissue culture flasks containing rpmi 1640 medium (irvine scientific, santa anna, ca) supplemented with 10% fetal bovine serum (fbs) (gemini bioproducts, calabasas, ca), penicillin (100 u / ml), streptomycin (0.1 mg / ml), 2 mm glutamine (jrh biosciences, lenexa, ks) and maintained at 37c in a humidified atmosphere containing 5% co2 in air . The cell lines were mycoplasma free and cells were utilized up to the tenth passage before thawing frozen stock cells from liquid n2 . For tumorigenesis experiments, injection in the right supra scapular area of c57bl/6 mice and tumor volumes were monitored three times each week . Five day established tumors were treated with intratumoral injection of 0.5 g of recombinant murine slc / ccl21 (pepro tech, rocky hill, nj) or murine serum albumin (utilized as an irrelevant protein for control injections) were administered three times per week for two weeks as previously described . 24 h prior to slc / ccl21 treatment, and then three times a week, mice were injected i.p . With 1 ml / dose of anti - ip-10/cxcl10, or anti - mig / cxcl9, or 100 g / dose of purified monoclonal anti - ifn or appropriate control antibodies (goat igg and rat igg) at equivalent doses for the duration of the experiment . In response to these antibodies, there was a significant reduction of the respective cytokines in vivo (figure 2). Mig / cxcl9, ip-10/cxcl10 and ifn were quantified using a modification of a double ligand method as previously described . Flow cytometric analyses for t cell and dc markers were performed on a facscan flow cytometer (becton dickinson, san jose, ca) in the university of california, los angeles, jonsson cancer center flow cytometry core facility . Cells were identified as lymphocytes or dc by gating based on forward and side scatter profiles . 10,000-gated events were collected and analyzed using cell quest software (becton dickinson). For staining, two or three fluorochromes (pe, fitc, percp) (pharmingen) were used to gate on the cd3, cd4 and cd8 t lymphocyte population or cd11c dc in single cell suspensions from tumor nodule . For cxcr3 expression, t cells statistical analyses of the data in figures 1, 2 and in table 1 were performed using the paired student t test . Abs to murine ifn, recombinant ifn were obtained from pharmingen (san diego, ca). Abs to murine mig / cxcl9, ip-10/cxcl10 and recombinant cytokine standards was purchased from r&d (minneapolis, mn). Polyclonal goat anti - murine mig / cxcl9 and anti - murine ip-10/cxcl10 specific anti - serum were produced and characterized as previously described . Anti - mouse ifn monoclonal (r4 - 462, american type culture collection, rockville, maryland, md) neutralizing antibody was purified by affinity chromatography from scid mice ascites, which was generated 34 weeks after intraperitoneal injection of 10r4 - 462 hybridoma cells per mouse . For flow cytometry, labeled antibodies (fitc, pe, percp) against the t cell surface markers to cd3, cd4, cd8 and against the dc markers, cd11c and dec 205 and the appropriate controls were purchased from pharmingen . A weakly immunogenic lung cancer, lewis lung carcinoma (3ll, h-2) obtained from atcc (crl-1642, llc) was utilized for assessment of cytokines important for slc / ccl21 mediated anti - tumor responses in vivo . The cells were routinely cultured as monolayers in 25 cmtissue culture flasks containing rpmi 1640 medium (irvine scientific, santa anna, ca) supplemented with 10% fetal bovine serum (fbs) (gemini bioproducts, calabasas, ca), penicillin (100 u / ml), streptomycin (0.1 mg / ml), 2 mm glutamine (jrh biosciences, lenexa, ks) and maintained at 37c in a humidified atmosphere containing 5% co2 in air . The cell lines were mycoplasma free and cells were utilized up to the tenth passage before thawing frozen stock cells from liquid n2 . For tumorigenesis experiments, injection in the right supra scapular area of c57bl/6 mice and tumor volumes were monitored three times each week . Five day established tumors were treated with intratumoral injection of 0.5 g of recombinant murine slc / ccl21 (pepro tech, rocky hill, nj) or murine serum albumin (utilized as an irrelevant protein for control injections) were administered three times per week for two weeks as previously described . 24 h prior to slc / ccl21 treatment, and then three times a week, mice were injected i.p . With 1 ml / dose of anti - ip-10/cxcl10, or anti - mig / cxcl9, or 100 g / dose of purified monoclonal anti - ifn or appropriate control antibodies (goat igg and rat igg) at equivalent doses for the duration of the experiment . In response to these antibodies, there was a significant reduction of the respective cytokines in vivo (figure 2). Mig / cxcl9, ip-10/cxcl10 and ifn were quantified using a modification of a double ligand method as previously described . Flow cytometric analyses for t cell and dc markers were performed on a facscan flow cytometer (becton dickinson, san jose, ca) in the university of california, los angeles, jonsson cancer center flow cytometry core facility . Cells were identified as lymphocytes or dc by gating based on forward and side scatter profiles . 10,000-gated events were collected and analyzed using cell quest software (becton dickinson). For staining, two or three fluorochromes (pe, fitc, percp) (pharmingen) were used to gate on the cd3, cd4 and cd8 t lymphocyte population or cd11c dc in single cell suspensions from tumor nodule . For cxcr3 expression, statistical analyses of the data in figures 1, 2 and in table 1 were performed using the paired student t test . Authors sh, lxz, mh, rkb and mdb contributed to the discussion of the article . Authors rms and smd together with ss conceived of the study, participated in its design and drafted the manuscript . We thank kimberly atianzar for excellent technical assistance in conducting the elisa measurements . National institutes of health grant r01 ca78654, ca85686, p50 ca90388, medical research funds from the department of veterans affairs, the research enhancement award program in cancer gene medicine and the tobacco - related disease research program of the university of california supported this work.
Neonatal bacterial meningitis continues to be the most common serious infection of the central nervous system (cns) in newborns with high morbidity and mortality despite the availability of effective bactericidal antibiotics over the last sixty years [1, 2]. This high morbidity and mortality are due to inadequate knowledge of the pathogenesis of this disease . E. coli is the most common gram - negative bacterium causing neonatal sepsis and meningitis . Bacterial meningitis in newborns is due to hematogenous spread of the pathogen to the meninges . The most important issue in the pathogenesis of e. coli meningitis is how circulating pathogens cross the blood - brain barrier (bbb), which is mainly composed of brain microvascular endothelial cells (bmecs) [3, 4]. Our previous studies showed that e. coli k1 invasion of human bmec was significantly greater with stationary - phase (sp) cultures than with exponential - phase cultures, suggesting that expression of e. coli k1 invasion - associated virulence genes is strongly regulated in a growth - phase - dependent manner . A nonsense mutation in the sp regulatory gene rpos was identified in e. coli k1 strains e44 and ihe3034 . Complementation with the wild type e. coli k12 rpos gene significantly enhanced ihe3034 invasion of bmec, but failed to improve the invasion activity of another e. coli k1 strain e44 . These studies suggest that the growth - phase - dependent invasion of bmec by ihe3034 is affected by rpos and that e44 carries a loss - of - function mutation in the rpos gene . Several virulence factors, including ibe (termed after invasion of brain endothelial cells) proteins [6, 7], ompa, yiijp, fimh, asla, and traj, have been identified in various strains of e. coli in the in vitro and in vivo models of the bbb as invasins . Most of those invasion genes are present in the e. coli k-12 genome [4, 16]. However, the ibea gene encoding a 50 kda protein has been found to be unique to some pathogenic e. coli k1 strains (e.g., c5 and rs218), while laboratory strains of e. coli k-12 (e.g., dh5 and hb101), as well as noninvasive e. coli (e.g., e412), lack ibea . Recently, vimentin has been identified as an ibea - binding protein on the surface of human bmec . Using the ibea gene as a probe, we have identified a 20.3 kb genomic locus as a genetic island of meningitic e. coli containing ibea (gima). This locus is situated between yjid and yjie, adjacent to the fim operon, and absent in nonpathogenic e. coli k12 strains . The first three operons (ptnipkc, cgldtec, gcxkrci) may be involved in energy metabolism and the last operon (iberat) contributes to e. coli k1 invasion of bmec . Our previous work showed that gima - mediated invasion of human endothelial cells is regulated by carbon source . This is consistent with the observations by others that carbon source modulates expression of virulence factors in several pathogenic bacteria . Ibea and ibet contribute to e. coli k1 invasion and adhesion [18, 19]. Our previous studies suggest that iber is a novel regulatory protein that is present in pathogenic e. coli k1 . It belongs to the ntrc / nifa family of transcriptional activators, carrying a sigma 54-interaction domain and showing significant sequence homology to various regulatory proteins for glycerol metabolism operon in citrobacter freundii (p45512), acetoacetate metabolism in e. coli k12, sigma l - dependent transcription in b. subtilis (p54529), nif - specific regulation in herbaspirillum seropedicae, dhar transcription in e. coli k12, and globe signal transduction in clostridium beijerinckii [4, 20]. However, it is unknown how iber contributes to the pathogenesis of meningitic infection by modulating the virulence of the pathogen . As e44 carries a nonsense mutation in the rpos gene and exhibits strong invasion activity in the sp, there we speculated that iber is an rpos - like regulator in sp gene expression in e44 . In order to dissect the regulation of sp gene expression in e44 that is associated with the pathogenesis of e. coli meningitis, a comparative proteomic analysis of an iber deletion mutant (br2) and its parent strain e44 our studies suggested that the iber gene was involved in regulating sp gene expression related to stress resistance and pathogenesis . The bacterial strain and plasmid vectors and their relevant characteristics are shown in table 1 . The mutant strains used in this study were derived from e44, which is a rifampin - resistant strain derived from a neonatal meningitis isolate, e. coli k1 rs218 (o18: k1: h7) [4, 21]. Sm10 (pir), dh5 (pir), and pcvd442 were used for making isogenic deletion mutants of iber and tnaa [6, 10, 11]. E. coli k12 strain mc4100 and its rpos insertion mutation tn10 mutant rh90 were used as positive and negative controls for rpos, respectively . Plasmid pstyabb, which carries the gene for monooxygenase, was used for indole assay . E. coli strains were grown at 37c in luria broth (lb; 1% tryptone, 0.5% yeast extract, 0.5% nacl) or brain heart infusion (bhi, difco laboratories, detroit, mich, usa) broth and were stored at 70c in lb plus 20% glycerol . When it was necessary, the medium was supplemented with ampicillin (100 g / ml) and rifampin (100 g / ml) for the positive selection of plasmids or bacterial strains (table 1). All genetic manipulations were done by using standard methods, as described elsewhere . Plasmid dna was extracted by using a plasmid mini kit (qiagen, calif, usa). Dna fragments were purified and were extracted from agarose gel slices, using qiaquick gel extraction kit (qiagen). Competent e. coli cells were made in 10% glycerol and were transformed by electroporation as described previously [6, 7]. To determine the role of the iber gene in the growth - phase - dependent e. coli k1 invasion of bmec, an isogenic deletion mutant of iber two pcr dna fragments, b (1.2 kb) and r (1.0 kb), flanking a 1.8-kb region to be deleted were produced from two pairs of primers (iber - s1/iber - b1 for b and iber - b2/iber - x2 for r, see table 2). The two fragments were ligated to make a 2.2 kb fragment (br) that carries an iber internal deletion . The br fragment was subcloned between sali and xbai sites on pcvd442, and the resulting recombinant plasmid was named pcbr2 . The mutants named br2 were obtained by mating e44 with sm10 pir that carries pcbr2 as described previously . We used pcr and dna sequencing to confirm the internal deletion in the iber deletion mutant br2 and the desired chromosomal gene iber of the mutant with primers iber - s1 and iber - x2 (table 2). Amplification was done by using the following cycle profile: 35 cycles at 94c for 1 minute, 58c for 1 minute, and 70c for 1.5 minutes . For the tnaa in - frame deletion briefly, 2 pcr dna fragments, ftn5 (1.0-kb) and ftn3 (1.4-kb), were made to flank a 1.3 kb region containing tnaa to be deleted, by using 2 primer pairs (tn - s1/tn - b1 for ftn5 and tn - b2/tn - x2 for ftn3, see table 2). Then the two fragments were ligated to make a 2.4 kb fragment (ftn53) that carried a tnaa internal deletion . The ftn53 fragment was subcloned into pcvd442 between sali and xbai sites to get the suicide plasmid pctna2 . To get the tnaa in - frame deletion mutant, tna44, conjugation and screening were carried out as described above . The tnaa gene deletion in the mutant tna44 was confirmed by pcr using the primers tn - s1 and tn - x2 (table 2). Human bmecs were routinely cultured in rpmi 1640 medium (mediatech, herndon, va, usa) containing 10% heat inactivated fetal bovine serum, 10% nu - serum, 2 mm glutamine, 1 mm sodium pyruvate, essential amino acids, vitamins, penicillin g (50 g / ml), and streptomycin (100 g / ml) at 37c in 5% co2 . The number of intracellular bacteria was determined on blood agar plates after the extracellular bacteria were killed by incubation of the monolayers with experimental medium containing gentamicin (100 g / ml) for 1 hour . Results were expressed as percent invasion (100 (number of intracellular bacteria recovered)/(number of bacteria inoculated)). Briefly, e. coli strain e44 and the iber deletion mutant br2 were cultured in bhi medium overnight without agitation . The bacterial cells were harvested at the stationary - phase (od = 2.53.0) by centrifugation at 6000 g for 10 minutes . Denaturing protein extraction (phenol extraction procedure) was performed according to saravanan and rose . The lyophilized pellets were dissolved in rehydration buffer (7 m urea, 2 m thiourea, 4% chaps, 0.2% ph 310 biolytes, 1% dtt; 1 mg dry pellets for 200 l buffer) and shaken on vortex for 1 hour at room temperature . The first and second dimensions of the page were performed at least in triplicate (to reduce the likelihood of differences based solely upon gel - to - gel variability) according to the standard protocols developed and defined by bio - rad . Solubilized total e. coli protein samples (200 l each) were loaded on 11 cm immobilized ph gradient (ipg) strips (ph 47). Rehydration / loading was done passively (no voltage) for 1 hour, followed immediately by 14 hours of active rehydration (50 v) at 20c . Isoelectric focusing of the ipg strips was performed at 20c using a 50 a current limit per strip to prevent damage to the strip and the instrument . Electrophoresis was carried out as follows: step 1, 250 v for 20 minutes; step 2, a rapid ramp to 8000 v; step 3, focusing at 8000 v for 55 000 vhr; step 4, hold at 400 v. due to latent ionic components in the sample the actual running voltage was only approximately 6500 v. after the first dimensional run was completed, the ipg strips were equilibrated in buffer i (6 m urea, 0.375 m tris - hcl ph 8.8, 2% sds, 20% glycerol, and 2%dtt) for 15 minutes and then in buffer ii (6 m urea, 0.375 m tris - hcl ph 8.8, 2% sds, 20% glycerol, and 2%dtt) for additional 15 minutes . The second dimension sds - page was performed with 15% resolving gels and 5% stacking gels (160 180 0.5 mm). The 2de gels were scanned at a 200 bpi resolution with typhoon scanner (amersham biosciences, nj, usa), and analyzed with imagemaster 2d platinum version 6.0 (ge healthcare bio - science, nj, usa). Only those protein spots having differences in density of 1.5-fold or greater between the groups were chosen . Moreover, all protein spots selected for analysis were shown to have significant difference in protein density (mean sem, p <.01) by software of spss 10.0 . Protein bands were excised from preparative coomassie blue - stained gels and washed several times with destaining solutions (25 mm nh4hco3 for 15 minutes and then with 50% acetonitrile containing 25 mm nh4hco3 for 15 minutes). Gel pieces were then dehydrated with 100% acetonitrile, dried, and then incubated with a reducing solution (25 mm nh4hco3 containing 10 mm dithiothreitol) for 1 hour at 56c and subsequently with an alkylating solution (25 mm nh4hco3 containing 55 mm iodoacetamide) for 45 minutes at room temperature . After reduction and alkylation, gels were washed several times with the destaining solutions and finally with pure water for 15 minutes before being treated again with 100% acetonitrile . Depending on the protein content, 2 - 3 l of 0.1 g/l modified trypsin (promega, wiss, usa, sequencing grade) in 25 mm nh4hco3 was added over the gel spots and incubated for 30 minutes . About 710 l of 25 mm nh4hco3 was then added to cover the gel spots and incubated at 37c overnight . The in - gel digestion products were extracted with formic acid / acetonitrile solutions followed by evaporation . Samples were desalted using mziptip c18 pipette tips (millipore, mass, usa) before ms / ms analysis . The sample was resuspended in 10 l of 0.1% formic acid, injected via an autosampler (surveyor, thermofinnigan, calif, usa) and subjected to reverse phase liquid chromatography using thermofinnigan surveyor ms - pump in conjunction with a biobasic 18 100 0.18 mm reverse - phase capillary column (thermofinnigan, calif, usa). Mass analysis was done using a thermofinnigan lcq deca xp plus ion trap mass spectrometer equipped with a nanospray ion source (thermofinnigan, calif, usa) employing a 4 cm metal emitter (proxeon, odense, denmark). Spray voltage of the mass spectrometer was set to 2.9 kv and capillary temperature was set at 190c . The column equilibrated for 5 minutes at 1.5 l / min with 95% solution a and 5% solution b (a, 0.1% formic acid in water; b, 0.1% formic acid in acetonitrile) followed by a linear gradient was initiated 5 minutes after sample injection ramping to 65% solution a over 45 minutes . Solution a was increased to 80% over the subsequent 5 minutes and held at 80% for 5 minutes, after which the column was reequilibrated back to 5% solution a (aqueous). A data - dependent acquisition mode was used where each of the top five ions for a given scan was subjected to ms / ms analysis . The protein identification was conducted with the ms / ms search software mascot 1.9 with confirmatory or complementary analyses using turbosequest as implemented in the bioworks 3.2 . E. coli genome sequences at the national center for biotechnology information (ncbi) were used as the primary search databases and searches were complemented with the ncbi nonredundant protein database . For determination of indole production, we followed the method of indole conversion into indigo as described previously [12, 27], with minor modifications . All strains e44, br2, tna44, mg1655, and rh90 were transformed with the pstyabb plasmid, which constitutively expresses the styab protein converting indole to indigo . The bacteria were incubated in m9 medium containing 0.4% glucose and 100 g / ml ampicillin overnight with shaking . Then the bacteria were collected by centrifugation and resuspended to od600 = 0.2 in bhi medium supplied with 100 g / ml ampicillin, and incubated at 37c without shaking . To determine the indigo formation at different time points, the samples were read at 600 nm to determine the indigo concentration by comparison to a standard curve . Bacteria were grown in bhi broth at 37c overnight without shaking, and collected by centrifugation . The number of cells was measured on the basis of their od at 600 nm . Bacteria were suspended and diluted to 10 cells / ml in pbs for the following assays . For heat shock, 100 l of bacteria was heated at 54c for 3 minutes . For alkali endurance, the bacterial suspension was mixed with equal volume of tris buffer (1 m, ph = 10.0) and 8 volumes of water (final concentration, 100 mm, ph 10.0) and incubated at 37c for 30 minutes . For acid endurance, 1/10 volume of the bacterial suspension was mixed with lb containing acetic acid (final concentration, 90 mm, ph 2.8) and incubated at 37c for 20 minutes . For high osmolarity challenge, bacteria were mixed with an equal volume of 4.8 m nacl (final concentration, 2.4 m) and incubated at 37c for 1 hour . For oxidative stress, bacteria were harvested and resuspended in an equal volume of pbs containing 10 m h2o2 incubated at 37c for 30 minutes . After exposure to these stresses, bacteria were diluted in 0.9% saline and plated in duplicate on lb agar plates . The surviving rate with stress was calculated from the ratio of the bacterial number under stress condition to the bacteria number under nonstress condition . The surviving rate without stress was calculated from the bacteria number grown on plates . The gene iber in e. coli k1 e44 is predicted as the only regulatory protein present in the iberat operon in gima by bioinformatics approaches . To determine the role of iber gene in the growth - phase - dependent invasion of bmec by meningitic e. coli k1, an isogenic in - frame deletion mutant of iber was made by chromosomal gene replacement with the recombinant suicide plasmid pcbr2 carrying a 2.2 kb dna fragment with iber internal deletion (figure 1(a)). The 2.2 kb dna fragment was generated by ligation of two pcr amplicons (1.2 and 1.0 kbs) flanking the 1.8 kb iber coding region . The iber deletion mutant was obtained by mating e44 with sm10 pir carrying pcbr2 . The mutant colony morphology on lb agar plates and growth rate in lb broth were the same as the parent strain e44 . The deletion of iber was confirmed by colony pcr and dna sequencing (figure 1(b)). In order to examine the virulence phenotype of the iber deletion mutant, a comparative study of the invasiveness of e44 (parent strain), br2, and the complemented br2 was carried out . As shown in figure 1(c), the relative invasion rate of br2 was significantly reduced as compared to that of e44 and the plasmid pwks1030 carrying the iber gene was able to complement the noninvasive phenotype of br2, suggesting that the iber gene contributes to the e. coli e44 invasion process . To determine the role of iber in regulating sp gene expressionof meningitic e. coli k1, the wild type e. coli e44 and its iber deletion mutant br2 were cultured in bhi broth overnight . The total proteins of each strain were extracted from the cells as described in section 2 . The experiment was performed three times with two sets of independently grown cultures . Only spots showing the same pattern in three independent runs were retained and quantified using the software imagemaster 2d platinum version 6.0 . Figures 2(a) and 2(b) showed the protein patterns of e44 and br2, respectively . All the upregulated spots and downregulated spots satisfying the criteria as mentioned above were marked on both the 2d maps . They were excised and identified by lc - ms / ms (table 3). Eight protein spots were found to be differentially expressed in br2 as compared to its parent strain e44 . Among them, 4 protein spots were significantly upregulated in br2 including elongation factor ef - tu (tufb, spot u1), glyceraldehyde-3-phosphate dehydrogenase a (gapa, spot u2), outer membrane protein 3a (ompa, spot u3), and alkyl hydroperoxide reductase (ahpc, spot u4), while 4 protein spots were of decreased abundance in br2, including dihydrolipoamide dehydrogenase (lpda, spot d1), tryptophanase (tnaa, spot d2), and two isoforms of outer membrane protein c (ompc, spot d3, and d4). Figure 3(a) showed the enlargements of each changed protein marked with black arrows and spot numbers . The relative ratios of each downregulated protein and upregulated protein were shown in figures 3(b) and 3(c). We classified the proteins into two main categories on the basis of their roles in the sp growth of e. coli cells: (a) response to environmental modifications (including tnaa, tufb, ompc, ompa, and ahpc) and (b) central metabolism (including lpda and gapa). Since iber was hypothesized as an sp - regulator contributing to the growth regulation and virulence of e44, its role in the invasion process and resistance to stress conditions should be further characterized . Tnaa, a tryptophanase, degrades tryptophan, resulting in the formation of indole, which has been proposed to act as an extracellular signal in stationary phase cells of e. coli [28, 29]. Production of indole, via the enzymatic activity of tnaa, is also induced during biofilm formation . Tnaa, which was controlled by rpos in other e. coli strains, is one of the most important transcriptional regulators for the gene expressions in sp cells . Tufb (ef - tu) is responsible for binding and transporting the appropriate codon - specified aminoacyl - trna to the aminoacyl (a) site of the ribosome [32, 33]. In addition to its function in translation elongation, elongation factor tu is implicated in protein folding and protection from stress like a chaperone molecule . Ompc, as well as ompf, is a porin protein present on the outer membrane of e. coli, responding to the osmotic challenge . Ompr, as a regulator, activates transcription of ompf and ompc, and changes the ratio of these two, so that the total level of porin proteins remains approximately constant [36, 37]. Ompf, which produces slightly wider pores (1.2 nm) than does ompc, predominates at low osmotic strength, whereas ompc (1.1 nm) predominates at high osmotic strength . In e. coli, the expression of ompc it has been proposed that the smaller pores formed by ompc could reduce the diffusion of larger hydrophobic and negatively charged molecules when bacteria encounter high osmolarity conditions as in the host compartments . Presumably, this protein is very important for the stress resistance of e. coli in the stationary phase . In this study, the downregulation of ompc resulting from the iber deletion might decrease resistance to high osmolarity . In addition, it had been reported that ompc is involved in invasion of epithelial cells by crohn's disease - associated e. coli strain lf82 and shigella flexneri [39, 40], suggesting that ompc might be involved in e44 invasion of the host tissue barriers . Ompa is a major protein in the outer membrane of both pathogenic and nonpathogenic e. coli . As shown in our previous study, the ompa - deletion mutant of e44 was significantly more sensitive than that of its parent strain to sds, cholate, acidic environment, and high osmolarity . Ompa is downregulated upon entry into sp by sigmae, which plays a central role in maintaining cell envelope integrity both under stress conditions and during normal growth [42, 43]. We demonstrated here that ompa was upregulated in the iber mutant br2 (figure 3), suggesting that ompa expression is suppressed upon entry into sp by iber in a manner similar to sigmae . Alkyl hydroperoxidase (ahpc) functions as a primary scavenger of endogenous h2o2 at a low (10 m) concentration . All of ahpc, katg, and kate genes are known to participate in the antioxidant defense mechanism against h2o2-induced stress in e. coli . It has been reported that sp - inducible rpos regulates kate gene expression and oxyr regulates ahpc and katg genes [45, 46]. Our previous study has demonstrated that e. coli k1 rs218 had a nonsense mutation in its rpos gene, resulting in a negligible kate activity, but no obvious difference in katg . In this study, the increase in ahpc expression indicated that the iber deletion led to an increased oxidative stress in sp compared with the wild type strain, suggesting that iber is involved in the resistance to oxidative stress upon entry into sp . Lipoamide dehydrogenase (lpda), which is the same as dihydrolipoamide dehydrogenase (dldh), makes up the e3 component of pyruvate dehydrogenase complex, 2-oxo glutarate dehydrogenase, and branched - chain 2-oxo acid dehydrogenase complexes . Dldh has been identified as virulence factors contributing to the pathogenesis of bacterial infections caused by mycobacterium tuberculosis and streptococcus pneumoniae because it enhances their survival within the host cells [47, 48]. As shown in figure 3, lpda was downregulated in the iber deletion mutant br2, suggesting that this enzyme might be involved in the virulence of meningitic e. coli k1 via enhancing the pathogen survival within the host . Recently, gapa in e. coli has been identified as one of a few proteins, which harbors functionally important thiol groups against oxidative stress . As gapa is upregulated in br2 (figure 3), iber may be involved in the negative control of gapa in sp . In summary, all these proteins contribute to growth - related carbon source metabolism or stress resistance . They are associated with the sp regulation . Among these proteins, tnaa is the most important one as it produces the signal molecule indole and is regulated by rpos . Since e44 carries a loss - of - function mutation in its rpos gene, there should be alternative signaling pathway(s) to complement the functional deficiency of rpos in this pathogenic e. coli strain . Our proteomic analyses showed that the tnaa expression was significantly affected by iber, which might be functionally equivalent to rpos . Therefore, our focus for further studies was placed on how tnaa is regulated by iber . To test our hypothesis that iber is an rpos - like regulator, the tnaa in - frame deletion mutant tna44 was generated with the same gene replacement approach that was used for the iber deletion mutant . Tna44 was obtained by mating e44 with sm10 pir carrying the recombinant suicide plasmid pctna2 which contains the truncated tnaa gene . Although overall growth rates did not differ between the mutants (br2 and tna44) and their parent strain e44 (figure 4(a)), the invasive capability of tna44 (19%) and br2 (35%) was significantly reduced as compared to that of e44 (100%) (figures 4(c)-4(d)). These data suggest that tnaa is an important downstream regulator that is required foriber - modulated e. coli k1 invasion . As our proteomics analysis had shown that tnaa expression was induced by iber in sp and the tnaa and iber deletion resulted in a deficiency in indole production in sp - cultures (figure 4(b)), we further tested whether the tnaa product indole, as an sp extracellular signal molecule, played a role in the process of invasion . Indole is converted to indigo (which is not further degraded in e. coli) by several monooxygenases, thus providing an easy method for its determination . A plasmid pstyabb, carrying the gene for styrene monooxygenase, was used to monitor the indole production through its conversion to indigo . The plasmid was transformed into e. coli strains e44, br2, and tna44, and the indole production was measured at different time points for these stains in bhi media (figure 4(b)). By contrast, the indole production was almost abolished in the tnaa deletion mutant and severely reduced in the iber deletion mutant (figure 4(b)). These results demonstrated that the indole production was controlled by iber through tnaa . To examine whether indole could compliment the noninvasive phenotype of tnaa and iber deletion mutants, indole was supplied in the bhi medium at 100 m to the tna44 and br2 sp cultures . The result showed that indole was able to significantly enhance the relative invasion rate of tna44 (19% to 69%) and br2 (35% to 65%) as compared to that of e44, suggesting that indole could partly compliment the noninvasive phenotype of tna44 (figure 4(c)) and br2 (figure 4(d)). Lacour and landini have shown that the rpos gene in e. coli k12 strain mg1655 controls the production of indole, which acts as a signal molecule in sp cells, via regulation of tnaa, the indole - producing enzyme . As tnaa is regulated by iber in e44, it is most likely that iber is a novel regulator to complement the functional deficiency of rpos in e44 . It has been reported that rpos is able to positively and negatively control expression of a large set of genes when bacteria enter into the sp [46, 50, 51]. During such transition, bacteria undergo physiological changes that allow their sp organisms to survive better in such insults as heat, high - osmotic environment, starvation, uv radiation, h2o2, and acid than their exponential counterpart [50, 52]. The loss of rpos resulted in the decrease of stress resistance and cell survival in the sp [5, 53]. Although our study showed that the loss of iber did not affect the growth rate in bhi medium, the survival rates of the iber deletion mutant br2 in the sp significantly decreased as compared to that of the wild type strain e44 even without any stress treatment (figure 5(a)). Our proteomics analysis also revealed that the most significant proteomic changes in the iber deletion mutant were related to bacterial response to environmental modifications . For example, ahpc, as a primary scavenger of endogenous h2o2, was upregulated in the iber deletion mutant, implying that the loss of iber resulted in the decreased survival rates of bacterial cells under an oxidative stress in the sp . Ompc, as a porin protein, was downregulated in br2, perhaps resulting in the decreased resistance to osmolarity stress . These results suggested that iber plays a regulatory role in response to stress conditions in e44 that carries a nonsense mutation in rpos . To examine the function of iber in response to stress environments, we performed several survival assays under different stress conditions, including heat shock (54c for 3 minutes), alkali endurance (tris, ph = 10 for 30 minutes), acid endurance (acetic acid, ph = 2.8 for 20 minutes), high osmolarity challenge (2.5 m nacl for 1 hour), and oxidative stress (10 m h2o2 for 30 minutes). In all the survival experiments, the wild type strain e44 showed higher survival rates than the iber deletion mutant, indicating that the iber gene is required for all these stress resistances (figure 5(b)). Especially in the heat shock assay, the loss of iber resulted in over 95% cells death, indicating that iber played a vital role in temperature sensitivity in this strain . In the other stress treatments, the iber deletion also significantly reduced the survival rates (more than 60% cell death) of br2 as compared to that of the wild type strain e44, suggesting that iber had a global regulatory role in the resistance to acid, alkali, high osmolarity and oxidative stress . In the survival assays for the e. coli control strain mg1655, the rpos deletion mutant rh90 also decreased the survival levels in these five stress conditions, showing the similar patterns like iber in response to stress environments (data not shown). Combining the proteomics analysis and the stress survival studies, we conclude that iber is an rpos - like regulator to control gene expression of proteins that are critical for stress - resistance and cell survival in the sp in e44, which carries a loss - of - function mutation in the rpos gene . Currently, most e. coli meningitis studies are done with sp cultures in which the pathogen invasion of human bmec is significantly greater than the log phase cultures . In most strains of e. coli, rpos plays a central role in regulating the sp regulatory genes for protecting cells against starvation and stress damage . Rpos, the major sp regulator, has also been shown to regulate the expression of microbial virulence genes in various bacteria including e. coli k1 (o157: h7), salmonella typhimurium, shigella flexneri, yersinia enterocolitica, vibrio cholera, and borrelia burgdorfer, [5, 53, 54]. Surprisingly, however, rpos was found to be inactive in meningitic strain e44 . The current proteomic studies may provide an answer to the long - standing question regarding the sp gene regulation in e44 . Combining the proteomics analysis, virulence determination, and the stress survival studies of the iber mutant br2, we have demonstrated for the first time that iber serves as an rpos - like regulator to control gene expression that is critical for stress - resistance and cell survival in the sp of e44 . Iber is not a structural homologue of rpos as iber and rpos do not share any significant sequence homology . Rpos (also known as,, or katf) is a global regulator in e. coli, which is the second principal subunit after the major factor . In e44, however, iber appears not to be a master regulator on the basis of its genomic prevalence and functional spectrum . The prevalence of the gimalocus carrying iber is highly dependent on the origin of the strain and on the subgroup it belongs to (a, b1, b2, and d) (4). In all the studies, where the presence of this locushas been analyzed, gima was found to be restricted to the b2 subgroup, a subgroup that includes strains with the highest virulence in mice and the highest level of virulence determinants (4). Our proteomic studies showed that a limited number of genes were regulated by iber, suggesting that iber is a regulator with a narrow functional spectrum . In other e. coli strains, either pathogenic or probiotic strains, functional heterogeneity of rpos in stress tolerance was widely observed [5, 53, 55]. Those studies have shown that some e. coli strains can maintain their stress tolerance capability or significantly modulate their stress resistance phenotype independent of their rpos genotypes . Such adaptation processes compromising the rpos - dependent stress responses may have significant impact on bacterial survival in environments, as well as in the host's stomach and intestine [53, 55]. Iber, a regulator in the gima regulon, may contribute to bacterial virulence adaptation process in e44 to complement the functional deficiency of rpos . Another significant finding of our proteomic studies is that iber in meningitic strain e44 is able to upregulate tnaa, which is controlled by rpos in other e. coli strains . The virulence determination of the tnaa mutant showed that tnaa and its product indole were required for e44 invasion of human bmec . The generation of indole, via the tryptophanase activity of tnaa, was also observed during the formation of biofilm in e. coli and other bacteria [28, 56]. In addition to the initiation of indole - mediated signaling, tnaa (tryptophanase) is able to catabolize tryptophan, cysteine, and serine to pyruvate [29, 56]. The three proteins significantly upregulated by iber are tnaa, lpda, and ompc, all of which are directly or indirectly involved in pyruvate metabolism . Ompc, an osmotically regulated porin, may facilitate nutrient uptake . On the other hand, the three operons (ptnipkc, cgldtec, and gcxkrci) in the gima regulon may also directly or indirectly contribute to pyruvate metabolism by converting dihydroxyacetone, glycerol, and glycerate to pyruvate . It has been shown that the capability to catabolize carbon source is an important parameter in the ability to persist and compete in stationary phase . This raises the possibility that signaling by indole, which is regulated by iber via tnaa, may play a critical role in the pathways that prepare the pathogens for a nutrient - poor environment (e.g., cns) when the carbon source becomes limited for energy production.
Today, education has been assigned as an industry for human resource development for different walks of life . The students constitute the heart of the education system and they are the most affected ones by the strengths and weaknesses of the education system . The students form an identical subgroup compared to others in the society . Some of the most common risks faced by a student during his / her college days are psychological, emotional, social, academic, and career risks . Stress is a very familiar condition faced by the students when they are unable to bear the risks involved in higher education . The main sources of stress are academic and time concerns, fear of failure, classroom interactions, and economic issues . Apart from this the parents have unlimited expectation from their children and therefore they impose their own desires on them . The impact of these influences results in a number of students reporting emotional problems, anxiety, stress, and other neurotic problems . They are often found in frightening, abusive, depressing, threatening, competitive, unpredictable, and confusing situations . The percentage of suicides is more among the college students compared to the other ones and these suicides majority of the college students strive and try to handle the stress in a positive way . Whereas, some of the students do not know, the ways to cope up with their problems and; therefore, adopt unhealthy ways . The coping could be at an emotional, cognitive, or social support seeking level . The emotional responsiveness grows out of the simple reflexes of planful emotional responsive behaviour, by the age of two or three years . Kopp (1989) described five types of behaviour: visual or physical avoidance, distraction, self - soothing, problem - oriented, and care - eliciting . A coping style can be effective at one age but may be totally unsuitable at another age . In the similar way, a particular coping style may be totally appropriate in one situation while inappropriate in another . This may be determined to a large part of cultural expectations on the other hand, individual vulnerabilities set limits to ones coping . Therefore, it is mandatory to understand that how coping ways adopted by college students differ from other students who experience the significant stress; their perception of handling the psycho - social problems effectively, might help in the planning of intervention services . Thus, the present study was made to understand the ways in which college students differ in coping with significant stress experienced by the students and to examine the different ways of coping strategies adopted by them . The present study examines the coping patterns adopted by the junior college students with respect to stress in the academic area, and to study the gender difference in coping patterns among junior college students . The universe considered for the study was, i puc and ii puc students, who were studying either in bachelor of arts, science, or commerce . In the investigation process, a coping checklist with 70 diverse coping pattern, prepared by rao et al ., was used . Yesno format and they described a broad range of behavioural, emotional, and cognitive response that may be used to handle stress; and a socio - demographic data sheet was also used . In order to compute the difference between the means of two groups (male and female), frequency, percentage, and t - test were used; and to compare the frequencies of certain variables in the two groups, chi - square was used . The study was conducted on i puc and ii puc college students . With regard to the socio - demographic characteristics of the college students, it was observed that majority of the students were aged between 1718 years, and number of females was slightly more than males . With respect to the religion, a majority of the students were hindu, and the least were islamic, compared to the students belonging to other religion; because most of the people from islam do not tend to pursue higher education . With regard to birth order of the child, majority of the students were middle born . It was also noted that, a majority of the respondents spoke other languages at home than kannada . This proves that respondents families belong to various states, but they have settled here . It was seen that most of the students were from nuclear families, which is a very common trend in urban areas . With respect to education background, a majority of the students were from well - educated families, with their fathers being postgraduate and mothers being educated up to higher secondary . The studies also revealed that, the fathers of the respondents were businessmen, whereas mothers were housewives . However, approximately one - third of the respondents mothers were employed . The coping strategies adopted by college students with regard to stressful events, i.e., academic area were assessed . The results revealed that most of the students (approximately 90%) used appraisal - focused coping strategies such as, going over the problem again and again, trying to understand it, to make the situation light, and refuse to get too serious about it . Also, majority of the college students reported that they frequently adopt problem - solving coping strategies such as, knowing what has to be done, so doubling the efforts and trying harder to make things work, analyzing the problem bit by bit, seeking reassurance and emotional support from family members, and coming up with different solutions to the problem . In the current study, it has been observed that the students used a combination of appraisal - focused and problem - focused coping strategies . Whereas, study by lazarus revealed that, problem - focused and emotion - focused coping strategies were used in virtually every stressful encounter . In the present study, it was found that the students used appraisal - focused coping strategies such as, analyzing the situation, cognitively redefining, and cognitively avoiding the situation . This proves that the students in adolescence stage were still confused and were not able to choose those coping strategies which were of positive approach to encounter any stress . The present study revealed that the students resorted to the coping strategies such as seeking sexual comfort (24%), taking analgesics or minor tranquilizers without medical advice (15%), to make themselves feel better by having a drink or two (10%), and to make themselves feel better by taking mood elevating drugs (10%). These findings were supported by the study by o. languhlin (1984), who also reported that resorting to drugs, alcohol, tranquilizers, and sex are more characteristics of inefficient coopers . The findings by mccormack (1996), stated that 23% to 36% of the students advocate drinking when under stress; however, even a small percentage of students using this coping strategy may form a high risk group . In the current study, the sample comprised college students belonging to 1 puc and ii puc, who were still in adolescence stage and these students were not fully exposed to the evils of the society . In the study, distribution of the coping strategies adopted by the college students was examined with respect to their gender background . The results also revealed that the students irrespective of their gender, used combination of problem - focused and emotion - focused coping strategies . It was found that, out of 70 items, 16 significant responses were used by the students . The coping strategies adopted by female students were, talking to a friend who can do something about the problem, analyzing the problem bit by bit, consoling themselves that things are not that bad and could have been worse, visit places of worship, go on a pilgrimage; while male students resorted to coping strategies such as, seeking reassurance and emotional support from family members, engage in vigorous physical exercises, talking to a friend who can do something about the problem, and taking a big chance or doing something very risky . It was observed that the female students used emotion - focused coping strategies and sought more social support than males . These findings were supported by the study done by frydenberg and lewis, (1991) and reported that females sought more social support and employed strategies related to, hoping for the best and wishful thinking, than males . Also, hobfall, et al ., (1994) reported that when faced with problems of professional and interpersonal situations, support seeking was employed more by women than men . The findings by mike and thoits (1993) suggested that men experience a stoic style of responding to stressors and women have an emotional expressive style . The distribution of coping strategies adopted by college students was examined with respect to different faculties, such as arts, science, and commerce . It was observed that the students used a combination of appraisal - focused and problem - focused coping strategies . Out of 70 responses, the frequently used coping strategies by the students belonging to different faculties were problem - focused, for example, pray to god,and knowing what has to be done, so doubling the efforts and trying harder to make things work, were displayed by more than 90% of the college students . Majority (90%) of the students belonging to science and arts faculties and 75% from commerce, used appraisal - focused coping strategies such as, console yourself that the things are not at all that bad, it could be worse, make light of the situation, refused to get too serious about it, refusing to believe that it happened . Also, 43% of the students from commerce and science faculties used emotion - focused coping strategies, whereas 35% from arts faculty used emotional - focused coping strategies, such as, preparing themselves for the worst to come, feeling that other people are responsible for what has happened, and trying to feel better by eating / nibbling . Overall, the total coping repertoires of students belonging to arts, science, and commerce faculties were 37, 38, and 35, respectively . With respect to gender, the total coping repertoire for male and female students, were 36 and 37, respectively . Murthy (1995) found in her study of working women that they have a coping repertoire of approximately 21 . Karen (2000) reported that the total coping repertoire of unsuccessful college students was 37 . The study was conducted on i puc and ii puc college students . With regard to the socio - demographic characteristics of the college students, it was observed that majority of the students were aged between 1718 years, and number of females was slightly more than males . With respect to the religion, a majority of the students were hindu, and the least were islamic, compared to the students belonging to other religion; because most of the people from islam do not tend to pursue higher education . With regard to birth order of the child, majority of the students were middle born . It was also noted that, a majority of the respondents spoke other languages at home than kannada . This proves that respondents families belong to various states, but they have settled here . It was seen that most of the students were from nuclear families, which is a very common trend in urban areas . With respect to education background, a majority of the students were from well - educated families, with their fathers being postgraduate and mothers being educated up to higher secondary . The studies also revealed that, the fathers of the respondents were businessmen, whereas mothers were housewives . However, approximately one - third of the respondents mothers were employed . The coping strategies adopted by college students with regard to stressful events, i.e., academic area were assessed . The results revealed that most of the students (approximately 90%) used appraisal - focused coping strategies such as, going over the problem again and again, trying to understand it, to make the situation light, and refuse to get too serious about it . Also, majority of the college students reported that they frequently adopt problem - solving coping strategies such as, knowing what has to be done, so doubling the efforts and trying harder to make things work, analyzing the problem bit by bit, seeking reassurance and emotional support from family members, and coming up with different solutions to the problem . In the current study, it has been observed that the students used a combination of appraisal - focused and problem - focused coping strategies . Whereas, study by lazarus revealed that, problem - focused and emotion - focused coping strategies were used in virtually every stressful encounter . In the present study, it was found that the students used appraisal - focused coping strategies such as, analyzing the situation, cognitively redefining, and cognitively avoiding the situation . This proves that the students in adolescence stage were still confused and were not able to choose those coping strategies which were of positive approach to encounter any stress . The present study revealed that the students resorted to the coping strategies such as seeking sexual comfort (24%), taking analgesics or minor tranquilizers without medical advice (15%), to make themselves feel better by having a drink or two (10%), and to make themselves feel better by taking mood elevating drugs (10%). These findings were supported by the study by o. languhlin (1984), who also reported that resorting to drugs, alcohol, tranquilizers, and sex are more characteristics of inefficient coopers . The findings by mccormack (1996), stated that 23% to 36% of the students advocate drinking when under stress; however, even a small percentage of students using this coping strategy may form a high risk group . In the current study, the sample comprised college students belonging to 1 puc and ii puc, who were still in adolescence stage and these students were not fully exposed to the evils of the society . In the study, distribution of the coping strategies adopted by the college students was examined with respect to their gender background . The results also revealed that the students irrespective of their gender, used combination of problem - focused and emotion - focused coping strategies . It was found that, out of 70 items, 16 significant responses were used by the students . The coping strategies adopted by female students were, talking to a friend who can do something about the problem, analyzing the problem bit by bit, consoling themselves that things are not that bad and could have been worse, visit places of worship, go on a pilgrimage; while male students resorted to coping strategies such as, seeking reassurance and emotional support from family members, engage in vigorous physical exercises, talking to a friend who can do something about the problem, and taking a big chance or doing something very risky . It was observed that the female students used emotion - focused coping strategies and sought more social support than males . These findings were supported by the study done by frydenberg and lewis, (1991) and reported that females sought more social support and employed strategies related to, hoping for the best and wishful thinking, than males . Also, hobfall, et al ., (1994) reported that when faced with problems of professional and interpersonal situations, support seeking was employed more by women than men . The findings by mike and thoits (1993) suggested that men experience a stoic style of responding to stressors and women have an emotional expressive style . The distribution of coping strategies adopted by college students was examined with respect to different faculties, such as arts, science, and commerce . It was observed that the students used a combination of appraisal - focused and problem - focused coping strategies . Out of 70 responses, the frequently used coping strategies by the students belonging to different faculties were problem - focused, for example, pray to god,and knowing what has to be done, so doubling the efforts and trying harder to make things work, were displayed by more than 90% of the college students . Majority (90%) of the students belonging to science and arts faculties and 75% from commerce, used appraisal - focused coping strategies such as, console yourself that the things are not at all that bad, it could be worse, make light of the situation, refused to get too serious about it, refusing to believe that it happened . Also, 43% of the students from commerce and science faculties used emotion - focused coping strategies, whereas 35% from arts faculty used emotional - focused coping strategies, such as, preparing themselves for the worst to come, feeling that other people are responsible for what has happened, and trying to feel better by eating / nibbling . Overall, the total coping repertoires of students belonging to arts, science, and commerce faculties were 37, 38, and 35, respectively . With respect to gender, the total coping repertoire for male and female students, were 36 and 37, respectively . Murthy (1995) found in her study of working women that they have a coping repertoire of approximately 21 . Karen (2000) reported that the total coping repertoire of unsuccessful college students was 37.
Dermatomyositis (dm) is an uncommon idiopathic inflammatory myopathy that primarily affects skeletal muscle and skin with well - characterized cutaneous findings . It has been well documented that dm carries an increased risk of malignancy and can present as a paraneoplastic syndrome to multiple types of underlying malignancies [3, 4]. However, 1530% of cases of dm are manifestations of paraneoplastic syndromes of an underlying malignancy . Here, we present a case of concurrent dm and breast cancer to highlight a rare presentation, progression and regression of symptoms, as well as key components of diagnosis and treatment . The patient is a 47-year - old polish premenopausal female who presented with complaints of a non - tender palpable left breast mass for 4 months at an outside institution . She underwent a diagnostic mammogram and ultrasound that reported a 2-cm irregular, hypoechoic mass at 12 o'clock . There was a 2.2-cm enhancing mass in the left breast at 12:00 that was consistent with the patient's biopsy - proven malignancy (fig ., she underwent a core needle biopsy, which revealed a poorly differentiated triple - negative invasive ductal carcinoma (fig . There is a 1.9 1.4 cm irregular mass in the left breast at 1112:00 6 cm from the nipple . There was a 2.2 cm enhancing mass in the left breast at 12:00 that was consistent with the patient's biopsy - proven malignancy . High power view of the left breast core biopsy (b). Left breast ultrasound . There is a 1.9 1.4 cm irregular mass in the left breast at 1112:00 6 cm from the nipple . The circled area revealed a 2-cm irregular mass with biopsy clip noted . Left breast on mri . There was a 2.2 cm enhancing mass in the left breast at 12:00 that was consistent with the patient's biopsy - proven malignancy . Two weeks after the biopsy, she complained of new onset pruritic, erythematous rash on her anterior chest, face and back of the hands (fig . She was complaining of diffuse myalgias, worse during menstruation . Owing to her worsening symptoms which included cuticular hypertrophy of the hands with associated erythema over the metacarpophalangeal and proximal interphalangeal joints bilaterally (fig . 5c), she was transferred to the bellevue hospital breast clinic and referred to the rheumatology clinic for management of her symptoms . Initial laboratories revealed moderately elevated laboratory values (table 1). At this time, she was presumptively diagnosed with paraneoplastic dm secondary to invasive ductal carcinoma of the left breast and treated with 20 mg prednisone daily . Her persistent and slowly progressing symptoms despite aggressive treatments prompted our decision to proceed with surgical intervention in hopes of preventing progression of her musculoskeletal manifestations . Postoperatively, she experienced rapid improvement in both cutaneous and musculoskeletal manifestations with visible clearing of her rash and return of her strength . She was maintained on prednisone with excellent symptomatic control . On her 6-month visit, we noted resolution of most of her rash, periorbital edema as well as myalgias and muscle weakness (fig . Table 1laboratory values prior to the surgery, 3 weeks postoperatively and 6 weeks postoperatively.preop3 weeks postop6 weeks postopwhite blood cell (10)9.312.67.8creatine kinase (u / l)2183302160aldolase (u / l)17.45.3myoglobin247ast (u / l)1177744alt (u / l)539130 figure 5:image of patient with facial rash and periorbital edema that initially developed during her workup (a). Images of the worsening rash on her chest and hands that initially developed during her workup (b and c). Image of patient with facial rash and periorbital edema that initially developed during her workup (a). Images of the worsening rash on her chest and hands that initially developed during her workup (b and c). Dm typically presents with progressive, symmetrical, proximal muscle weakness and characteristic skin lesions such as helitrope rash, gottron's papules, gottron's sign, the v - sign and shawl sign . Additional cutaneous manifestations that have become more commonly recognized include vasculopathic changes (i.e. Telangiectasias and livedo reticularis), cuticular overgrowth (mechanic's hands) and poikiloderma . Initial presentation follows a bimodal distribution between ages 515 and 4564 and tends to progress over a 3- to 6-month period before the patient will seek medical attention . Contrary to this typical presentation, the above patient experienced rapid progression of symptoms over a much shorter period and predominately reported skin manifestations with subsequent rapid development of muscular deterioration . The diagnosis and classification of dm commonly follows the criteria set forth by bohan and peter in 1975 (table 2) [1, 2]. Initial evaluation for suspected dm should include such investigations as serum creatine kinase, aldolase, aspartate aminotransferase, alanine aminotransferase and/or lactate dehydrogenase . Serum creatine kinase is the most sensitive muscle enzyme in the acute phase of the disease as it is released in the serum during muscle damage . Serum inflammatory markers (e.g. Erythrocyte sedimentation rate and c - reactive protein) may also be elevated during the active phase of the disease . Table 2bohan and peter criteria for diagnosis of dermatomyositis.individual criteria symmetric proximal muscle weaknessmuscle biopsy evidence of myositisincrease in serum skeletal muscle enzymescharacteristic electromyographic patternstypical rash of dermatomyositisdiagnostic criteria definitive5 plus any three of 14probably5 plus any two of 14possible5 plus any one of 14 bohan and peter criteria for diagnosis of dermatomyositis . Symmetric proximal muscle weakness muscle biopsy evidence of myositis increase in serum skeletal muscle enzymes characteristic electromyographic patterns typical rash of dermatomyositis definitive5 plus any three of 14 probably5 plus any two of 14 possible5 plus any one of 14 previous studies have shown that up to 30% of dm patients had underlying malignancies [6, 7]. Malignancies in dm patients have been found to be highest in those over 45 years of age, commonly diagnosed within the first year of initial presentation, and most strongly associated with lung, ovarian, gastric, pancreatic and colorectal origin . Malignancy can precede, occur concurrently or develop after the diagnosis of dm . In our patient, the symptoms of dm occurred 2 weeks following her diagnosis of breast cancer . The goals of therapy in patients with dm are to improve the patient's ability to carry out activities of daily living by increasing muscle strength and improving myalgias as well as treating the extramuscular manifestations (including rash and arthralgias). There has been report of variable influence of the treatment of the underlying malignancy on the clinical course of the dm [9, 10]. In our patient, the complete surgical excision of the underlying breast cancer resulted in dramatic improvement in both her cutaneous manifestations and her musculosckeletal symptoms . Given the high frequency of malignancy in patients with a diagnosis of dm, any woman over the age of 45 with newly diagnosed dm should prompt a thorough physical examination, including a breast examination, as well as further workup for other malignancies (e.g. Ovarian or colorectal cancer). Early diagnosis of dm as a paraneoplastic syndrome allows for initiation of systemic therapy for symptom control and also treatment of the underlying malignancy itself, which may aid in control of the dm symptoms.
Taken together, malaria (caused by plasmodium species), human african trypanosomiasis (hat, caused by trypanosoma brucei), chagas disease (caused by t. cruzi), and leishmaniasis (caused by leishmania species) represent diseases with an estimated combined burden of over 87 million disability - adjusted life years . While progress has certainly been made toward the discovery of new hits, much work remains to translate these hits into promising chemical lead series that may be advanced for drug development . Target repurposing, wherein inhibitors of human homologues of essential parasite proteins are assessed for efficacy against parasite cells . We have recently initiated the repurposing of a variety of kinase inhibitors for parasitic diseases and have also described the use of established human phosphodiesterase inhibitors as starting points for lead discovery . We have described the discovery of neu-617 (1), a derivative of the approved human cancer drug lapatinib (2), which acts as a potent, orally bioavailable growth inhibitor of t. brucei that showed a modest effect in a mouse model of bloodstream infection . Based on the close phylogenetic relationship between the kinomes of the trypanosomatid parasites (t. brucei, t. cruzi, and leishmania major), we hypothesized that all the pathogens would be susceptible to these inhibitors . We have therefore tested 1, along with the other analogs synthesized during the course of our hit - to - lead optimization program, against the two other kinetoplastid parasites . Based on our previous observation that related chemotypes show activity against plasmodium falciparum, we also screened this set of compounds against cultures of drug - sensitive and drug - resistant strains of malaria . In doing so, we noted differential structure activity relationships for these compounds growth inhibitory properties against each of the parasites, thus launching a multiparasite optimization campaign from the same lead series . In this paper, we describe these cross - pathogen assay results and outline our evaluation of replacements for the quinazoline scaffold of 1 for each of the parasites . We posit that such a cross - screening approach can be a highly fruitful method for identification of new protozoan parasite growth inhibitors . First, in order to ascertain antiparasitic activities of our 66 previously reported analogs, we measured their growth inhibitory activities against t. cruzi (intracellular amastigotes), l. major (promastigote and intracellular amastigote life stages), and p. falciparum (d6, drug sensitive strain). The structures of the most potent compounds for each parasite are shown in figure 1 and table 1, and the complete set of screening data for these is tabulated in the supporting information (tables s1s7). Program progression from 2 to four sem values within 50% . % inh at 10 m . Control compounds: t. brucei, suramin; t. cruzi, benznidazole; l. major, amphotericin b; p. falciparum, chloroquine . From these experiments, we observed that 15 morpholinosulfonamide compounds were identified with activity against l. major promastigotes: seven of these were submicromolar inhibitors and two were also submicromolar against intracellular amastigotes (the most relevant life stage for human infections of leishmaniasis). The des - fluoro analog neu-551 (3) showed the highest activity against promastigotes (ec50 = 0.50 m). We initially tested compounds against t. cruzi at a single concentration and found that 32 compounds inhibited proliferation> 65% at 10 m concentrations; of these 32 compounds, the homopiperazinyl sulfonamide - substituted tail was preferred (neu-628 (4), ec50 = 0.51 m). The active compounds against p. falciparum (drug - sensitive d6 strain) all contained a basic nitrogen at the end of the tail group with neu-627 (5) being the most potent with an ec50 of 27 nm . Though we note that the number of headgroup replacement analogs tested was far smaller and less diverse than the tail variations, the lapatinib headgroup 3-chloro-4-((3-fluorobenzyl)oxy)aniline showed optimal potency against all of the parasites, except for both leishmania life stages . Previous efforts focused on the head (4-benzyloxyanilines) and tail (denoted as r in figure 1) regions of lapatinib, while maintaining the central quinazoline scaffold (highlighted in blue in figure 1). Thus, we planned a broader evaluation of other bicyclic aromatic replacements for the quinazoline scaffold that would test the important features of the chemotype for activity . These scaffold replacements were selected for exploration of the requisite nitrogen atom positioning in the heteroaromatic ring (see the heterocycle precursors highlighted in figure 2, dihalides 69). In addition, the cyanoquinoline and thienopyrimidine scaffolds (dihalides 1012) were selected in order to include other established tyrosine kinase inhibitor chemotypes . For a matched exploration of these scaffolds, we elected to maintain the four permutations of head / tail combinations that displayed the most potency in each parasite (shown in figure 1) in the new analogs that we prepared . We first set out to synthesize the requisite dihalogenated scaffolds shown in figure 2 . The syntheses of 6- and 7-bromo-4-chloroquinolines 6a and 6b were carried out via the gould was devised to draw out essential interactions with either nitrogen of the quinazoline scaffold . The requisite cyanoquinoline template 12 was prepared using established protocols similar to that of the 4-chloroquinolines . Though few examples of cinnoline or phthalazine based kinase inhibitors exist in the literature, we opted to prepare analogues utilizing both heterocycles to test the required positioning of the two nitrogen atoms on the scaffold . Preparation of 6-bromo-4-chlorocinnoline 8a commenced with the bromination of o - aminoacetophenone 13 followed by diazotization of the amine and in situ cyclization to the 6-bromocinnolin-4(1h)-one 15 (scheme 1). We hypothesized that this side reaction occurred via chloride displacement under the acidic conditions of the reaction (scheme s1, see supporting information). A 10:1 product ratio of 8a to 8b could be achieved through the use of thf as solvent and 3 equiv of pocl3 . The regiomeric 7-bromo-4-chlorocinnoline 8c was prepared in a similar fashion starting instead with the acylation of 3-bromoaniline 16 to 2-amino-4-bromoacetophenone 17, followed by diazotization and chlorination . Reagents and conditions: (a) nbs, ch2cl2, 10 c 23 c, o.n . ; (b) nano2, aq . Hcl, h2o, 75 c, o.n . ; (c) pocl3, thf, reflux, 2 h; (d) (i) bcl3, (ch2cl)2, 0 c; (ii) alcl3, ch3cn, 80 c, o.n . ; (iii) aq . Hcl, 80 c, 30 min; (b) nano2, aq . Hcl, h2o, 75 c, o.n . ; (c) pocl3, thf, reflux, 2 h. the 7-bromo-1-chlorophthalazine template 9a was synthesized as shown in scheme 2 . Aryl bromination of phthalide 19 was carried out in acidic medium with nbs to produce a separable mixture of 4- and 6-bromophthalide 20 . Benzylic bromination was achieved using a modification of an established procedure, and the resulting dibromides 21a, b were converted to the corresponding bromophthalazinone 22 upon treatment with hydrazine . As with 15, 22a was converted to the dichlorinated side product 9b on treatment with neat pocl3, though dilution minimized this side reaction . Acetonitrile with 3 equiv of pocl3 provided a> 10:1 ratio of the desired chlorination product 9a . The 6-bromo-1-chlorophthalazine scaffold 9c could be synthesized in an identical manner starting with the commercially available 5-bromophthalide 20b . Reagents and conditions: (a) nbs, h2so4, cf3co2h, 23 c, 40 h; (b) nbs, aibn, chcl3, reflux, 1 h; (c) n2h4, i - proh, reflux, 1.5 h; (d) pocl3, ch3cn, reflux, 3 h. amination of the dihalogenated quinoline, isoquinoline, cyanoquinoline, and thienopyrimidine scaffolds was carried out by applying methods similar to those we previously reported (scheme 3). However, low yields (20%) resulted when these conditions were applied to the cinnoline and phthalazine templates . Switching the solvent to toluene and using 4 equiv of amine improved yields to 5478% . Suzuki couplings using the requisite boronates and 57 mol% pd(pph3)4 or 1 mol% pd(oac)2 provided the final analogs . Yields of the cinnoline or phthalazine products were drastically improved by switching to 2.5 mol% pdcl2(pph3)2 or 5 mol% pd(oac)2 with extended reaction times . A higher catalyst loading of pd(oac)2 was employed in lieu of pdcl2(pph3)2 in situations where the pph3o byproduct could not be separated from the products . Reagents and conditions: (a) 1.1 equiv of amine, i - proh, reflux, o.n . ; (b) 4 equiv of amine, toluene, reflux, 2.5 h; (c) 4 equiv of amine, toluene, 50 c, o.n . ; (d) arbpin, et3n, pd(oac)2, 1:1 h2o / etoh, mw, 120 c, 13 h; (e) arbpin, et3n, pdcl2(pph3)2, 1:1 h2o / etoh, mw, 120 c, 1 h; (f) arbpin, aq . Na2co3, pd(pph3)4, 3:2 glyme / etoh, n2, 85 c, 712 h. with this set of new analogs that are matched to the previously described antiparasitic agents, we set out measuring these analogs activities against t. brucei, t. cruzi, l. major, and p. falciparum, and counter - screened against host cells (nih 3t3 and hepg2 cell lines) using assays that we previously reported . A small number of compounds showed activity against nih 3t3 cells; these are summarized in the supporting information, table s8 . All sem values within 25% unless noted otherwise . % inh at 5 m . Compound 1 remained the most potent t. brucei growth inhibitor among this set of analogs . A pair of quinoline - based compounds, 50 and 52, each with a basic aliphatic amine on the tail group show double digit nanomolar potency (79 and 87 nm, respectively). The potency of these compounds seems to be driven by the aliphatic amine in the tail region rather than by the placement of nitrogen atoms in the scaffold, because the analogous isoquinoline analogue 58 is approximately equipotent (100 nm) to 50 . The quinoline analogs 50, 51, and 52 displayed submicromolar potency against intracellular amastigotes of t. cruzi . Most other analogs showed a complete loss of activity, perhaps due to issues with penetration into both the host cell and the parasite . (with intracellular parasites, we note that compensating effects from interactions with host cell targets cannot be ruled out .) However, the 3-cyanoquinoline analogue 83, shows a 5-fold improvement in potency over the matched original quinazoline analog 4 . In the headgroup region, the 3-fluoro substituent affords a 10-fold increase in potency over the matched des - fluoro analog (cf . The quinoline 51 displayed a slight drop in potency compared with the quinazoline 4, and the matched isoquinoline 59 is 10-fold less potent . Taken together with the cyanoquinoline analog 83, this data demonstrates the apparent importance of two h - bond acceptor motifs in the scaffold . In the case of l. major, 47 showed a 2.5-fold increase in potency from the matched quinazoline analogue 3 (0.20 versus 0.50 m respectively) against the promastigote life stage of l. major . The corresponding isoquinoline compound 55 showed a complete loss in activity, reinforcing the essentiality of the n1 atom of the quinazoline . Interestingly, there was no activity observed against the amastigote life stage of the parasite for 47 . In contrast, the 7-substituted quinoline 52 displayed submicromolar potency against both life stages (0.40 m against promastigotes and 0.89 m versus amastigotes). Compared with the quinazoline compound 4, the analogous cinnoline 68 displayed increased potency against amastigotes (0.24 m) albeit with a significant decrease in potency against the promastigote form . This lack of correlation of compounds between promastigote and amastigote life stages of the parasite has been reported by us and others recently . Other compounds displaying modest potency include 76 and 91, both containing the n - methylhomopiperazinyl sulfonamide tail . In p. falciparum cultures, three isoquinoline compounds with a basic amine at the terminus of the tail region showed modest potency highlighted by 57 being nearly equipotent to the parent quinazoline 5 (40 vs 27 nm). Though the matching quinoline 49 showed a complete loss in activity, its regioisomer 50 was slightly more active than 5 (ec50 = 19 nm). The four quinoline analogs without a basic amine on the tail group were within 2-fold of 5 with 48 being the most potent with an ec50 = 16 nm . Similarly, all four thienopyrimidine analogs with a basic tail group amine (8992) were potent, within 4-fold of 5 . Among these compounds there was no preference for either thienopyridine isomer; the n - methylpiperazinyl tail group was 2.5- to 4-fold more potent than the n - methylhomopiperazinyl tail on each scaffold . Among the phthalazines, 74 and 76, both with a basic nitrogen on the tail and substituted at the 6-position (regiomeric to 5) were equipotent to 5 . Also requiring a basic amine on the tail region, the cinnoline analogs with piperazinyl and homopiperazinyl moieties (6568) showed modest to high potency against p. falciparum d6 strain . Excitingly, the 7-substituted isomers (66 and 68) were highly potent antiplasmodial agents (14 and 3 nm, respectively). Since we observed that the most potent compounds against p. falciparum were 4-aminoquinolines (similar to the prototypical antimalarial agent chloroquine), we tested them against drug resistant parasite strains w2 and c235 . We were gratified to observe that all of the compounds tested are similarly active against drug resistant strains of p. falciparum (table s9, supporting information), suggesting that despite the shared scaffold, these analogs likely display a mechanism of action or mechanism of resistance that differs from those of chloroquine . Our studies demonstrate which of these scaffolds are the most promising for further optimization on a parasite - by - parasite basis ., it is clear that the quinoline scaffold is significantly active across all four pathogens . We note that cinnoline (c) and quinoline (a) generally provide the most potent antiplasmodial agents, and the 7-position substitutions are slightly favored in both of these templates . For t. brucei, the 7-substituted quinolines and 6-substituted isoquinolines appear optimal (noting that, despite the differential numbering of the scaffolds, these two particular scaffolds present the same relative regiochemistry). Analogous diagrams for the other pathogens are also provided in figure 3 (and a diagram for leishmania promastigotes is in the supporting information (figure s1). A summary of the preferred bicyclic scaffold for each parasite is shown in figure 3e, noting that none appear to be markedly better than the others against leishmania intracellular amastigotes . Bar chart showing range of potencies for each scaffold (as labeled in tables 2 and 3) for (a) malaria, (b) t. brucei, (c) l. major (amastigotes), and (d) t. cruzi . Regiochemical information is shown by the suffix number (e.g., scaffold a-7 is the 7-substituted quinoline scaffold). Quinolines are shown in blue, isoquinolines in green, cinnolines in yellow, phthalazines in orange, cyanoquinolines in red, thieno[3,2-d]pyrimidines in purple, thieno[2,3-d]pyrimidines in light gray, and quinazolines in dark gray . For all parasites except for t. brucei, we also note a preference for tail substituents with a basic amine present . Considering only the most potent compounds (ec50 0.1 m for the extracellular parasites t. brucei and p. falciparum; ec50 1 m for the intracellular parasites l. major and t. cruzi), we note that the n - methylpiperizine and n - methylhomopiperazine are preferred among analogs that were tested, over the less - basic morpholine or nonbasic morpholinosulfonamide substituents (table 4). At this point, we cannot discern whether this is an effect that is mediated by the biological target(s) involved in proliferation inhibition, by parasite permeation, or by a combination of both; this will be a matter for further investigation . Potent compounds are ec50 0.1 m . Potent compounds are ec50 1 m . We have previously observed that molecules bearing these head and tail combinations possess problematic physicochemical properties, likely due to the molecular size and lipophilicity . The intent of the present work was to explore the central scaffold functionality, rather than to address these shortcomings . Nonetheless, we selected five representative compounds from among the most potent to be tested for their physicochemical properties (table 5). Not unexpectedly, compounds tested were> 99% plasma protein bound with limited thermodynamic aqueous solubility (<1 m). Such properties are undoubtedly a result of the high molecular weights and clogp values; these issues remain the focus of ongoing efforts, which are now focused on specific scaffolds for each pathogen . In summary, through a cross - parasite screening campaign of existing quinazoline t. brucei proliferation inhibitors, we uncovered new hits against three other protozoan parasites . Encouraged by that initial success, we focused on exploring the central heterocycle scaffold, an exercise that has uncovered additional highly potent compounds . Scaffold variations have revealed heteroatom positioning essential for activity within this chemotype, while variations to regiochemical attachment points have introduced a new set of regioisomers with multiparasite potency . Thus, we have established a new lead series for each of these protozoan parasites, which can be now advanced in parallel for drug discovery against four different parasitic diseases . With this in mind, further optimization of physicochemical properties and cellular selectivity of each is ongoing, with a specific focus on the head and tail regions . (st . Louis, mo), fisher scientific, frontier scientific services, inc . (newark, de), or matrix scientific (columbia, sc) and used as received . Boronic acids and esters and aniline reagents were purchased, except for those whose syntheses are listed in the supporting information . Reaction solvents were dried by passage through alumina columns on a purification system manufactured by innovative technology (newburyport, ma). Nmr spectra were obtained with varian nmr systems, operating at 400 or 500 mhz for h acquisitions as noted . Lcms analysis was performed using a waters alliance reverse - phase hplc, with single - wavelength uv visible detector and lct premier time - of - flight mass spectrometer (electrospray ionization). All newly synthesized compounds that were submitted for biological testing were deemed> 95% pure by lcms analysis (uv and esi - ms detection) prior to submission for biological testing . Preparative lcms was performed on a waters fraction lynx system with a waters micromass zq mass spectrometer (electrospray ionization) and a single - wavelength uv yields reported for products obtained by preparative hplc represent the amount of pure material isolated; impure fractions were not repurified . In a flame - dried 250 ml round - bottom flask were added 6-bromocinnolin-4(1h)-one (1.00 g, 4.44 mmol), anhydrous tetrahydrofuran (45 ml), and phosphorus oxychloride (1.25 ml, 13.41 mmol). The mixture was refluxed for 1 h at which point a deep green / blue solution had resulted . The solution was cooled to 0 c and was quenched by the dropwise addition of sat . The mixture was allowed to warm to room temperature and stir for an additional 1 h. water (50 ml) was added, and the mixture was extracted with dichloromethane (3 100 ml). Nahco3 (50 ml), washed with brine (50 ml), dried over na2so4, concentrated on to silica, and purified by flash column chromatography using a gradient of 15% methanol in dichloromethane to yield an inseparable 10:1 mixture of 8a and 8b as a brown solid in 85% yield . H nmr (500 mhz, cdcl3) 9.36 (s, 1 h), 8.43 (d, j = 8.8 hz, 1 h), 8.36 (d, j = 2.0 hz, 1 h), 7.98 (dd, j = 9.3, 2.0 hz, 1 h). Lcms found 242.9 [m + h]. In a flame - dried 25 ml round - bottom flask were added 7-bromocinnolin-4(1h)-one (166 mg, 0.74 mmol), anhydrous tetrahydrofuran (7 ml), and phosphorus oxychloride (0.2 ml, 2.15 mmol). The mixture was refluxed for 1 h at which point a deep green / blue solution had resulted . The solution was cooled to 0 c and was quenched by the dropwise addition of sat . . The mixture was allowed to warm to room temperature and stir for an additional 1 h. water (12 ml) was added, and the mixture was extracted with dichloromethane (3 25 ml). Nahco3 (20 ml), washed with brine (20 ml), and dried over na2so4 to yield 8c as a dark brown solid in 92% yield . H nmr (500 mhz, cdcl3) 9.39 (s, 1 h), 8.76 (d, j = 2.0 hz, 1 h), 8.09 (d, j = 9.3 hz, 1 h), 7.95 (dd, j = 9.0, 1.7 hz, 1 h). Lcms found 242.9 [m + h]. In a flame - dried 25 ml round - bottom flask were added 7-bromophthalazin-1(2h)-one (205 mg, 0.91 mmol), anhydrous acetonitrile (9 ml), and phosphorus oxychloride (0.3 ml, 3.22 mmol). The mixture was refluxed for 2 h, then cooled to 0 c, diluted with dichloromethane (20 ml), and quenched with a dropwise addition of sat . After 1 h, the layers were separated and the aqueous was extracted with dichloromethane (2 30 ml). The combined organic layers were washed with sat . Nahco3 (25 ml), washed with brine (20 ml), dried over na2so4, and concentrated to yield 9a as an orange solid in 91% yield . H nmr (500 mhz, cdcl3) 9.45 (s, 1 h), 8.498.51 (m, 1 h), 8.10 (dd, j = 8.8, 2.0 hz, 1 h), 7.91 (d, j = 8.8 hz, 1 h). Lcms found 242.9 [m + h]. In a flame - dried 50 ml round - bottom flask were added 6-bromophthalazin-1(2h)-one (402 mg, 1.78 mmol), anhydrous acetonitrile (18 ml), and phosphorus oxychloride (0.5 ml, 5.36 mmol). The mixture was refluxed for 2 h, then cooled to 0 c, diluted with dichloromethane (40 ml), and quenched with a dropwise addition of sat . The biphasic mixture was stirred vigorously and allowed to warm to room temperature . After 1 h, the layers were separated, and the aqueous was extracted with dichloromethane (2 50 ml). The combined organic layers were washed with sat . Nahco3 (40 ml), washed with brine (30 ml), dried over na2so4, and concentrated to yield 29c as a yellow solid in 94% yield . H nmr (500 mhz, cdcl3) 9.39 (s, 1 h), 8.178.21 (m, 2 h), 8.10 (dd, j = 8.8, 2.0 hz, 1 h). In a 250 ml round - bottom flask were added 1-(2-amino-5-bromophenyl)ethanone (8.34 g, 39.0 mmol), water (30 ml), and conc . The mixture was cooled to 0 c in an ice bath and allowed to stir for 15 min until a suspension resulted . Aqueous sodium nitrite (2 m, 20 ml, 40.0 mmol) was then added dropwise with an addition funnel . The resulting solution was allowed to warm to room temperature over 1.5 h and was stirred at room temperature overnight, then refluxed for 6 h. the mixture was cooled to room temperature, water (200 ml) was added, and the mixture was extracted with ethyl acetate (3 200 ml). The combined organic layers were then washed with brine (50 ml), dried over sodium sulfate, filtered, and concentrated onto silica . The crude product was then purified by flash column chromatography using a gradient of 110% methanol in dichloromethane to yield 15 as a dark brown solid in 82% yield . H nmr (500 mhz, dmso - d6) 14.09 (br . S., 1 h), 8.09 (d, j = 2.2 hz, 1 h), 7.92 (dd, j = 8.8, 2.2 hz, 1 h), 7.79 (s, 1 h), 7.71 (d, j = 9.1 hz, 1 h). In a 50 ml round - bottom flask were added 1-(2-amino-4-bromophenyl)ethanone (712 mg, 3.33 mmol), water (3 ml), and conc . The mixture was cooled to 0 c in an ice bath and allowed to stir for 15 min until a suspension resulted . Aqueous sodium nitrite (2 m, 1.84 ml, 3.68 mmol) was then added dropwise with an addition funnel . The resulting solution was allowed to warm to room temperature over 1.5 h and was stirred at room temperature overnight, then refluxed for 6 h. the mixture was cooled to room temperature, water (35 ml) was added, and the mixture was extracted with ethyl acetate (3 40 ml). The combined organic layers were then washed with brine (20 ml), dried over sodium sulfate, filtered, and concentrated onto silica . The crude product was then purified by flash column chromatography using a gradient of 2050% ethyl acetate in hexanes, then ethyl acetate to yield 18 as a light brown solid in 26% yield . H nmr (500 mhz, dmso - d6) 13.49 (s, 1 h), 7.92 (d, j = 8.8 hz, 1 h), 7.76 (s, 1 h), 7.73 (d, j = 2.0 hz, 1 h), 7.53 (dd, j = 8.8, 2.0 hz, 1 h). In a 100 ml round - bottom flask was dissolved isobenzofuran-1(3h)-one (4.01 g, 29.9 mmol) in trifluoroacetic acid (14 ml, 182 mmol) and sulfuric acid (6.5 ml, 122 mmol). N - bromosuccinimide (7.95 g, 1.49 mmol) was added portionwise over 8 h, and the solution was stirred at room temperature for an additional 87 h. the solution was diluted with water (40 ml) and ethyl acetate (40 ml). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 50 ml). The combined organic layers were washed with brine (25 ml), dried over na2so4, and concentrated onto silica . The crude product was then purified by flash column chromatography using 1020% ethyl acetate in hexanes to yield 20a as white solid in 57% yield . H nmr (500 mhz, cdcl3) 7.98 (d, j = 1.5 hz, 1 h), 7.77 (dd, j = 8.3, 1.5 hz, 1 h), 7.40 (d, j = 8.3 hz, 1 h), 5.27 (s, 2 h). Lcms found 212.9 [m + h]. In a 50 ml round - bottom flask n - bromosuccinimide (958 mg, 5.38 mmol), 2,2-azobis(2-methylpropionitrile) (75 mg, 0.46 mmol), and chloroform (23 ml). The mixture was refluxed for 2.5 h, then cooled to room temperature and quenched with sat . The organic layer was removed, washed with water (20 ml), washed with brine (15 ml), and concentrated onto silica . The crude product was purified by flash column chromatography using a gradient of 510% ethyl acetate in hexanes to yield 21a as a white solid in 61% yield . H nmr (500 mhz, cdcl3) 8.06 (d, j = 1.5 hz, 1 h), 7.90 (dd, j = 8.1, 1.7 hz, 1 h), 7.52 (d, j = 8.3 hz, 1 h), 7.37 (s, 1 h). Were added 5-bromoisobenzofuran-1(3h)-one (499 mg, 2.34 mmol), n - bromosuccinimide (421 mg, 2.37 mmol), 2,2-azobis(2-methylpropionitrile) (38 mg, 0.23 mmol), and chloroform (10 ml). The mixture was refluxed for 2.5 h, then cooled to room temperature and quenched with sat . The organic layer was removed, washed with water (10 ml), washed with brine (5 ml), and concentrated onto silica . The crude product was purified by flash column chromatography using 10% ethyl acetate in hexanes to yield 21b as a white solid in 49% yield . H nmr (500 mhz, cdcl3) 7.747.83 (m, 3 h), 7.36 (s, 1 h). Gcms found 289.8 [m]. In a 25 ml round - bottom flask was dissolved 3,6-dibromoisobenzofuran-1(3h)-one (143 mg, 0.49 mmol) in ethanol (5 ml). Hydrazine monohydrate (0.12 ml, 2.48 mmol) was then added via a syringe, and the solution was refluxed for 1.5 h. the solution was cooled to room temperature, and ice water (15 ml) was added to the reaction mixture . The precipitate was vacuum filtered and dried under a vacuum overnight to yield 22a as a white solid in 56% yield . H nmr (500 mhz, dmso - d6) 12.82 (br . S., 1 h), 8.39 (s, 1 h), 8.30 (d, j = 2.0 hz, 1 h), 8.11 (dd, j = 8.5, 2.2 hz, 1 h), 7.90 (d, j = 8.3 hz, 1 h). In a 25 ml round - bottom flask was dissolved 3,5-dibromoisobenzofuran-1(3h)-one (302 mg, 1.04 mmol) in ethanol (10 ml). Hydrazine monohydrate (0.25 ml, 5.18 mmol) was then added via a syringe, and the solution was refluxed for 1.5 h. the solution was cooled to room temperature, and ice water (30 ml) was added to the reaction mixture . The precipitate was vacuum filtered and dried under a vacuum overnight to yield 22b as a white solid in 73% yield . H nmr (500 mhz, dmso - d6) 12.78 (br . S., 1 h), 8.33 (s, 1 h), 8.23 (d, j = 2.0 hz, 1 h), 8.12 (d, j = 8.3 hz, 1 h), 8.00 (dd, j = 8.3, 2.0 hz, 1 h). To a solution of the appropriate aryl chloride (1 equiv) in 2-propanol (0.15 m) was added 3-chloro-4-((3-fluorobenzyl)oxy)aniline or 4-(benzyloxy)-3-chloroaniline (1.1 equiv). The formed precipitate was collected by vacuum filtration to obtain the desired products . To a solution of the appropriate aryl chloride (1 equiv) in 2-propanol (0.15 m) the combined organic layers were washed with water, washed with brine, dried over na2so4, and concentrated . The crude products were purified by flash column chromatography to obtain the desired products . A solution of the appropriate 4-chlorocinnoline (1 equiv) and 3-chloro-4-((3-fluorobenzyl)oxy)aniline or 4-(benzyloxy)-3-chloroaniline (4 equiv) in toluene (0.1 m) was refluxed for 2.5 h and cooled to room temperature . Triethylamine (4 equiv) was added, and the mixture was returned to reflux for an additional 30 min . The mixture was cooled back to room temperature, and the formed yellow precipitate was vacuum filtered, washed with ethyl acetate, concentrated onto silica, and purified by flash column chromatography . A solution of the appropriate 1-chlorophthalazine (1 equiv) and 3-chloro-4-((3-fluorobenzyl)oxy)aniline or 4-(benzyloxy)-3-chloroaniline (4 equiv) in anhydrous toluene (0.2 m) was heated at 50 c overnight . The combined organic layers were washed with brine, dried over na2so4, concentrated onto silica, and purified by flash column chromatography . Synthesized by general procedure b. flash column chromatography: 2050% ethyl acetate in hexanes to yield 23 as a light brown solid in 90% yield . H nmr (500 mhz, dmso - d6) 8.99 (s, 1 h), 8.63 (s, 1 h), 8.46 (d, j = 5.4 hz, 1 h), 7.80 (d, j = 1.5 hz, 2 h), 7.437.51 (m, 2 h), 7.277.36 (m, 4 h), 7.19 (td, j = 8.7, 2.2 hz, 1 h), 6.80 (d, j = 5.4 hz, 1 h), 5.26 (s, 2 h). Lcms found 456.8 [m + h]. Synthesized by general procedure b. flash column chromatography: 2070% ethyl acetate in hexanes to yield 24 as a brown solid in 70% yield . H nmr (500 mhz, dmso - d6) 8.98 (s, 1 h), 8.63 (s, 1 h), 8.45 (d, j = 5.4 hz, 1 h), 7.80 (s, 2 h), 7.50 (d, j = 7.3 hz, 2 h), 7.397.46 (m, 3 h), 7.35 (t, j = 7.3 hz, 1 h), 7.30 (s, 2 h), 6.79 (d, j = 5.4 hz, 1 h), 5.23 (s, 2 h). Synthesized by general procedure b. flash column chromatography: 2050% ethyl acetate in hexanes to yield 25 as a tan colored solid in 82% yield . H nmr (399 mhz, dmso - d6) 9.05 (s, 1 h), 8.43 (d, j = 5.1 hz, 1 h), 8.31 (d, j = 8.8 hz, 1 h), 8.05 (d, j = 2.2 hz, 1 h), 7.67 (dd, j = 8.8, 2.2 hz, 1 h), 7.417.52 (m, 2 h), 7.257.37 (m, 4 h), 7.18 (td, j = 8.6, 2.6 hz, 1 h), 6.76 (d, j = 5.9 hz, 1 h), 5.25 (s, 2 h). Synthesized by general procedure b. flash column chromatography: 2050% ethyl acetate in hexanes to yield 26 as an off - white solid in 83% yield . H nmr (500 mhz, dmso - d6) 9.04 (s, 1 h), 8.43 (d, j = 4.9 hz, 1 h), 8.31 (d, j = 9.3 hz, 1 h), 8.05 (d, j = 2.0 hz, 1 h), 7.67 (dd, j = 9.0, 2.2 hz, 1 h), 7.49 (d, j = 6.8 hz, 2 h), 7.397.46 (m, 3 h), 7.35 (t, j = 7.3 hz, 1 h), 7.30 (s, 2 h), 6.75 (d, j = 5.4 hz, 1 h), 5.23 (s, 2 h). Synthesized by general procedure b. flash column chromatography: 1030% ethyl acetate in hexanes to yield 27 as a pale red - brown solid in 97% yield . H nmr (500 mhz, dmso - d6) 9.24 (s, 1 h), 8.80 (s, 1 h), 8.07 (d, j = 2.4 hz, 1 h), 8.02 (d, j = 5.9 hz, 1 h), 7.82 (dd, j = 8.8, 1.5 hz, 1 h), 7.727.79 (m, 2 h), 7.417.49 (m, 1 h), 7.277.35 (m, 2 h), 7.137.23 (m, 3 h), 5.21 (s, 2 h). Synthesized by general procedure b. flash column chromatography: 1030% ethyl acetate in hexanes to yield 28 as a light red solid in 93% yield . H nmr (500 mhz, dmso - d6) 9.22 (s, 1 h), 8.80 (s, 1 h), 8.07 (d, j = 2.4 hz, 1 h), 8.02 (d, j = 5.9 hz, 1 h), 7.81 (dd, j = 8.8, 1.5 hz, 1 h), 7.737.78 (m, 2 h), 7.48 (d, j = 7.3 hz, 2 h), 7.40 (t, j = 7.6 hz, 2 h), 7.33 (t, j = 7.3 hz, 1 h), 7.21 (d, j = 8.8 hz, 1 h), 7.15 (d, j = 5.9 hz, 1 h), 5.18 (s, 2 h). Lcms found 439.2 [m + h]. Synthesized by general procedure b. flash column chromatography: 1030% ethyl acetate in hexanes to yield 29 as a salmon colored solid in 91% yield . H nmr (500 mhz, dmso - d6) 9.27 (s, 1 h), 8.45 (d, j = 8.8 hz, 1 h), 8.08 (dd, j = 12.0, 2.2 hz, 2 h), 8.01 (d, j = 5.4 hz, 1 h), 7.75 (ddd, j = 13.8, 9.2, 2.4 hz, 2 h), 7.46 (m, j = 5.9 hz, 1 h), 7.277.35 (m, 2 h), 7.107.23 (m, 3 h), 5.22 (s, 2 h). Synthesized by general procedure b. flash column chromatography: 1030% ethyl acetate in hexanes to yield 30 as a burnt orange solid in 81% yield . H nmr (500 mhz, dmso - d6) 9.24 (s, 1 h), 8.45 (d, j = 8.8 hz, 1 h), 8.10 (d, j = 2.0 hz, 1 h), 8.05 (d, j = 2.4 hz, 1 h), 8.01 (d, j = 5.9 hz, 1 h), 7.76 (dd, j = 9.0, 2.2 hz, 1 h), 7.72 (dd, j = 8.8, 2.4 hz, 1 h), 7.48 (d, j = 6.8 hz, 2 h), 7.41 (t, j = 7.3 hz, 2 h), 7.34 (t, j = 7.8 hz, 1 h), 7.21 (d, j = 9.3 hz, 1 h), 7.13 (d, j = 5.9 hz, 1 h), 5.19 (s, 2 h). Synthesized by general procedure c. flash column chromatography: 5% methanol in dichloromethane to yield an inseparable 10:1 mixture of 31 and dichloro side product as a vibrant yellow solid in 54% yield . H nmr (500 mhz, dmso - d6) 9.33 (s, 1 h), 8.78 (s, 1 h), 8.70 (d, j = 1.5 hz, 1 h), 8.14 (d, j = 8.8 hz, 1 h), 7.97 (dd, j = 9.3, 1.5 hz, 1 h), 7.54 (d, j = 2.0 hz, 1 h), 7.48 (td, j = 8.3, 6.3 hz, 1 h), 7.40 (dd, j = 8.8, 2.4 hz, 1 h), 7.307.37 (m, 3 h), 7.20 (td, j = 9.3, 2.0 hz, 1 h), 5.29 (s, 2 h). Synthesized by general procedure c. flash column chromatography: 5% methanol in dichloromethane to yield an inseparable 10:1 mixture of 32 and dichloro side product as a yellow solid in 68% yield . H nmr (500 mhz, dmso - d6) 9.32 (s, 1 h), 8.78 (s, 1 h), 8.71 (d, j = 2.0 hz, 1 h), 8.14 (d, j = 9.3 hz, 1 h), 7.97 (dd, j = 9.0, 1.7 hz, 1 h), 7.477.55 (m, 3 h), 7.44 (t, j = 7.6 hz, 2 h), 7.337.41 (m, 3 h), 5.26 (s, 2 h). Synthesized by general procedure c. flash column chromatography: 5% methanol in dichloromethane to yield 33 as a brown solid in 55% yield with 84% purity . H nmr (500 mhz, dmso - d6) 9.43 (s, 1 h), 8.76 (s, 1 h), 8.41 (d, j = 2.0 hz, 1 h), 8.35 (d, j = 9.3 hz, 1 h), 7.90 (dd, j = 9.0, 2.2 hz, 1 h), 7.55 (d, j = 2.4 hz, 1 h), 7.49 (m, j = 6.3 hz, 1 h), 7.41 (dd, j = 8.8, 2.4 hz, 1 h), 7.317.37 (m, 3 h), 7.20 (td, j = 8.4, 2.7 hz, 1 h), 5.29 (s, 2 h). Synthesized by general procedure c. flash column chromatography: 5% methanol in dichloromethane to yield 34 as a metallic bronze colored solid in 57% yield with 85% purity . H nmr (500 mhz, dmso - d6) 9.42 (s, 1 h), 8.75 (s, 1 h), 8.41 (d, j = 2.0 hz, 1 h), 8.35 (d, j = 8.8 hz, 1 h), 7.90 (dd, j = 9.0, 2.2 hz, 1 h), 7.54 (d, j = 2.4 hz, 1 h), 7.51 (d, j = 7.3 hz, 2 h), 7.43 (t, j = 7.6 hz, 2 h), 7.40 (dd, j = 8.8, 2.4 hz, 1 h), 7.337.38 (m, 2 h), 5.26 (s, 2 h). Synthesized by general procedure d. flash column chromatography: 520% ethyl acetate in dichloromethane to yield 35 as a yellow solid in 69% yield . H nmr (500 mhz, dmso - d6) 9.21 (s, 1 h), 9.13 (s, 1 h), 8.87 (s, 1 h), 8.16 (d, j = 2.4 hz, 1 h), 8.11 (dd, j = 8.8, 1.5 hz, 1 h), 7.99 (d, j = 8.8 hz, 1 h), 7.81 (dd, j = 8.8, 2.4 hz, 1 h), 7.47 (td, j = 7.9, 6.1 hz, 1 h), 7.297.36 (m, 2 h), 7.25 (d, j = 9.3 hz, 1 h), 7.17 (td, j = 8.7, 2.2 hz, 1 h), 5.24 (s, 2 h). Synthesized by general procedure d. flash column chromatography: 520% ethyl acetate in dichloromethane to yield 36 as a light greenish brown solid in 78% yield . H nmr (500 mhz, dmso - d6) 9.20 (s, 1 h), 9.14 (s, 1 h), 8.88 (s, 1 h), 8.15 (d, j = 2.4 hz, 1 h), 8.13 (dd, j = 8.5, 1.7 hz, 1 h), 8.00 (d, j = 8.8 hz, 1 h), 7.80 (dd, j = 9.0, 2.7 hz, 1 h), 7.50 (d, j = 7.3 hz, 2 h), 7.42 (t, j = 7.6 hz, 2 h), 7.35 (t, j = 7.3 hz, 1 h), 7.27 (d, j = 8.8 hz, 1 h), 5.21 (s, 2 h). Synthesized by general procedure d. flash column chromatography: 520% ethyl acetate in dichloromethane to yield 37 as a light brown solid in 64% yield . H nmr (500 mhz, dmso - d6) 9.42 (s, 1 h), 9.09 (s, 1 h), 8.62 (d, j = 9.3 hz, 1 h), 8.33 (d, j = 2.0 hz, 1 h), 8.19 (d, j = 2.4 hz, 1 h), 8.16 (dd, j = 9.0, 2.2 hz, 1 h), 7.83 (dd, j = 8.8, 2.4 hz, 1 h), 7.47 (td, j = 7.8, 5.9 hz, 1 h), 7.32 (m, j = 7.3 hz, 2 h), 7.25 (d, j = 9.3 hz, 1 h), 7.17 (td, j = 8.5, 2.0 hz, 1 h), 5.24 (s, 2 h). Synthesized by general procedure d. flash column chromatography: 530% ethyl acetate in dichloromethane to yield 38 as a green - gray colored solid in 75% yield . H nmr (500 mhz, dmso - d6) 9.25 (s, 1 h), 9.09 (s, 1 h), 8.51 (d, j = 8.8 hz, 1 h), 8.33 (d, j = 2.0 hz, 1 h), 8.17 (dd, j = 8.8, 2.0 hz, 1 h), 8.14 (d, j = 2.9 hz, 1 h), 7.78 (dd, j = 9.0, 2.7 hz, 1 h), 7.49 (d, j = 6.8 hz, 2 h), 7.42 (t, j = 7.6 hz, 2 h), 7.35 (t, j = 7.3 hz, 1 h), 7.26 (d, j = 8.8 hz, 1 h), 5.21 (s, 2 h). Synthesized by general procedure a, collected as a yellow solid in 80% yield . H nmr (400 mhz, dmso - d6) 9.03 (s, 1h), 8.85 (br, s, 1h), 8.23 (d, j = 8.8 hz, 1h), 7.73 (d, j = 8.8, 1h), 7.59 (d, j = 2.4, 1h), 7.457.50 (m, 1h), 7.307.39 (m, 4h), 7.187.22 (m, 1h), 5.30 (s, 2h). Synthesized by general procedure a, collected as a yellow solid in 52% yield . H nmr (500 mhz, dmso - d6) ppm 9.02 (s, 1h), 8.82 (br, s, 1h), 8.22 (d, j = 9.0 hz, 1h), 7.72 (d, j = 8.5 hz, 1h), 7.57 (s, 1h), 7.5 (d, j = 7.0 hz, 2h), 7.43 (t, j = 7.5 hz, 2h), 7.35 (m, 3h), 5.26 (s, 2h). H nmr (400 mhz, dmso - d6) ppm 10.64 (s, 1 h), 8.71 (s, 1 h), 7.87 (d, j = 2.9 hz, 1 h), 7.74 (s, 1 h), 7.57 (dd, j = 8.8, 2.2 hz, 1 h), 7.47 (m, 1 h), 7.31 (m, 3 h), 7.19 (td, j = 8.1, 2.2 hz, 1 h), 5.28 (s, 2 h). H nmr (500 mhz, dmso - d6) ppm 9.7 1 (s, 1 h), 8.54 (s, 1 h), 7.89 (d, j = 2.0 hz, 1 h), 7.68 (s, 1 h), 7.58 (dd, j = 8.8, 2.9 hz, 1 h), 7.49 (d, j = 7.8 hz, 2 h), 7.42 (t, j = 7.6 hz, 2 h), 7.35 (t, j = 7.3 hz, 1 h), 7.26 (d, j = 8.8 hz, 1 h), 5.23 (s, 2 h). H nmr (500 mhz, dmso - d6) 9.80 (s, 1h), 8.49 (s, 1h), 8.12 (s, 1h), 8.02 (d, j = 2.4 hz, 1h), 7.67 (dd, j = 2.7, 9.03 hz, 1h), 7.437.49 (m, 1h), 7.247.35 (m, 3h), 7.18 (dt, j = 2.4, 8.6 hz, 1h), 5.24 (s, 2h). H nmr (500 mhz, dmso - d6) ppm 9.94 (s, 1 h), 8.49 (s, 1 h), 8.19 (s, 1 h), 8.02 (d, j = 2.4 hz, 1 h), 7.68 (dd, j = 8.8, 2.4 hz, 1 h), 7.49 (m, 2 h), 7.41 (m, 2 h), 7.34 (m, 1 h), 7.27 (d, j = 9.3 hz, 1 h), 5.21 (s, 2 h). Lcms found 445.9 [m + h]. In a 25 ml microwave vial equipped with a stir bar were added aryl bromide (1 equiv), boronic ester (1.3 equiv), 1:1 water / ethanol (0.04 m), triethylamine (3 equiv), and palladium(ii) acetate (0.01 m in acetone, 1 mol%). The vial was sealed with a septum, and the contents were irradiated to and held at 120 c with stirring for 1 h. the reaction mixture was cooled to room temperature, diluted with water (8 ml), and extracted with dichloromethane (3 8 ml). Naoh (1 m, 2 5 ml), water (5 ml), and brine (5 ml). The crude product was purified by flash column chromatography . In a 25 ml microwave vial equipped with a stir bar were added aryl bromide (1 equiv), boronic ester (1.3 equiv), 1:1 water / ethanol (0.04 m), triethylamine (3 equiv), and bis(triphenylphosphine)palladium(ii) chloride (2.5 mol%). The vial was sealed with a septum, and the contents were irradiated to and held at 120 c with stirring for 1 h. the reaction mixture was cooled to room temperature, diluted with water (8 ml), and extracted with dichloromethane (3 8 ml). Naoh (1 m, 2 5 ml), water (5 ml), and brine (5 ml). The organic layer was then dried over na2so4 and concentrated on to silica . The crude product was purified by flash column chromatography . In a 25 ml microwave vial equipped with a stir bar were added aryl bromide (1 equiv), boronic ester (1.3 equiv), 1:1 water / ethanol (0.04 m), triethylamine (3 equiv), and palladium(ii) acetate (5 mol%). The vial was sealed with a septum, and the contents were irradiated to and held at 120 c with stirring for 3 h. the reaction mixture was cooled to room temperature, diluted with water (8 ml), and extracted with dichloromethane (3 8 ml). Naoh (1 m, 2 5 ml), water (5 ml), and brine (5 ml). The crude product was purified by flash column chromatography . To a solution of the appropriate aryl iodide (1 equiv) in 3:2 dimethoxyethane / ethanol (0.05 m) 2 m na2co3 (6 equiv), and pd(pph3)4 (5 mol%). The mixture was purged with nitrogen and heated at 85 c for 7 h. the mixture was cooled to room temperature and filtered, and the filtrate was concentrated . The residue was dissolved in ethyl acetate, washed with water, washed with brine, dried over na2so4, and purified by flash column chromatography . To a solution of the appropriate aryl bromide (1 equiv) in 3:2 dimethoxyethane / ethanol (0.05 m) 2 m na2co3 (6 equiv), and pd(pph3)4 (7 mol%). The mixture was heated at 85 c for 12 h, then cooled to room temperature, and the solvents were removed under reduced pressure . The residue was purified by silica column chromatography (hexanes / ethyl acetate) and then by reverse phase chromatography (water / acetonitrile) unless otherwise mentioned . Synthesized by general procedure a. flash column chromatography: 25% methanol in dichloromethane, isolated as a yellow solid in 27% yield . H nmr (500 mhz, dmso - d6) ppm 9.10 (br . S., 1 h), 8.61 (d, j = 2.0 hz, 1 h), 8.43 (d, j = 5.4 hz, 1 h), 8.04 (dd, j = 8.8, 2.0 hz, 1 h), 7.92 (d, j = 8.8 hz, 1 h), 7.457.52 (m, 2 h), 7.39 (t, j = 7.8 hz, 1 h), 7.297.37 (m, 6 h), 7.20 (td, j = 8.8, 2.0 hz, 1 h), 7.00 (dd, j = 8.1, 1.7 hz, 1 h), 6.77 (d, j = 5.4 hz, 1 h), 5.28 (s, 2 h), 3.753.81 (m, 4 h), 3.193.26 (m, 4 h). Lcms found 540.1 [m + h]. Synthesized by general procedure a. flash column chromatography: 25% methanol in dichloromethane, isolated as a yellow solid in 24% yield . H nmr (500 mhz, dmso - d6) ppm 8.99 (br . S., 1 h), 8.47 (d, j = 5.4 hz, 1 h), 8.41 (d, j = 8.8 hz, 1 h), 8.12 (d, j = 2.0 hz, 1 h), 7.88 (dd, j = 8.8, 1.5 hz, 1 h), 7.457.51 (m, 2 h), 7.267.40 (m, 7 h), 7.20 (td, j = 8.8, 2.4 hz, 1 h), 7.02 (dd, j = 8.3, 2.0 hz, 1 h), 6.76 (d, j = 5.4 hz, 1 h), 5.27 (s, 2 h), 3.743.82 (m, 4 h), 3.203.27 (m, 4 h). Synthesized by general procedure a. flash column chromatography: 05% methanol in dichloromethane, isolated as a tan colored solid in 65% yield . H nmr (500 mhz, dmso - d6) ppm 9.14 (s, 1 h), 8.78 (d, j = 2.0 hz, 1 h), 8.47 (d, j = 5.4 hz, 1 h), 8.17 (d, j = 8.8 hz, 2 h), 8.11 (dd, j = 8.8, 2.0 hz, 1 h), 7.98 (d, j = 8.8 hz, 1 h), 7.88 (d, j = 8.3 hz, 2 h), 7.51 (d, j = 7.3 hz, 2 h), 7.48 (d, j = 1.5 hz, 1 h), 7.43 (t, j = 7.6 hz, 2 h), 7.327.39 (m, 3 h), 6.80 (d, j = 5.4 hz, 1 h), 5.25 (s, 2 h), 3.613.70 (m, 4 h), 2.882.97 (m, 4 h). Synthesized by general procedure a. flash column chromatography: 25% methanol in dichloromethane, isolated as a tan colored solid in 63% yield . H nmr (500 mhz, dmso - d6) ppm 9.02 (s, 1 h), 8.50 (m, j = 5.4 hz, 2 h), 8.25 (d, j = 1.5 hz, 1 h), 8.18 (d, j = 8.8 hz, 2 h), 7.97 (dd, j = 8.8, 1.5 hz, 1 h), 7.87 (d, j = 8.3 hz, 2 h), 7.51 (d, j = 7.3 hz, 2 h), 7.47 (d, j = 2.0 hz, 1 h), 7.44 (t, j = 7.6 hz, 2 h), 7.36 (m, j = 6.8 hz, 1 h), 7.307.34 (m, 2 h), 6.79 (d, j = 5.4 hz, 1 h), 5.24 (s, 2 h), 3.623.70 (m, 4 h), 2.902.97 (m, 4 h). Synthesized by general procedure a. flash column chromatography: 510% methanol in dichloromethane, isolated as a yellow solid in 40% yield . H nmr (500 mhz, dmso - d6) ppm 9.19 (br . S., 1 h), 8.78 (d, j = 2.0 hz, 1 h), 8.47 (d, j = 5.4 hz, 1 h), 8.16 (d, j = 8.8 hz, 2 h), 8.12 (dd, j = 8.8, 2.0 hz, 1 h), 7.98 (d, j = 8.8 hz, 1 h), 7.87 (d, j = 8.8 hz, 2 h), 7.457.52 (m, 2 h), 7.307.38 (m, 4 h), 7.20 (td, j = 8.7, 2.7 hz, 1 h), 6.81 (d, j = 5.4 hz, 1 h), 5.28 (s, 2 h), 2.95 (br . S., 4 h), 2.352.43 (m, 4 h), 2.15 (s, 3 h). Synthesized by general procedure a. flash column chromatography: 510% methanol in dichloromethane, isolated as a yellow solid in 33% yield . H nmr (500 mhz, dmso - d6) ppm 9.10 (br . S., 1 h), 8.50 (d, j = 8.3 hz, 2 h), 8.25 (d, j = 2.0 hz, 1 h), 8.16 (d, j = 8.3 hz, 2 h), 7.97 (dd, j = 8.8, 2.0 hz, 1 h), 7.87 (d, j = 8.3 hz, 2 h), 7.457.52 (m, 2 h), 7.297.38 (m, 4 h), 7.20 (td, j = 8.5, 2.0 hz, 1 h), 6.80 (d, j = 5.4 hz, 1 h), 5.28 (s, 2 h), 2.96 (br . S., 4 h), 2.40 (br . Then prep hplc: 595% acetonitrile in water, isolated as a yellow solid in 10% yield . H nmr (500 mhz, dmso - d6) ppm 8.77 (d, j = 2.0 hz, 1 h), 8.46 (d, j = 5.4 hz, 1 h), 8.27 (s, 1 h), 8.078.15 (m, 3 h), 7.97 (d, j = 8.8 hz, 1 h), 7.92 (d, j = 8.3 hz, 2 h), 7.457.52 (m, 2 h), 7.307.38 (m, 4 h), 7.20 (td, j = 8.5, 2.4 hz, 1 h), 6.80 (d, j = 5.4 hz, 1 h), 5.28 (s, 2 h), 3.36 (m, j = 5.2, 2.5, 2.5 hz, 2 h), 3.33 (t, j = 6.3 hz, 2 h), 2.532.57 (m, 2 h), 2.47 (m, j = 5.9 hz, 2 h), 2.22 (s, 3 h), 1.74 (quin, j = 5.9 hz, 2 h). Synthesized by general procedure a. flash column chromatography: 520% methanol in dichloromethane, isolated as a brown solid in 32% yield . H nmr (500 mhz, dmso - d6) ppm 9.06 (s, 1 h), 8.478.52 (m, 2 h), 8.23 (d, j = 2.0 hz, 1 h), 8.12 (d, j = 8.3 hz, 2 h), 7.96 (dd, j = 8.8, 2.0 hz, 1 h), 7.91 (d, j = 8.3 hz, 2 h), 7.457.52 (m, 2 h), 7.297.37 (m, 4 h), 7.20 (td, j = 8.5, 2.4 hz, 1 h), 6.79 (d, j = 4.9 hz, 1 h), 5.27 (s, 2 h), 3.39 (m, j = 3.4 hz, 2 h), 3.313.35 (m, 2 h), 2.63 (br . S., 2 h), 2.58 (br . S., 2 h), 2.29 (br . S., 3 h), 1.741.83 (m, 2 h). Synthesized by general procedure a. flash column chromatography: 3050% ethyl acetate in hexanes, isolated as a biege solid in 59% yield . H nmr (500 mhz, dmso - d6) ppm 9.27 (s, 1 h), 8.71 (s, 1 h), 8.06 (d, j = 2.4 hz, 1 h), 8.03 (dd, j = 8.3, 1.5 hz, 1 h), 7.98 (d, j = 5.4 hz, 1 h), 7.88 (d, j = 8.8 hz, 1 h), 7.76 (dd, j = 8.8, 2.4 hz, 1 h), 7.47 (td, j = 8.1, 6.3 hz, 1 h), 7.40 (t, j = 7.8 hz, 1 h), 7.297.36 (m, 4 h), 7.23 (d, j = 9.3 hz, 1 h), 7.157.21 (m, 2 h), 7.02 (dd, j = 8.3, 2.0 hz, 1 h), 5.23 (s, 2 h), 3.753.82 (m, 4 h), 3.203.26 (m, 4 h). Synthesized by general procedure a. flash column chromatography: 3050% ethyl acetate in hexanes, isolated as an orange solid in 81% yield . H nmr (500 mhz, dmso - d6) ppm 9.21 (s, 1 h), 8.56 (d, j = 8.8 hz, 1 h), 8.13 (d, j = 2.4 hz, 1 h), 8.11 (d, j = 2.0 hz, 1 h), 8.00 (d, j = 5.4 hz, 1 h), 7.94 (dd, j = 8.8, 1.5 hz, 1 h), 7.78 (dd, j = 8.8, 2.4 hz, 1 h), 7.47 (td, j = 8.0, 6.3 hz, 1 h), 7.357.41 (m, 2 h), 7.307.35 (m, 2 h), 7.28 (d, j = 7.8 hz, 1 h), 7.207.25 (m, 2 h), 7.18 (td, j = 8.8, 2.4 hz, 1 h), 7.03 (dd, j = 8.1, 2.2 hz, 1 h), 5.23 (s, 2 h), 3.763.81 (m, 4 h), 3.213.25 (m, 4 h). Lcms found 540.2 [m + h]. Synthesized by general procedure a. flash column chromatography: 2050% ethyl acetate in hexanes, isolated as an orange solid in 55% yield . H nmr (500 mhz, dmso - d6) ppm 9.35 (s, 1 h), 8.86 (s, 1 h), 8.18 (d, j = 8.8 hz, 2 h), 8.12 (dd, j = 8.8, 1.5 hz, 1 h), 8.04 (d, j = 2.4 hz, 1 h), 8.03 (d, j = 5.9 hz, 1 h), 7.96 (d, j = 8.3 hz, 1 h), 7.90 (d, j = 8.3 hz, 2 h), 7.76 (dd, j = 9.3, 2.9 hz, 1 h), 7.50 (d, j = 6.8 hz, 2 h), 7.42 (t, j = 7.3 hz, 2 h), 7.35 (t, j = 7.3 hz, 1 h), 7.25 (d, j = 9.3 hz, 1 h), 7.22 (d, j = 5.9 hz, 1 h), 5.21 (s, 2 h), 3.633.70 (m, 4 h), 2.902.96 (m, 4 h). Synthesized by general procedure a. flash column chromatography: 3050% ethyl acetate in hexanes, isolated as an orange solid in 81% yield . H nmr (500 mhz, dmso - d6) ppm 9.26 (s, 1 h), 8.65 (d, j = 9.3 hz, 1 h), 8.25 (d, j = 1.5 hz, 1 h), 8.16 (d, j = 8.8 hz, 2 h), 8.12 (d, j = 2.9 hz, 1 h), 8.018.07 (m, 2 h), 7.88 (d, j = 8.3 hz, 2 h), 7.78 (dd, j = 9.0, 2.7 hz, 1 h), 7.50 (d, j = 6.8 hz, 2 h), 7.42 (t, j = 7.3 hz, 2 h), 7.35 (t, j = 7.3 hz, 1 h), 7.27 (d, j = 5.9 hz, 1 h), 7.23 (d, j = 9.3 hz, 1 h), 5.20 (s, 1 h), 3.623.69 (m, 4 h), 2.892.97 (m, 4 h). Synthesized by general procedure a. flash column chromatography: ethyl acetate, isolated as a cream colored solid in 53% yield . H nmr (500 mhz, cdcl3) ppm 8.118.14 (m, 2 h), 7.797.89 (m, 7 h), 7.54 (dd, j = 8.8, 2.4 hz, 1 h), 7.36 (td, j = 7.8, 5.9 hz, 1 h), 7.28 (s, 1 h), 7.217.27 (m, 2 h), 7.18 (d, j = 5.9 hz, 1 h), 7.03 (td, j = 8.3, 2.4 hz, 1 h), 6.97 (d, j = 8.8 hz, 1 h), 5.15 (s, 2 h), 3.08 (br . S., 4 h), 2.49 (t, j = 4.6 hz, 4 h), 2.28 (s, 3 h). Synthesized by general procedure a. flash column chromatography: ethyl acetate, isolated as a pale orange solid in 52% yield . H nmr (500 mhz, cdcl3) ppm 8.14 (d, j = 5.9 hz, 1 h), 8.02 (d, j = 8.8 hz, 1 h), 7.95 (d, j = 2.0 hz, 1 h), 7.837.91 (m, 5 h), 7.76 (dd, j = 8.5, 1.7 hz, 1 h), 7.50 (dd, j = 8.8, 2.4 hz, 1 h), 7.37 (td, j = 7.8, 5.9 hz, 1 h), 7.227.27 (m, 2 h), 7.21 (d, j = 5.9 hz, 1 h), 7.007.07 (m, 2 h), 6.98 (d, j = 8.8 hz, 1 h), 5.16 (s, 2 h), 3.11 (br . S., 4 h), 2.52 (t, j = 4.6 hz, 4 h), 2.29 (s, 3 h). Synthesized by general procedure a. flash column chromatography: 5% methanol in dichloromethane, isolated as an orange solid in 73% yield . H nmr (500 mhz, dmso - d6) ppm 9.36 (s, 1 h), 8.85 (s, 1 h), 8.098.14 (m, 3 h), 8.05 (d, j = 2.4 hz, 1 h), 8.02 (d, j = 5.9 hz, 1 h), 7.917.97 (m, 3 h), 7.77 (dd, j = 8.8, 2.9 hz, 1 h), 7.47 (td, j = 8.1, 6.3 hz, 1 h), 7.297.36 (m, 2 h), 7.24 (d, j = 9.3 hz, 1 h), 7.22 (d, j = 5.4 hz, 1 h), 7.18 (td, j = 9.3, 2.0 hz, 1 h), 5.24 (s, 2 h), 3.353.38 (m, 2 h), 3.313.35 (m, 2 h), 2.542.58 (m, 2 h), 2.472.49 (m, 2 h), 2.23 (s, 3 h), 1.75 (quin, j = 5.8 hz, 2 h). Synthesized by general procedure a. flash column chromatography: 5% methanol in dichloromethane, isolated as a pale yellow solid in 68% yield . H nmr (500 mhz, dmso - d6) ppm 9.26 (s, 1 h), 8.64 (d, j = 8.8 hz, 1 h), 8.23 (d, j = 1.5 hz, 1 h), 8.13 (d, j = 2.4 hz, 1 h), 8.10 (d, j = 8.3 hz, 2 h), 8.008.06 (m, 2 h), 7.92 (d, j = 8.3 hz, 2 h), 7.79 (dd, j = 9.0, 2.7 hz, 1 h), 7.47 (td, j = 8.3, 6.3 hz, 1 h), 7.297.35 (m, 2 h), 7.27 (d, j = 5.9 hz, 1 h), 7.22 (d, j = 9.3 hz, 1 h), 7.18 (td, j = 8.7, 2.7 hz, 1 h), 5.23 (s, 2 h), 3.36 (m, j = 4.9, 2.4, 2.4 hz, 2 h), 3.313.35 (m, 2 h), 2.532.58 (m, 2 h), 2.462.49 (m, 2 h), 2.23 (s, 3 h), 1.75 (quin, j = 5.8 hz, 2 h). Synthesized by general procedure b. flash column chromatography: 210% methanol in dichloromethane, isolated as a yellow solid in 67% yield . S., 1 h), 8.30 (s, 1 h), 8.26 (d, j = 4.9 hz, 1 h), 7.95 (dd, j = 9.0, 1.2 hz, 1 h), 7.487.59 (m, 1 h), 7.327.41 (m, 2 h), 7.29 (t, j = 7.8 hz, 1 h), 7.177.24 (m, 2 h), 7.087.17 (m, 3 h), 7.02 (td, j = 8.4, 2.2 hz, 1 h), 6.876.94 (m, 2 h), 5.13 (s, 2 h), 3.763.82 (m, 4 h), 3.093.16 (m, 4 h). Synthesized by general procedure b. flash column chromatography: 28% methanol in dichloromethane, then 50100% ethyl acetate in hexanes, isolated as a yellow solid in 38% yield . S., 1 h), 8.31 (d, j = 8.8 hz, 1 h), 7.96 (d, j = 8.8 hz, 1 h), 7.387.50 (m, 3 h), 7.237.35 (m, 5 h), 7.15 (d, j = 8.8 hz, 1 h), 7.06 (m, j = 8.3, 2.0 hz, 2 h), 5.24 (s, 2 h), 3.903.96 (m, 4 h), 3.273.32 (m, 4 h). Synthesized by general procedure a. flash column chromatography: 2% methanol and 18% acetone in dichloromethane, then 1% methanol in ethyl acetate, isolated as a yellow solid in 8% yield . S., 2 h), 8.098.40 (m, 4 h), 7.91 (d, j = 7.8 hz, 2 h), 7.487.60 (m, 3 h), 7.44 (t, j = 7.6 hz, 3 h), 7.37 (t, j = 7.3 hz, 2 h), 5.26 (s, 2 h), 3.613.70 (m, 4 h), 2.872.99 (m, 4 h). Synthesized by general procedure b. flash column chromatography: 28% methanol in dichloromethane, then 10100% ethyl acetate in dichloromethane, isolated as a yellow solid in 71% yield . S., 1 h), 8.40 (d, j = 8.8 hz, 1 h), 8.05 (d, j = 8.3 hz, 2 h), 8.01 (d, j = 8.8 hz, 1 h), 7.95 (d, j = 8.3 hz, 2 h), 7.52 (d, j = 7.3 hz, 2 h), 7.48 (br . S., 1 h), 7.43 (t, j = 7.6 hz, 2 h), 7.36 (t, j = 7.3 hz, 1 h), 7.30 (d, j = 8.8 hz, 1 h), 7.17 (d, j = 8.8 hz, 1 h), 5.25 (s, 2 h), 3.773.82 (m, 4 h), 3.073.12 (m, 4 h). Synthesized by general procedure a. flash column chromatography: 510% methanol in dichloromethane, then 1030% methanol in ethyl acetate, isolated as a yellow solid in 15% yield . H nmr (500 mhz, dmso - d6) 9.81 (s, 1 h), 9.01 (s, 1 h), 8.82 (s, 1 h), 8.218.34 (m, 4 h), 7.88 (d, j = 8.3 hz, 2 h), 7.61 (d, j = 2.0 hz, 1 h), 7.437.53 (m, 2 h), 7.297.40 (m, 3 h), 7.20 (t, j = 8.8 hz, 1 h), 5.29 (s, 2 h), 2.95 (br . Synthesized by general procedure b. flash column chromatography: 510% methanol in dichloromethane, isolated as a yellow solid in 50% yield . S., 1 h), 8.39 (d, j = 8.8 hz, 1 h), 7.938.04 (m, 5 h), 7.48 (br . S., 1 h), 7.42 (td, j = 8.1, 5.9 hz, 1 h), 7.31 (d, j = 7.8 hz, 2 h), 7.26 (d, j = 9.8 hz, 1 h), 7.16 (d, j = 8.8 hz, 1 h), 7.06 (td, j = 8.5, 2.0 hz, 1 h), 5.25 (s, 2 h), 3.483.52 (m, 2 h), 3.46 (t, j = 6.3 hz, 2 h), 2.732.77 (m, 2 h), 2.692.73 (m, 2 h), 2.40 (s, 3 h), 1.95 (dt, j = 11.4, 5.8 hz, 2 h). Lcms found 618.1 [m + h]. Synthesized by general procedure b. flash column chromatography: 210% methanol in dichloromethane, isolated as a yellow solid in 61% yield s., 1 h), 8.62 (s, 1 h), 8.31 (br . S., 1 h), 8.14 (d, j = 8.8 hz, 1 h), 8.02 (d, j = 8.3 hz, 2 h), 7.94 (d, j = 8.8 hz, 2 h), 7.48 (br . S., 1 h), 7.387.45 (m, 2 h), 7.237.33 (m, 3 h), 7.16 (d, j = 8.8 hz, 1 h), 7.06 (td, j = 8.4, 2.2 hz, 1 h), 5.25 (s, 2 h), 3.473.50 (m, 2 h), 3.45 (t, j = 6.6 hz, 2 h), 2.702.74 (m, 2 h), 2.662.70 (m, 2 h), 2.38 (s, 3 h), 1.891.96 (m, 2 h). Synthesized by general procedure b. flash column chromatography: 515% methanol in dichloromethane, isolated as a yellow solid in 52% yield . S., 1 h), 8.39 (d, j = 8.8 hz, 1 h), 7.938.04 (m, 5 h), 7.48 (br . S., 1 h), 7.42 (td, j = 8.1, 5.9 hz, 1 h), 7.31 (d, j = 7.8 hz, 2 h), 7.26 (d, j = 9.8 hz, 1 h), 7.16 (d, j = 8.8 hz, 1 h), 7.06 (td, j = 8.5, 2.0 hz, 1 h), 5.25 (s, 2 h), 3.483.52 (m, 2 h), 3.46 (t, j = 6.3 hz, 2 h), 2.732.77 (m, 2 h), 2.692.73 (m, 2 h), 2.40 (s, 3 h), 1.95 (dt, j = 11.4, 5.8 hz, 2 h). Lcms found 632.1 [m + h]. Synthesized by general procedure c. flash column chromatography: 4080% ethyl acetate in hexanes, then prep hplc 3050% acetonitrile in water, isolated as a yellow solid in 9% yield . H nmr (500 mhz, dmso - d6) 9.27 (s, 1 h), 9.14 (s, 1 h), 8.78 (s, 1 h), 8.27 (dd, j = 8.3, 1.5 hz, 1 h), 8.16 (d, j = 2.4 hz, 1 h), 8.09 (d, j = 8.3 hz, 1 h), 7.82 (dd, j = 9.0, 2.7 hz, 1 h), 7.407.50 (m, 2 h), 7.39 (s, 1 h), 7.307.36 (m, 3 h), 7.27 (d, j = 8.8 hz, 1 h), 7.18 (td, j = 8.7, 2.2 hz, 1 h), 7.07 (dd, j = 8.3, 2.0 hz, 1 h), 5.25 (s, 2 h), 3.763.82 (m, 4 h), 3.223.27 (m, 4 h). Synthesized by general procedure c. flash column chromatography: 4080% ethyl acetate in hexanes, then prep hplc 3050% acetonitrile in water, isolated as a pale yellow solid in 14% yield . H nmr (500 mhz, dmso - d6) 9.24 (s, 1 h), 9.16 (s, 1 h), 8.62 (d, j = 8.3 hz, 1 h), 8.308.36 (m, 2 h), 8.21 (d, j = 2.4 hz, 1 h), 7.84 (dd, j = 9.0, 2.7 hz, 1 h), 7.47 (td, j = 7.8, 5.9 hz, 1 h), 7.387.43 (m, 2 h), 7.297.36 (m, 3 h), 7.26 (d, j = 9.3 hz, 1 h), 7.18 (td, j = 8.7, 2.2 hz, 1 h), 7.06 (dd, j = 8.3, 2.0 hz, 1 h), 5.25 (s, 2 h), 3.753.83 (m, 4 h), 3.213.28 (m, 4 h). Synthesized by general procedure c. flash column chromatography: 50100% ethyl acetate in hexanes, then prep hplc 3050% acetonitrile in water, isolated as a yellow solid in 9% yield . H nmr (500 mhz, dmso - d6) 9.36 (s, 1 h), 9.18 (s, 1 h), 8.93 (s, 1 h), 8.36 (dd, j = 8.3, 1.5 hz, 1 h), 8.20 (d, j = 8.3 hz, 2 h), 8.17 (d, j = 8.3 hz, 1 h), 8.15 (d, j = 2.9 hz, 1 h), 7.94 (d, j = 8.3 hz, 2 h), 7.82 (dd, j = 9.0, 2.7 hz, 1 h), 7.50 (d, j = 7.3 hz, 2 h), 7.42 (t, j = 7.6 hz, 2 h), 7.35 (t, j = 7.3 hz, 1 h), 7.30 (d, j = 9.3 hz, 1 h), 5.22 (s, 2 h), 3.633.69 (m, 4 h), 2.902.98 (m, 4 h). Synthesized by general procedure c. flash column chromatography: 5100% ethyl acetate in hexanes, then prep hplc 3050% acetonitrile in water, isolated as a yellow solid in 23% yield . H nmr (500 mhz, dmso - d6) 9.28 (s, 1 h), 9.19 (s, 1 h), 8.70 (d, j = 8.8 hz, 1 h), 8.45 (d, j = 2.0 hz, 1 h), 8.41 (dd, j = 8.5, 1.7 hz, 1 h), 8.20 (d, j = 2.4 hz, 1 h), 8.18 (d, j = 8.3 hz, 2 h), 7.91 (d, j = 8.3 hz, 2 h), 7.83 (dd, j = 8.8, 2.4 hz, 1 h), 7.50 (d, j = 6.8 hz, 2 h), 7.42 (t, j = 7.3 hz, 2 h), 7.35 (t, j = 6.8 hz, 1 h), 7.27 (d, j = 8.8 hz, 1 h), 5.21 (s, 2 h), 3.633.69 (m, 4 h), 2.912.97 (m, 4 h). Synthesized by general procedure b. flash column chromatography: 37% methanol in dichloromethane, then prep hplc 595% acetonitrile in water, isolated as an orange solid in 39% yield . S., 1 h), 9.18 (s, 1 h), 8.91 (s, 1 h), 8.35 (d, j = 7.3 hz, 1 h), 8.128.21 (m, 6 h), 7.93 (d, j = 8.3 hz, 2 h), 7.83 (d, j = 6.8 hz, 1 h), 7.47 (td, j = 8.3, 6.3 hz, 1 h), 7.307.36 (m, 2 h), 7.28 (d, j = 8.8 hz, 1 h), 7.18 (td, j = 8.7, 2.2 hz, 1 h), 5.25 (s, 2 h), 2.96 (br . S., 4 h), 2.352.43 (m, 4 h), 2.15 (s, 3 h). Synthesized by general procedure b. flash column chromatography: 310% methanol in dichloromethane, then prep hplc 595% acetonitrile in water, isolated as a dull yellow solid in 28% yield . H nmr (500 mhz, dmso - d6) 9.28 (s, 1 h), 9.20 (s, 1 h), 8.70 (d, j = 8.3 hz, 1 h), 8.47 (d, j = 2.0 hz, 1 h), 8.42 (dd, j = 8.5, 1.7 hz, 1 h), 8.21 (d, j = 2.9 hz, 1 h), 8.168.20 (m, 3 h), 7.91 (d, j = 8.3 hz, 2 h), 7.84 (dd, j = 9.0, 2.7 hz, 1 h), 7.48 (td, j = 8.3, 6.3 hz, 1 h), 7.307.36 (m, 2 h), 7.27 (d, j = 9.3 hz, 1 h), 7.18 (td, j = 8.7, 2.7 hz, 1 h), 5.25 (s, 2 h), 2.96 (br . S., 4 h), 2.39 (t, j = 4.6 hz, 4 h), 2.15 (s, 3 h). Synthesized by general procedure b. flash column chromatography: 510% methanol in dichloromethane, then prep hplc 595% acetonitrile in water, isolated as an orange solid in 29% yield . H nmr (500 mhz, meoh - d4) 8.94 (s, 1 h), 8.73 (s, 1 h), 8.25 (s, 2 h), 8.20 (dd, j = 8.5, 1.2 hz, 1 h), 8.008.06 (m, 3 h), 7.95 (d, j = 8.3 hz, 2 h), 7.88 (d, j = 2.4 hz, 1 h), 7.59 (dd, j = 8.8, 2.4 hz, 1 h), 7.38 (td, j = 8.1, 5.9 hz, 1 h), 7.28 (d, j = 7.8 hz, 1 h), 7.23 (d, j = 9.8 hz, 1 h), 6.997.06 (m, 2 h), 5.18 (s, 2 h), 3.603.66 (m, 2 h), 3.47 (t, j = 6.6 hz, 2 h), 3.223.28 (m, 4 h), 2.77 (s, 3 h), 2.142.21 (m, 2 h). Synthesized by general procedure b. flash column chromatography: 515% methanol in dichloromethane, then prep hplc 595% acetonitrile in water, isolated as a light yellow solid in 29% yield . H nmr (500 mhz, meoh - d4) 8.99 (s, 1 h), 8.51 (d, j = 8.3 hz, 1 h), 8.25 (s, 1 h), 8.178.22 (m, 2 h), 7.97 (s, 4 h), 7.91 (d, j = 2.9 hz, 1 h), 7.587.63 (m, 1 h), 7.39 (td, j = 7.8, 5.9 hz, 1 h), 7.29 (d, j = 7.8 hz, 1 h), 7.24 (d, j = 9.3 hz, 1 h), 7.007.08 (m, 2 h), 5.20 (s, 2 h), 3.543.59 (m, 2 h), 3.47 (t, j = 6.6 hz, 2 h), 2.973.04 (m, 4 h), 2.60 (s, 3 h), 2.06 (quin, j = 6.3 hz, 2 h). Synthesized by general procedure d. flash column chromatography: 030% methanol in dichloromethane, isolated as a yellow solid in 20% yield . H nmr (400 mhz, dmso - d6) 9.91 (br, s, 1h), 8.73 (s, 1h), 8.55 (s, 1h), 8.18 (d, j = 8.8 hz, 1h), 7.98 (d, j = 8.8 hz, 1h), 7.55 (d, j = 2.4 hz, 1h), 7.457.50 (m, 1h), 7.377.41 (m, 1h), 7.307.35 (m, 6h), 7.177.22 (m, 1h), 7.02 (d, j = 8.0 hz, 1h), 5.29 (s, 2h), 3.763.79 (m, 4h), 3.203.23 (m, 4h). Lcms found 565.0 [m + h]. Synthesized by general procedure d. flash column chromatography: 020% ethyl acetate in hexanes, isolated as a yellow solid in 50% yield . H nmr (400 mhz, dmso - d6) 9.99 (s, 1h), 8.87 (s, 1h), 8.55 (s, 1h), 8.24 (d, j = 8.8 hz, 1h), 8.14 (d, j = 8.0 hz, 2h), 8.01 (d, j = 8.8, 1h), 7.85 (d, j = 8.4 hz, 2h), 7.467.51 (m, 3h), 7.39 (t, j = 7.2 hz, 2h), 7.297.34 (m, 3h), 5.22 (s, 2h), 3.603.63 (m, 4h), 2.872.89 (m, 4h). Lcms found 611.1 [m + h]. Synthesized by general procedure d. flash column chromatography: 030% methanol in dichloromethane, isolated as a yellow solid in 16% yield . H nmr (400 mhz, dmso - d6) 10.01 (br, s, 1h), 8.89 (s, 1h), 8.59 (s, 1h), 8.27 (d, j = 8.8 hz, 1h), 8.16 (d, j = 8.0 hz, 2h), 8.05 (d, j = 8.8 hz, 1h), 7.88 (d, j = 8.0 hz, 2h), 7.55 (br, s, 1h), 7.457.70 (m, 1h), 7.317.35 (m 4h), 7.177.21 (m, 1h), 5.30 (s, 2h), 2.95 (br, s, 4h), 2.38 (br, 4h), 2.14 (s, 3h). Synthesized by general procedure d. flash column chromatography: 030% methanol in dichloromethane, isolated as a yellow solid in 32% yield . H nmr (400 mhz, cdcl3) 8.71 (s, 1h), 8.14 (d, j = 8.8 hz, 1h), 8.08 (d, j = 2.4 hz, 1h), 7.99 (dd, j = 8.8, 2.2 hz, 1h), 7.807.82 (m, 2h), 7.66 (d, j = 8.8 hz, 2h), 7.47 (s, 1h), 7.41 (d, j = 2 hz, 1h), 7.367.39 (m, 1h), 7.23 (s, 1h), 7.197.20 (m, 1h), 7.17 (d, j = 2 hz, 1h), 7.05 (dd, j = 8.0,2.4 hz, 1h), 7.01 (d, j = 8.8 hz, 1h), 5.19 (s, 2h), 3.363.40 (m, 4h), 2.562.62 (m, 4h), 2.33 (s, 3h), 1.811.85 (m, 2h). Synthesized by general procedure e, isolated as a yellow solid in 12% yield . H nmr (500 mhz, dmso - d6) ppm 9.68 (s, 1 h), 8.59 (s, 1 h), 8.01 (d, j = 2.4 hz, 1 h), 7.94 (s, 1 h), 7.67 (dd, j = 8.8, 2.4 hz, 1 h), 7.47 (m, 1 h), 7.40 (m, 1 h), 7.37 (d, j = 7.8 hz, 1 h), 7.32 (m, 2 h), 7.25 (m, 2 h), 7.18 (td, j = 9.3, 2.4 hz, 1 h), 7.08 (dd, j = 7.8, 2.4 hz, 1 h), 5.25 (s, 2 h), 3.78 (m, 4 h), 3.23 (m, 4 h), lcms found 547.0, [m + h]. H nmr (500 mhz, dmso - d6) 9.64 (s, 1h), 8.49 (s, 1h), 8.18 (s, 1h), 8.05 (d, j = 2.93 hz, 1h), 7.71 (dd, j = 2.44, 8.79 hz, 1h), 7.447.50 (m, 1h), 7.137.41 (m, 7h), 7.04 (dd, j = 1.95, 8.30 hz, 1h), 5.25 (s, 2h), 3.78 (t, j = 4.7 hz, 4h), 3.21 (t, j = 4.7 hz, 4h). Synthesized by general procedure e, isolated as a yellow solid in 8% yield . H nmr (400 mhz, dmso - d6) ppm 8.61 (s, 1 h), 9.83 (s, 1 h), 8.15 (m, 3 h), 7.98 (s, 1 h), 7.88 (d, j = 8.8 hz, 2 h), 7.65 (m, 1 h), 7.49 (m, 2 h), 7.42 (t, j = 7.3 hz, 2 h), 7.35 (m, 1 h), 7.27 (d, j = 8.8 hz, 1 h), 5.23 (s, 2 h), 3.65 (m, 4 h), 2.94 (m, 4 h). Synthesized by general procedure e, isolated as a yellow solid in 40% yield . H nmr (500 mhz, dmso - d6) ppm 9.79 (s, 1 h), 8.52 (s, 1 h), 8.41 (s, 1 h), 8.03 (d, j = 2.0 hz, 1 h), 7.97 (d, j = 8.3 hz, 2 h), 7.88 (d, j = 8.3 hz, 2 h), 7.68 (dd, j = 8.8, 2.4 hz, 1 h), 7.49 (d, j = 7.8 hz, 2 h), 7.42 (t, j = 7.6 hz, 2 h), 7.35 (t, j = 7.3 hz, 1 h), 7.29 (d, j = 9.3 hz, 1 h), 5.22 (s, 2 h), 3.65 (m, 4 h), 2.94 (m, 4 h). Synthesized by general procedure e, isolated as a yellow solid in 36% yield . H nmr (400 mhz, dmso - d6) ppm 9.84 (s, 1 h), 8.62 (s, 1 h), 8.14 (m, 3 h), 7.99 (d, j = 2.2 hz, 1 h), 7.87 (d, j = 8.8 hz, 2 h), 7.66 (dd, j = 9.2, 2.6 hz, 1 h), 7.47 (m, 1 h), 7.32 (m, 2 h), 7.26 (d, j = 8.8 hz, 1 h), 7.19 (td, j = 8.8, 2.2 hz, 1 h), 5.26 (s, 2 h), 2.95 (m, 4 h), 2.38 (m, 4 h), 2.14 (s, 3 h). Synthesized by general procedure e, isolated as a yellow solid in 25% yield . H nmr (500 mhz, dmso - d6) ppm 9.79 (s, 1 h), 8.52 (s, 1 h), 8.41 (s, 1 h), 8.05 (d, j = 2.4 hz, 1 h), 7.95 (d, j = 8.3 hz, 2 h), 7.87 (d, j = 8.3 hz, 2 h), 7.69 (dd, j = 8.8, 2.4 hz, 1 h), 7.47 (m, 1 h), 7.32 (m, 2 h), 7.28 (d, j = 8.8 hz, 1 h), 7.18 (td, j = 8.5, 2.4 hz, 1 h), 5.24 (s, 2 h), 2.95 (m, 4 h), 2.37 (m, 4 h), 2.14 (s, 3 h). Synthesized by general procedure e, isolated as a pale yellow solid in 4% yield . The product was separated by silica column chromatography (dichloromethane / methanol) and purified by hplc to obtain the formic acid salt . H nmr (500 mhz, dmso - d6) ppm 9.82 (s, 1 h), 8.61 (s, 1 h), 8.22 (s, 1 h), 8.068.13 (m, 3 h), 7.99 (d, j = 2.4 hz, 1 h), 7.91 (d, j = 8.8 hz, 2 h), 7.66 (dd, j = 9.0, 2.7 hz, 1 h), 7.47 (td, j = 8.3, 6.3 hz, 1 h), 7.297.36 (m, 2 h), 7.26 (d, j = 8.8 hz, 1 h), 7.18 (td, j = 8.7, 2.2 hz, 1 h), 5.26 (s, 2 h), 3.35 (dt, j = 5.1, 2.3 hz, 2 h), 3.32 (t, j = 6.1 hz, 2 h), 2.532.58 (m, 2 h), 2.48 (d, j = 5.9 hz, 2 h), 2.23 (s, 3 h), 1.74 (quin, j = 5.9 hz, 2 h). Synthesized by general procedure e, isolated as a pale yellow solid in 10% yield . The product was separated by silica column chromatography (dichloromethane / methanol) and purified by hplc to obtain the formic acid salt . H nmr (500 mhz, cdcl3/cd3od) ppm 8.46 (s, 1 h), 8.10 (s, 1 h), 7.89 (m, 5 h), 7.60 (m, 1 h), 7.39 (m, 1 h), 7.28 (d, j = 7.8 hz, 1 h), 7.24 (d, j = 9.3 hz, 1 h), 7.04 (m, 2 h), 5.20 (s, 2 h), 3.57 (m, 2 h), 3.45 (t, j = 6.6 hz, 2 h), 3.05 (m, 4 h), 2.63 (s, 3 h), 2.08 (m, 2 h).
Acute appendicitis is considered as the most frequent cause of acute abdomen in young patients with a slight predominance in males . Although its clinical diagnosis is simple, 20 - 30% of appendicitis cases are reported to be gangrenous or perforated because of delayed diagnosis, which can lead to increased morbidity and mortality . Additionally, up to 10% of patients especially older ones, immunocompromised patients, and pregnant women are diagnosed with atypical presentations such as appendiceal mass which requires long hospital admissions, intravenous antibiotics, and often needs percutaneous drainage . Using colonoscopy for the diagnosis and treatment of asymptomatic acute appendicitis and appendiceal abscess are rare . In unusual appendicitis cases, especially in the presence of abdominal comorbidities, colonoscopy is used; although colonoscopy is not considered as the first line intervention because of complications risk such as bleeding or perforation . Furthermore non - invasive tests such as ultrasonography and computed tomography with more sensitivity can be used . We report an atypical case of appendiceal abscess, which was diagnosed during colonoscopy . A 30-year - old man referred for evaluation to our center with epigastric pain from three days prior to his evaluation . The pain was nonspecific and colicky distributed in periumblical region, did not radiate to any site and was unrelated to bowel movements . He had experienced also nausea, anorexia and intermittent vomiting . There was a history of marijuana and alcohol usage from several years ago . Upon physical examination, he was ill with restlessness . Other laboratory values such as hemoglobin, platelets, serum amylase, erythrocyte sedimentation rate, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and electrolytes were normal . His workup on admission including; chest x - ray, abdominal x - ray, and abdominal ultrasonography were reported normal . An upper gastrointestinal endoscopy was performed after four days of admission that was normal . Within second week after admission, the appendiceal orifice was discovered in the floor of cecum which was draining pus into the lumen (figure 1). Purulent discharge from the appendiceal orifice before (up) and after (down) pressing by forceps (arrow) result of the biopsies of the mucosa at the base of the appendix showed mild nonspecific inflammation with some necrotic materials and staphylococcus organism . The biopsies of the mucosa at terminal ileum, left and right colon demonstrated mild nonspecific inflammation . A second abdominal ultrasonography was performed that showed a fluid collection (about 70 40 mm) in favor of appendiceal abscess in right lower quadrant (figure 2). The patient was scheduled for spiral abdomen - pelvic computed tomographic scan which showed a hypodense well defined collection with air fluid level (about 70 40 30 mm) in right lower quadrant . The collection was extended to pelvic cavity associated with inflammation of the surrounding fat in favor of appendiceal abscess (figure 3). Ultrasonography showing a fluid collection in favor of appendiceal abscess computed tomographic scan showing a hypodense well defined collection with air fluid level associated with inflammation of surrounding fat in favor of appendiceal abscess (arrow). One week after antibiotic therapy a third abdominal ultrasonography was performed that showed a smaller fluid collection (about 40 15 mm) around the appendix . Three weeks after antibiotic therapy the patient was asymptomatic with normal white blood cell count . On the 5th week after antibiotic therapy, exploratory laparotomy did not show any mass or collection in the appendiceal region and elective appendectomy was performed . After 3 months of follow - up the patient was well without any complaint or physical finding . Acute appendicitis is one of the most frequent causes of acute abdomen . Despite the use of scores in the radiological evaluation, and laparoscopic advances, in a cohort study of patients with appendiceal abscess or phlegmon due to acute appendicitis concluded that initial conservative treatment should be considered . Colonoscopy is reported as a diagnostic and therapeutic approach for delayed or atypical presentations of acute appendicitis . Our patient is one of these unusual cases among whom complicated acute appendicitis was incidentally diagnosed by colonoscopy . Although the appendiceal orifice is routinely examined during colonoscopy, however there are only few published reports describing acute appendicitis diagnosis in the appendiceal orifice . In a review article, khawaja fi recommended that colonoscopy should only be done in selected patients with atypical appendicitis or uncertain diagnosis of appendiceal perforation . Benatta ma reported an atypical case of pericecal appendiceal abscess diagnosed and treated at colonoscopy, while initial presentation was without any suggestive symptoms of appendicitis . Wade ad et al reported an atypical case of appendicitis in a patient with ulcerative colitis diagnosed during colonoscopy . In this report christoffersen mmw et al reported an atypical case of periappendicular abscess diagnosed and treated at colonoscopy, while the patient was admitted for colonoscopy polyp control without any suggestive symptoms . Kawahara y et al reported a case of appendiceal abscess with draining pus from appendiceal into the lumen diagnosed at colonoscopy and ct scan . In this report because of spontaneous drainage of abscess into the lumen, the appendiceal abscess had resolved by the time of surgery . Kapral c et al . Reported a case of pericecal abscess that treated by drainage at colonoscopy and antibiotic therapy . They intubated the appendix by sphincteroltomy catheter which was placed into the abscess cavity . Petro m reported a case of the earliest stage of acute appendicitis in an asymptomatic patient undergoing colonoscopy for evaluation of colorectal cancer . For evaluation of the role of colonoscopy in the diagnosis of atypical appendicitis, a retrospective study in 21 patients performed by chang hs et al . Some of acute appendicitis cases are reported to be complicated, due to delayed diagnosis . This unusual case highlights a patient with an appendiceal abscess diagnosed by colonoscopy . Because of spontaneous drainage of abscess into the lumen of colon and antibiotic therapy, although the diagnostic and therapeutic role of colonoscopy in these patients is uncertain, however, it s use can be selected in special situations in the future.
They possess binding sites specific to carbohydrates and have the capacity to interact with molecules of biological fluids and those present in cellular surfaces . Lectins can substitute natural linkers and activate cellular responses through different roads of intracellular signalling or endocytosis of formed compounds [3, 4]. Camaratu or camaratuba bean (cratylia mollis) is forage of the northeast semi - arid region from brazil . Molecular forms of c. mollis seed lectin (cramoll 1, cramoll 2, cramoll 3, and cramoll 4) have been highly purified and characterized (de souza et al ., cramoll 1, cramoll 2, and cramoll 4 specifically bind glucose / mannose, while cramoll 3 is a galactose - specific glycoprotein . Preparations containing isoforms (cramoll 1,4) and isolectins (cramoll 1,2,3), as well as cramoll 1, have been studied in structural analyses and for the most several biotechnological applications (de souza et al ., 2003) [612]. The use of nuclear technique for the evaluation of biomolecules and medicines although old is not widely used even nowadays . Since the discovery of the element technetium, in 1947 scintigraphy imaging provides a dimensional method for exactly locating gamma emitters in a noninvasive procedure under in vivo conditions . For the characterization of drugs, and biomolecules, molecular imaging techniques are extremely helpful to follow biodistribution in experimental animal studies . In this study, the isolectins cramol 1,4 were radiolabeled with tc-99 m for the evaluation of the biodistribution and in order to forecast a possible use as radiopharmaceutical in healing process . The labeling processes were made using 150 l of cramol (isoform 1,4) solution incubated with stannous chloride (sncl2) solutions (80 l / ml) (sigma - aldrich) for 20 minutes at room temperature . Then, this solution was incubated with 100 ci (approximately 300 l) of technetium-99 m (ipen / cnen) for other 10 minutes in order to label the cramol 1,4 (here just called cramol) with tc-99 m . In order to characterize the labeled cramol, thin - layer chromatography (tlc) was made using paper whatman no . The tlc was performed using 2 l of a labeled sample in acetone (proquimios) as a mobile phase . The radioactivity of the strips was verified in a gamma counter (packard, cobra ii) as described in table 1 . The institutional review board and the animal ethics committee approved the study protocol for this study . The labeled samples (3,7 mbq/0,2 ml) planar images were obtained 1 hour after injection at a millennium gamma camera (ge healthcare, cleveland, usa). Counts were acquired for 5 min in a 15% window centered at 140 kev (figure 1). Then, animals were sacrificed, their were organs removed and weighted, and the radioactivity uptake was counted in a gamma counter (packard - cobra ii). Results were expressed as the percentage of injected dose per organ and percent of injected dose per gram of tissue (table 2, figures 2 and 3)., the results showed that the acetone can be used for this purpose . In a triplicate test, the free pertechnetate was eluted to the top of the chromatograms, and the cramol stayed in the bottom . Moreover, the results observed in the chromatogram elucidated the question about the efficacy of the labeling process, corroborating that the cramol was efficiently labeled with tc-99 m . Tc - cramol scintigraphy 1 h after injection shows liver, kidneys, and bladder . As we can see, the great part of the lectin was processed by the liver . This is absolutely normal especially for injectable drugs, since almost all the drugs are first processed by the hepatic system (figure 2). Otherwise, when dissecting, weighting and counting the organs, a suspicious data appeared . As a protein, we were expecting that its clearance would be made by the kidney, and in fact, it occurred . However, the rate between the bowel and the kidney is over the double . In this case, the clearance time would be reduced and the reabsorption of the drug could be facilitated by the microvilli of the bowel . In this case a dosage error could occur or even a toxic reaction due its accumulation in the bowel . In both cases, further studies should be made to clarify this mechanism and the relevance of that information in a developed drug based on lectin similar to the cramol 1,4 . It is interesting to notice, analyzing both figures 1 and 2, that the lectin cannot pass the hematoencephalic barrier . And in this case, the lectin does not represent a risk for the brain function . The radiolabeling of the cramol showed that this lectin has an uncommon behavior specially related to the clearance mechanism that we believe must be more studied . It also showed that the nuclear technique can be very helpful for the development of drugs and medicines.
The comingling of swine from numerous premises with varied management practices and their interaction with large numbers of exhibitors and visitors make agricultural fairs an ideal setting for the intra- and inter - species transmission of influenza a viruses (iavs) between swine and human populations . The frequency with which intra- and inter - species iav transmission occurs in these settings is likely due to a myriad of factors, including but not limited to, management practices, iav strain, and animal and/or human population immunity . Swine is a host species in which reassortment of iav genomic segments may lead to emergent novel strains, since they are susceptible to infection from swine, human and avian influenza a viruses . For this reason, limiting the bidirectional zoonotic transmission of these viruses at agricultural fairs is important for public and animal health . The association between human and swine influenza was reported after respiratory disease similar to the human disease was noted in pigs during the 1918 human spanish flu pandemic . Iav subsequently became established in the united states swine population with the relatedness of the swine and human viruses being established in 1931 . For nearly 80 years, classical swine influenza h1n1 virus was the dominant endemic iav strain in the north america swine population . In 1998, triple reassortant h3n2 iavs containing polymerase basic 1 (pb1), hemagglutinin (ha) and neuraminidase (na) gene segments from human iav lineages, polymerase basic 2 (pb2) and polymerase acidic (pa) genes from avian lineages, and nucleoprotein (np), matrix (m) and non - structural (ns) gene segments from swine lineages, emerged in north american swine . Subsequently, this lineage became established in us and canadian swine herds and has resulted in an increased rate of genetic and antigenic change among swine - origin iavs . Reported cases of humans contracting iav infections directly or indirectly from pigs have been historically sporadic and these variant iavs showed limited capability for sustained human - to - human transmission . However, the emergence of the influenza a(h1n1)pdm09 virus, a strain containing gene segments from north american and european swine lineages, illustrated the pandemic potential of swine lineage iavs crossing the species barrier to humans . While a(h1n1)pdm09 rapidly spread worldwide and became endemic in the human population, sequencing of this virus has to date failed to elucidate any virulence or adaptation markers that would explain its human - to - human transmission efficiency, highlighting our inability to predict iavs with pandemic potential . While the origin of the a(h1n1)pdm09 virus remains unknown, the virus was introduced into the north american swine population in 2009 and has since reassorted with other swine - origin iavs . Epidemiological data show that zoonotic transmission of iav from swine to humans has been documented at unprecedented levels in recent years . More than 320 human cases of infection with variant iavs were reported to the centers for disease control 20112012 and likely thousands more h3n2v cases went unreported during those years . These zoonotic iavs contained seven genes from contemporary north american swine lineage iav and one gene (m) derived from the h1n1pdm09 virus . The majority of the cases were epidemiologically linked to swine exposure occurring at agricultural fairs across several states . Within ohio, 107 h3n2v cases documented during 2012 resulted in eleven hospitalizations and one fatality . We recovered iav from exhibition swine at 10 of 40 (25%) ohio fairs sampled during 2012 . Genomic analyses of h3n2 iav isolates recovered from pigs at one agricultural fair in the state during 2012 demonstrated> 99% nucleotide similarity to h3n2v isolates recovered from concurrent human cases, providing molecular confirmation of zoonotic iav transmission . This record number of variant influenza a cases created the need for a one health' effort to minimize intra- and bidirectional inter - species iav transmission at swine exhibitions . The reason to prevent iav infections among swine at fairs is clear; however, the paucity of scientific evidence makes it difficult for veterinary officials to make sound recommendations to protect public and animal health . In the present study, we investigate fair specific risk factors contributing to the emergence of influenza a virus in exhibition swine that could be altered to mitigate the risk of iav transmission in these settings . As part of an ongoing active iav surveillance project, swine nasal swabs and associated metadata about management practices were collected at 40 ohio fairs in 2012 . Sample size was selected to provide a 95% probability of detecting iav infection if greater than 15% of the pigs at each fair were infected . All pigs sampled in this study were from junior fair market swine shows occurring at agricultural fairs . For the outcome of interest, a fair was considered positive if viable iav was recovered from one or more pigs at the fair . Data collection focused on fair level variables possibly contributing to the presence or absence of iav in the pigs at each exhibition . Junior fair shows are limited to local exhibitors approximately nine years of age through 19 years of age participating in 4-h, ffa or another youth organization, whereas open - class shows are generally open to all participants regardless of age, affiliation or residence . Classification of swine included market swine (pigs bred, raised and intended for food purposes) and breeding swine (gilts, sows and/or boars being raised for breeding purposes). Terminal swine shows are those in which all participating livestock are consigned to harvest immediately following the exhibition and partial terminal shows usually require the champion animals to be harvested following the exhibition and other pigs may or may not go to harvest . To account for the variability of arrival and departure procedures among fairs enrolled in this study, the length of the swine exhibition was defined as the number of days between the required arrival deadline for the pigs and the day the pigs were sampled . Study team members calculated the area per pig (ft / pig) from the recorded size of the pens and the number of pigs per pen . While on the fairgrounds, study team members also documented if there was an easily identifiable and operational hand - wash and/or hand - sanitizer station within close proximity to the swine barn(s). These sanitation stations were used by study team members to determine if they were functional . Additional variables included the number of pigs at the fair, number of swine exhibitors, fair attendance (number of people) and vaccine requirements, all of which were reported to the study team by the fair organizers . Fair officials also reported if there was a commingling event, such as a pre - fair animal identification or weighing session, during the weeks or months prior to the fair . Exhibition directors also reported if there were other pigs besides the exhibition swine on the fairgrounds (i.e., petting zoo, pig races, educational displays). The commercial swine inventory was retrieved from united states department of agriculture's 2007 census of agriculture . The county human population was defined as the value reported in the 2010 us census report . Weather data were collected from the national weather service's weather station nearest to each fairground . Data were analyzed using stata version 11.1 (statacorp, college station, tx, usa). Univariate analysis was performed to calculate unadjusted odd ratios to identify factors contributing to the presence of iav in pigs at fairs . Exact logistic regression was used for multivariable modeling using a forward stepwise model building approach . Influenza a virus was recovered from pigs at 10 of the 40 fairs included in the investigation . The presence or absence of iav infection among the pigs at the fairs could not be associated with county population, county swine inventory and number of people attending the fair (table 1). All iav - positive fairs and 27 of 30 (90%) negative fairs were mixed sex (barrows and gilts) market swine exhibitions . The average space per pig at the studied fairs was 12.8 ft / pig . Properly functioning hand - wash and/or hand - sanitizer stations were available at 25 of 40 (62.5%) fairs . Average daily mean temperature was almost 4 c higher for fairs with iav - positive pigs (table 1). Pre - fair tag - in and/or weigh - in events were rather common with 23 out of 40 (57.5%) of the enrolled fairs holding one of these events . For every increase of 20 pigs at a fair, the odds ratio of iav infection in the pigs increased by 1.27 times (table 2). The results of the current study provide the first look at fair - level risk factors associated with iav infections in swine at agricultural fairs . While it is likely impossible to completely prevent iav transmission at swine exhibitions, these data can be used to develop and evaluate mitigation strategies to reduce the impact of intra- and inter - species iav infections at swine exhibitions . Just like all other agriculture biosecurity programs, mitigation strategies which are practical, user - friendly, low cost and do not dramatically alter the fair experience for exhibitors and visitors are most likely to be considered, implemented and maintained . Not surprisingly, larger pig shows appear to be more likely to have iav - infected pigs than smaller swine exhibitions (table 2). Larger swine exhibitions tend to also have open - class and breeding swine shows in addition to junior market shows . While open - class shows were common among our studied fairs (16 of the 40), only 5 of the 40 (12.5%) fairs in this study had a breeding show; 4 of those 5 (80%) fairs had iav - infected pigs at the fair . The small number of fairs with breeding shows in this study makes analysis of this risk factor problematic; however, the finding is enough to warrant concern given that breeding swine are intended to leave the exhibition and enter a herd for progeny production . This fair - to - farm movement of pigs is a disease introduction risk for the receiving herd and a potential method to disseminate iav strains across a larger geographic area . Previous research has shown that environmental stressors (heat, lack of space, noise) on pigs can affect the course of various diseases in commercial swine operations . The average space per pig at the studied fairs was well above 68 ft / finishing pig common throughout the swine industry . The results indicate that heat stress could be a contributing factor to iav infections in exhibition swine; however, caution must be used when interpreting this result because the vast majority of the fairs with iav - positive pigs occurred in a 4-week period during the middle of summer . This trend of mid - summer iav activity in ohio's exhibition swine was also observed in the previous three years and could be more related to animal and/or people movement between these fairs than the weather . Environmental temperature failed to meet the selection criteria for inclusion in the final multivariable model . While the majority of fairs had hand hygiene stations, their presence at fairs was not linked to the occurrence of iav among pigs at the respective fairs . The large number of h3n2v infections linked to swine exposure at agricultural fairs during 2012 suggests that hand hygiene stations also had minimal impact on zoonotic transmission of iav in these settings . However, hand hygiene stations are critically important in protecting human health by controlling zoonotic diseases transmitted via direct contact at these venues . This would limit the time for iav to spread among susceptible pigs and decrease the time humans are exposed to iav - infected pigs . The length of the exhibitions in the current study was similar between iav - positive and -negative fairs . Active recruitment of fairs with more diverse management practices is needed to study the impact of a shortened swine exhibition . No matter the length of the exhibition time, the disposition of the pigs following the show must be considered . The majority of fairs in this study (65%) had terminal junior market shows . The practice of having a terminal show was not associated with decreased odds of iav; however, sending all the pigs to harvest at the end of each fair is expected to help protect subsequent fairs by decreasing the potential for fair - to - fair spread of iav . Mandated vaccinations were almost non - existent with only one fair in the current study requiring the pigs be vaccinated for erysipelothrix rhusiopathiae prior to the fair; no fairs required the pigs to receive iav vaccination before arrival . Use of iav vaccine in exhibition swine to decrease the risk of iav infections in swine and humans has been one of the most debated topics following the h3n2v outbreak of 2012 . There are currently several commercially available swine influenza vaccines in the united states, all of which are universally indicated to reduce clinical signs of disease in pigs but appear to have limited efficacy against 2012 h3n2v strains . Although their impact on intra- and inter - species transmission dynamics remains unclear, it is expected that iav vaccines will impart at least partial immunity to circulating strains of iav, which should decrease viral shedding during a fair . An unintended consequence of iav vaccine use may be vaccine - associated enhanced respiratory disease, which has been reported in swine vaccinated with swine influenza virus vaccines that are mismatched to circulating strains . Furthermore, decrease of clinical signs may hamper recognition and response to active iav infections in exhibition pigs . Some of the major pitfalls of mandated iav vaccination lie with the practical application of vaccines in this setting . The logistics of vaccine distribution to swine exhibitors prior to the fair becomes difficult because most exhibition swine are raised by youth exhibitors in small, dispersed herds (<10 pigs per herd). Commercial vaccines are usually sold in 50 doses per bottle adding to the cost per vaccinated pig in these small herds . Because youth exhibitors and their family members may not be proficient at administering vaccines, agriculture education advisors often volunteer to assist the youth with this task, a practice that is time - consuming and increases the risk of infectious agents being transmitted farm to farm . Additionally, problematic is that most iav vaccines labeled for swine require a booster dose given 24 weeks later to provide optimal protection, which can be difficult for youth exhibitors and their family members to accomplish . Tagging or weighing events are frequently used as a way for exhibitors to declare ownership of their pig(s) prior to the fair . These pre - fair events could provide an opportunity for mass vaccination of pigs prior to the fair, but the application of such events can facilitate disease spread between animals . Even in properly vaccinated pigs, the immunity stimulated by current iav vaccines the strains used for commercial swine influenza vaccines are irregularly updated and the constant genetic and antigenic change occurring in contemporary swine - origin iavs makes viral antigens unpredictable and difficult vaccine targets . The reason for the increase in the number of reported h3n2v cases during 20112013 remains unclear, but the strain of iav is thought to be a major contributing factor . The swine - origin h3n2 iav isolates recovered from these human cases contains the matrix gene from the a(h1n1)pdm09 virus, a recently emerged genomic constellation that increases replication and transmission in cell culture and animal models . Epidemiological data modeling indicate that children are most susceptible to h3n2v, likely due to lack of strain - specific immunity . Additionally, current seasonal trivalent inactivated iav vaccine provides little to no protection against h3n2v strains . The limited ability for human - to - human transmission of h3n2v has minimized the impact of these recent zoonotic transmission events, but the outbreak has illustrated the importance of swine exhibitions in zoonotic iav transmission . It is nearly impossible to predict the iav strain that will infect the swine at fairs because iav reassortment events and novel strain generation frequently occur in modern swine populations . Agricultural fairs provide a pathway for human exposure to these ever changing viruses; thus, blanket iav prevention, without regard for strain, is needed for swine at fairs to decrease zoonotic iav transmission and protect public health . While iav can infect pigs at any fair, the data presented here indicate that special attention should be paid to large pig shows where the likelihood of iav among the pigs is much higher . The results presented herein are based on one year of data from a limited geographic area of the united states . Additional assessments of swine exhibitions in multiple states across several years are needed to provide more comprehensive evaluations of risk factors contributing to the problem . These data provide a critical first step toward developing effective iav mitigation strategies in swine populations that benefit fairs, exhibitors, visitors and the swine industry . This information will serve as a baseline for measuring the acceptance and effectiveness as mitigation strategies are developed and implemented.
Turmeric (curcuma longa l.) of family zingiberaceae is industrially as well as pharmaceutically important crop extensively cultivated in india . It is a vegetatively propagated, polyploid crop commonly known as golden spice . Because of its multipurpose use as food preservative, spice, therapeutic agent and natural dye, it is acquiring more importance in cosmetics, food and pharmaceutical industries, . In turmeric number of cultivars has been developed with different characteristics like high curcumin content, high essential oil content, rhizome yield and disease tolerance etc . But their asexual mode of reproduction and poor genetic makeup has creates confusion towards authentic identification . Single nucleotide polymorphism (snp) study could be useful to solve these problems by comparing sequence of each variety with each other . In this current study, we conducted reference based transcriptome assembly of suvarna, a well - known turmeric cultivar which could latter on used to get potential snps by comparing with other varieties towards proper identification . Fresh mature rhizomes of suvarna variety has been collected from centre for biotechnology, siksha o anusandhan university, bhubaneswar, india . These rhizomes were washed properly with distilled water and suspended in rna later solution for further analysis . Rna extraction and library preparation has been done by using illumina truseq rna library tool kit . 75 bp paired - end sequencing has been done using illumina nextseq500 sequencing platform which developed5 gb of raw data . We have precisely given detail information about the reference based transcriptome assembly of suvarna cultivar (table 1). The transcriptome assembly of suvarna developed 42994 numbers of transcripts, with median transcript length 773 and n50 value 1216 . We annotated the transcripts using various databases like go, kegg, kog, plantcyc etc . As per our understanding transcriptome analysis of cultivar suvarna has been done for the first time and this result could be efficiently used for development of markers such as ssrs and snps for identification of suvarna from its closely similar turmeric cultivars . Fresh mature rhizomes of suvarna variety has been collected from centre for biotechnology, siksha o anusandhan university, bhubaneswar, india . These rhizomes were washed properly with distilled water and suspended in rna later solution for further analysis . Rna extraction and library preparation has been done by using illumina truseq rna library tool kit . 75 bp paired - end sequencing has been done using illumina nextseq500 sequencing platform which developed5 gb of raw data . We have precisely given detail information about the reference based transcriptome assembly of suvarna cultivar (table 1). The transcriptome assembly of suvarna developed 42994 numbers of transcripts, with median transcript length 773 and n50 value 1216 . We annotated the transcripts using various databases like go, kegg, kog, plantcyc etc . As per our understanding transcriptome analysis of cultivar suvarna has been done for the first time and this result could be efficiently used for development of markers such as ssrs and snps for identification of suvarna from its closely similar turmeric cultivars.
The sense of coherence (soc), which was proposed by antonovsky is based on the salutogenic model of health . The soc is composed of three factors; (1) the stimuli derived from internal and external environments in the course of living are structured, predictable, and explicable (comprehensibility); (2) resources are available to meet the demands posed by these stimuli (manageability); and (3) such demands are challenges, worthy of investment and engagement (meaningfulness). Antonovsky defined the soc as a personality dimension that is hypothesized to influence stress recognition style, facilitate stress management, and contribute to overall well - being . People with a strong soc have a high ability to cope with stress and maintain good physical and mental health . To the best of our knowledge, six studies have investigated the association between soc, job stress, and mental health . Findings from these studies suggest that soc modified the effect of job stress on mental health . To the best of our knowledge, only four studies have investigated the relationship between the soc and depressive symptoms, one used the brief job stress questionnaire and the other three used general health questionnaire . These studies suggested that a weak soc was a strong predictor of mental distress, including depressive symptoms . Nurses are exposed to high - stress work environments, including irregular work schedules, shift work, and interaction with patients and other hospital staff members . Therefore, preventing mental distress, including depressive state, is an important issue for nurses . The aim of this study was to investigate the relationship between depressive state, job stress, and soc among nurses in japanese general hospital . In february 2013, supervisors distributed a questionnaire to all nurses (n = 710) in a general hospital with 611 beds in an urban area in japan . Nurse specialties included intensive care, pediatrics, surgery, oncology, and emergency medicine . An explanation of the nature of the survey accompanied the questionnaire, which was anonymous and voluntary . The study was approved by both the local direction board and the committee for the prevention of physical disease and mental illness among health care workers at the general hospital . The questionnaire collected data on age, hours of work (full - time or part - time), shift work, overtime hours per week, and job rank (manager, middle manager or staff nurse). We measured job stress using the japanese version of the effort - reward imbalance (eri) scale (23 items) translated by tsutsumi et al . The eri consists of three subscales; effort (6 items), reward (11 items) and over - commitment (6 items). The reward subscale is further divided into three subgroups: esteem, job security, and promotion . The eri model indicates that job stress is related to high effort with low reward . Four eri ratios (eri, effort - esteem imbalance, effort - promotion imbalance, and effort - job security imbalance) were calculated based on the total scores according to tsutsumi et al . The soc scale consisted of 29 items assessing comprehensibility, manageability, and meaningfulness . Scores for each item ranged from 1 to 7 points, and the total score was calculated as the soc score . We measured depressive state using the k6 short screening questionnaire that was developed in accordance with the world health organization translation guidelines . The k6 consists of six items on depression and anxiety, each of which is measured on a 5-point scale (0 - 4). Higher scores indicate a more depressive state . The k6 was translated into japanese and showed good validity with the diagnostic and statistical manual of mental disorders, fourth edition, mood, and anxiety disorders in a community sample . We used pearson's correlation to investigate the relationship among age, work related - factors, job stress, soc, and depressive state . To examine factors with a significant effect on depressive state, stepwise multiple regression analyses were conducted with the k6 total score as the dependent variable and variables related to the depressive state as independent variables . Completed questionnaires were returned by 420 out of 710 nurses (response rate, 59.2%). Male nurses were excluded from the analysis because only 28 of the 42 (66.7%) male nurses responded . Subjects with missing values for job stress, soc, or depressive state were also excluded (n = 43). The final sample for analysis consisted of 348 female nurses (52.1%), including nurses who were managers and middle managers . Overtime was reported in hours per week, which was voluntary but limited to 45 h / month . Shift work categories included: no shift work, shift work with rotation to night shift, or characteristics of study subjects table 2 shows the pearson's correlation coefficients between age, overtime hours, eri ratios, soc, and the depressive state . Depressive state moderately correlated with three eri ratios, over - commitment, and soc . Pearson's coreration between work environments, eri ratios, over - commitment, soc and depressive state table 3 shows the influence of work - related factors, eri ratios, and soc on depressive state . Soc, over - commitment, effort - esteem ratio, and age were significantly correlated with depressive state (= 0.46, p <0.001; = 0.27, p <0.001; = 0.16, p <0.001, = 0.10, p <0.01, respectively). The coefficient of multiple determination (r) was 0.56 (f = 106.56 p <0.001). We found that soc, over - commitment, effort - esteem ratio, and age were predictors of depressive state among female nurses in the general hospital . The soc was inversely associated with depressive state, which was similar to previous studies . The present findings contribute to the literature investigating the relationship between depressive state, job stress, and soc . Strength of our study is that we investigated the independent contribution of three ratios of eri (effort - esteem, effort - job security, and effort - promotion), over - commitment, and soc to the depressive state . According to our previous study, the present findings suggest that a depressive state of nurses was associated with not only job stress but also both over - commitment and soc . Age correlated positively with soc [table 2], which was also similar to a previous study by harri who suggested that soc tends to increase with age . Previous studies suggested that an increasing soc of workers may reduce negative job stress responses and mental health problems . Intervention support enhancing the soc could be effective in preventing nurses from experiencing a depressive state . First, the sample size was small and included nurses from only one general hospital . Our findings provide insight into some factors associated with a depressive state among nurses in a general hospital . From a practical perspective, the influence of soc on a depressive state should be considered for health care professionals . Intervention support such as group cognitive psychotherapy to strengthen comprehensibility, manageability, and meaningfulness may help nurses cope better with job stress and reduce their risk of depression.
Huntington's disease (hd) is an autosomal dominant, progressive neurodegenerative disorder characterized by chorea, subcortical dementia and psychiatric symptoms . The causal hd gene has been identified as it15, which has cag repeats in the open reading frame (orf, in exon 1) just following the 5end of the gene . It leads to the translation of a protein, huntingtin, whose function is unknown . Huntingtin forms intraneuronal inclusion bodies which lead to neuronal dysfunction followed by neuronal death due to apoptosis . The therapeutic strategies for hd are diverse, ranging from the classical drug therapies to the modern molecular strategies . One of the treatment options is the use of drugs with neuroprotective potential, like lithium . Lithium has demonstrated diverse molecular effects reversing well - described pathophysiological changes such as increased oxidative stress, apoptosis, inflammation, environmental stress, glial dysfunction, neurotrophic factor deficiency, excitotoxicity as well as mitochondrial and endoplasmic reticulum disruption . Lithium acts by inhibiting a protein- kinase called glycogen synthase kinase 3 (gsk3) that has important actions on the intracellular signal transmission by protein phosphorylation . Inhibition of this enzyme appears to have a neuroprotector effect in neurodegenerative diseases such as amyotrophic lateral sclerosis, spinocerebellar ataxia type 1 and huntington's disease . We report three patients with huntington's disease, who showed good response to treatment with low dose lithium . Mrs . A, a 48-year - old homemaker, presented in 2008 with a three - year history of involuntary movements of the trunk and the hands, which had worsened over the previous 7 - 8 months . Family history was not available . On examination, ocular saccades were slow; she had generalized choreiform movements involving hands legs, lips and the trunk; dystonia of hands and feet and ataxic gait . A genetic analysis for huntington's disease was performed after obtaining written informed consent which showed cag repeat expansion . Patient was started on lithium 150 mg, once daily, along with carbamazepine 200 mg twice a day . Patient is on regular follow up for the last four years and is maintaining the same improvement . B, a 58-year - old male, presented in 2001 with a history of abnormal movements ., the movements were disabling, irregular, would worsen with anxiety and disappear during sleep . Patient had a significant family history of hd; involving both parents and 7 out of 9 siblings, totally 9 affected first - degree relatives . On examination, patient was noted to have severe generalized chorea involving the hands, legs, trunk and the neck . Patient was admitted in 2009 with a worsening of movements over the previous 6 months and weight loss of about 6 kg . All investigations including prostate specific antigen and chest x - ray were normal and the weight loss was attributed to nutritional causes . Family members reported that the patient was able to eat well and had no behavioral symptoms . The patient has been on follow up for the last three years and maintains status quo . C, a 51-year - old lady presented in 2002 with history of psychiatric symptoms for the previous 8 years . Onset was sudden with symptoms of being anxious, accusing the family members of neglect, and being violent toward family members . Patient also had history of non - rhythmic quasi - purposive movements of hands which she attributed to anxiety . Family history revealed that her mother had movement disorder and an early death . On examination, patient c had choreic movements of the limbs, anxiety and depressive symptoms . A diagnosis of hd was made which was confirmed by genetic analysis, which showed expanded cag repeats in the huntingtin's locus . Patient was initially started on carbamazepine 400 mg and clonazepam 4 mg / day . Over the next one year, patient developed psychotic symptoms like delusions of reference and persecution toward her family members and was also started on quetiapine up to 300 mg . Two years later, patient had sudden deterioration in symptoms with worsening of chorea and onset of cognitive decline memory disturbances and apraxia, which made her completely dependent for all activities of daily living . During this time, she also developed significant mood swings . For the last 3 years, patient has been on a combination of lithium 300 mg, carbamazepine 800 mg, quetiapine 300 mg and clonazepam 2 mg / day . Patient's husband reported no further deterioration over 3 years in cognition or movements in the patient . However, she continued to be dependent on husband for activities of daily living and eventually died due to cardiac arrest . Mrs . A, a 48-year - old homemaker, presented in 2008 with a three - year history of involuntary movements of the trunk and the hands, which had worsened over the previous 7 - 8 months . Family history was not available . On examination, ocular saccades were slow; she had generalized choreiform movements involving hands legs, lips and the trunk; dystonia of hands and feet and ataxic gait . A genetic analysis for huntington's disease was performed after obtaining written informed consent which showed cag repeat expansion . Patient was started on lithium 150 mg, once daily, along with carbamazepine 200 mg twice a day . Patient is on regular follow up for the last four years and is maintaining the same improvement . B, a 58-year - old male, presented in 2001 with a history of abnormal movements ., the movements were disabling, irregular, would worsen with anxiety and disappear during sleep . Patient had a significant family history of hd; involving both parents and 7 out of 9 siblings, totally 9 affected first - degree relatives . On examination, patient was noted to have severe generalized chorea involving the hands, legs, trunk and the neck . Patient was admitted in 2009 with a worsening of movements over the previous 6 months and weight loss of about 6 kg . All investigations including prostate specific antigen and chest x - ray were normal and the weight loss was attributed to nutritional causes . Family members reported that the patient was able to eat well and had no behavioral symptoms . The patient has been on follow up for the last three years and maintains status quo . C, a 51-year - old lady presented in 2002 with history of psychiatric symptoms for the previous 8 years . Onset was sudden with symptoms of being anxious, accusing the family members of neglect, and being violent toward family members . Patient also had history of non - rhythmic quasi - purposive movements of hands which she attributed to anxiety . Family history revealed that her mother had movement disorder and an early death . On examination, a diagnosis of hd was made which was confirmed by genetic analysis, which showed expanded cag repeats in the huntingtin's locus . Patient was initially started on carbamazepine 400 mg and clonazepam 4 mg / day . Over the next one year, patient developed psychotic symptoms like delusions of reference and persecution toward her family members and was also started on quetiapine up to 300 mg . Two years later, patient had sudden deterioration in symptoms with worsening of chorea and onset of cognitive decline memory disturbances and apraxia, which made her completely dependent for all activities of daily living . During this time, she also developed significant mood swings . For the last 3 years, patient has been on a combination of lithium 300 mg, carbamazepine 800 mg, quetiapine 300 mg and clonazepam 2 mg / day . Patient's husband reported no further deterioration over 3 years in cognition or movements in the patient . However, she continued to be dependent on husband for activities of daily living and eventually died due to cardiac arrest . Huntington's disease (hd) is an autosomal dominant progressive neurodegenerative disorder caused by an expanded polyglutamine (poly q) tract within huntingtin protein and is characterized by neuronal loss mainly in the striatum and the cortex . Genetically, hd is caused by a mutation in the gene located on chromosome4p 16.3 . The disease - causing mutation in the first exon of huntingtin (htt) gene is the expansion of a polymorphic cag repeat that is completely penetrant once it has reached 40 triplets . Neuropathologically, the mutant huntington protein (mhtt) abnormally interacts with a variety of proteins and accumulates in the neuronal nucleus forming an inclusion body which is the hallmark of hd and leads to eventual cell death . In addition to the well - documented use of lithium as a mood stabilizing drug, primarily in the treatment of bipolar disorder, recent in vitro and in vivo studies in animal models have increasingly indicated that lithium can be used in the treatment of chronic neurodegenerative diseases like huntington's disease . Various pharmacological and gene manipulation studies support the notion that lithium's main mechanisms of action may be its ability to inhibit gsk3[68] as well as induce brain - derived neurotrophic factor - mediated (bdnf) signaling, leading to enhanced cell survival by alteration of a wide variety of downstream effectors . Lithium is shown to promote the action of anti - apoptotic proteins, such as heat - shock protein (hsp), insulin like growth factor (igf), vascular endothelial growth factor (vegf) and other neurotrophic factors . Lithium can directly or indirectly modulate proteins involved in neuronal survival / differentiation which may account for its neuroprotective effects. [1012] by inhibiting n - methyl - d - aspartate receptor - mediated calcium influx, lithium also contributes to calcium homeostasis and suppresses calcium - dependent activation of pro - apoptotic signaling pathways . Lithium is found to enhance autophagy of toxic aggregates in huntington's disease in various models of hd . It acts by increasing mtor - independent autophagy by inhibiting inositol monophosphatase (impase) and reducing inositol and ip3 levels . Induction of autophagy by lithium leads to enhanced clearance of mutant huntingtin fragments associated with huntington's disease (hd). These mechanisms might allow therapeutic doses of lithium to protect neuronal cells from diverse insults that would otherwise lead to massive cell death . Lithium also stimulates proliferation of stem cells, including neuronal and astroglial progenitor cell proliferation in the sub ventricular zone, hippocampus, striatum and forebrain protecting against apoptosis . The stimulation of endogenous neural stem cells and neurogenesis may explain why lithium increases brain cell density and volume in patients with bipolar disorder . In this report, we have described the use of lithium to prevent the progression of the huntington's disease . All patients reported here were started on low doses of lithium after explaining the rationale, possible side - effects and off label use of the drug . The patients were on regular follow up and reported no worsening of either psychiatric symptoms or choreic movements for a significant period . In these patients, lithium was started in order to alleviate the distress caused by the choreic movements and mood swings . Though rating of the movements was not done, there appears be some benefit in prescribing lithium to patients with huntington's disease for slowing the progression of chorea and preventing mood swings . This report signifies the use of lithium as a neuroprotective agent . It may be beneficial to explore the possibility of use of other psychotropic drugs in the treatment of neurodegenerative disorders
Walking ability is impaired in patients with peripheral arterial disease (pad) due to decreased blood flow to the skeletal muscle tissue used during walking . It has been demonstrated that walking distance is associated with the degree of impairment in the affected leg as measured by the ankle - brachial index (abi) and the time to onset of claudication pain [16]. Likewise, it has been shown that walking exercise in patients with pad prolongs the onset of claudication pain thus allowing the patient to walk longer but does not change the abi [79]. Recently, several investigators have used near - infrared spectroscopy (nirs) to dynamically study the effect of walking on oxygen desaturation of the gastrocnemius muscle of the impaired leg during exercise [2, 1014]. Investigators noted a marked decline in skeletal muscle tissue oxygenation (sto2) during walking exercise [2, 1014]. Gardner et al . Also identified that the rate of decline in calf muscle sto2 was significantly associated with initial claudication time and absolute claudication time in patients with pad . Three of the above studies examined the changes in tissue oxygenation, using nirs, in patients with pad before and after an exercise training program [1214]. Used a pretest - post - test design and reported greater exercise duration and lower mean exercise sto2 in 15 patients after a 3-month treadmill and calf exercise training program . Studied 55 patients with pad who were given the option to participate in a structured or unstructured home exercise program for 34 2 weeks . At the end of the study, those patients in the structured exercise program had an increased oxyhemoglobin area under the curve when compared to baseline; similar changes were not identified in the unstructured exercise group . Lastly, tew et al . Studied calf - tissue oxygenation and treadmill walking response time after randomization to 12 weeks of arm - crank ergometry or a control group in 57 patients with intermittent claudication . Investigators identified that walking distance and time to nadir sto2 increased in those assigned to arm - cranking and there were no changes in the control group . No studies however compared leg muscle responses after a traditional walking program compared to a walking - with - poles program of rehabilitation in patients with claudication . The purpose of this study was to determine if there were differences in calf muscle sto2 parameters before and after 12 weeks of a traditional walking program versus a walking - with - poles exercise program . We reasoned that by using the poles to offload the legs, patients would have less claudication pain and be able to walk longer distance thus achieving a greater training effect . We hypothesized that the calf muscle tissue deoxygenation would be less in both groups after 12 weeks of exercise training than at baseline and that the decrease would be greater in those patients assigned to the walking - with - poles group . These data represent a subanalysis of data from a larger clinical trial comparing traditional walking to walking with poles . Patients with pad were screened and recruited to participate in a randomized clinical trial designed to compare the effects of a traditional walking training program versus walking - with - poles exercise training program . A total of 2296 patients were screened for eligibility, 146 enrolled in the study . Patients were recruited using radio and newspaper advertising, posted fliers, and letters of invitation sent to patients at the university and va hospitals . For the purposes of this analysis, we included only those participants with an abi of 0.90 (n = 91 patients). Of those 91 patients, 3 did not have sto2 analyses on their baseline progressive treadmill test due to equipment malfunction and 3 had questionable data resulting in a final sample of 85 patients . Of those 85 patients, 45 were assigned to the walking - with - poles group and 40 patients were assigned to the traditional walking group . The major study was a randomized clinical trial to examine the effects of a traditional walking program compared to walking with poles on walking endurance and perceived physical function in patients with pad . Our primary outcome focused on changes the patients realized walking on the constant work rate treadmill test whereas this analysis used data from the progressive treadmill test . At two minutes on the constant work rate test, the speed and incline increased to 85% of the patient's workload on the progressive treadmill test resulting in a sudden decline in sto2 . Due to the gentle nature of the progressive treadmill protocol used, we were able to detect more subtle changes in sto2 and onset of claudication pain . Patients were randomized using computer - generated permuted blocks and group assignment was managed by the study statistician . The training programs for both groups were identical except that one group exercised with poles and the other group did not . Patients assigned to the walking - with - poles group received training on the use of the poles prior to beginning the exercise program . Additionally, patients were coached on proper poling mechanics during the training period if needed . The training program incorporated interval training whereby exercise consisted mostly of low - to - moderate intensity training at the start of the program and progressed to moderate - to - high intensity . Exercise intensity was determined by heart rate associated with a percentage of oxygen uptake obtained during the metabolic exercise treadmill tests . Exercise duration and intensity was adjusted every three weeks . During the first weeks, patients walked for 30 minutes (20% light intensity, 60% moderate intensity, and 20% hard intensity). By weeks 1012, patients walked for 55 minutes (10% light intensity, 45% moderate intensity, and 45% hard intensity). Light intensity was defined as 2544% peak vo2, moderate intensity was 4559% peak vo2, and hard intensity was 6084% vo2 peak . The program was designed so that patients walked on the treadmill two days / week and outdoors or in the hospital corridors one day / week . The exercise interval was adjusted based on the patient's pain tolerance . For example, some patients could only walk for one to two minutes at the higher intensities . In order for them to achieve 20% total time at the harder intensities, demographic information, height, weight, comorbid conditions, and medication history were obtained from the patient and reviewed in the patient medical record . The abi was measured in the more severely affected leg at baseline and 12 weeks after training . The location of the signal was recorded and used consistently throughout the study for that individual subject . After the patient rested comfortably in a supine position for 15 minutes, doppler ultrasound was used to measure the systolic pressure in the right and left arms and in the ankle of the most severely affected leg . The arm with the highest systolic pressure patients were tested using a gentle treadmill protocol that was developed for patients with pad [6, 16]. Increases in percent grade occurred every 30 seconds, and, after the first 6 minutes, speed increased every 3 minutes . Initial claudication time was defined as the time during the treadmill test when the subject experienced pain in the affected leg . The absolute walking time was defined as the time walked by the patient on the progressive treadmill test . Peak oxygen uptake was measured using the medgraphics cpx / d system (medical graphics corp, st . The metabolic cart was calibrated using a 3 l syringe prior to each test and the analyzers were calibrated using references gasses and room air . Patients breathed through a mouthpiece and their nose was clipped with a standard nose clip . Oxygen uptake was averaged over 30 s and the highest value at peak exercise was recorded . Skeletal muscle tissue oxygenation was measured prior to, during, and after exercise using the nirs spectrometer (inspectra 325, hutchinson technology, inc ., a 25 mm probe attached to an optical cable was used to noninvasively measure the percentage of hemoglobin oxygen saturation of the tissue beneath the near - infrared light . The inspectra machine was calibrated prior to each test according to known high and low reference standards . The nirs probe was attached to the medial section of the gastrocnemius muscle of the patient's most severely affected leg . The probe was placed into a specially designed adhesive housing unit to reduce ambient light . The probe was then secured to the leg to avoid excessive motion and possible tripping . The sto2 values used in this analysis were the sto2 value recorded after 2 minutes of standing prior to exercise (sto2 rest), time walked when the patient reached the nadir sto2 value (time to nadir sto2 value), sto2 value at peak exercise (sto2 peak), and absolute and relative drop in sto2 (absolute drop in sto2 = rest sto2 minimal sto2 value, relative drop in sto2 = [rest sto2 nadir sto2 value]/rest sto2). Ratings of perceived leg pain were obtained every minute using the borg ratio scale . The initial claudication time is the time that patients either notified the staff of the onset of pain or the time they first reported pain when asked every minute . The two groups were compared at baseline using the t - test for independent samples and mann whitney u test . Regression analyses were used to compute the initial decline in slopes for sto2 values (sto2 value regressed over time). Pearson r correlations were completed to examine relationships between the variables . Paired t - tests independent t - tests were used to examine differences between groups . Intent - to - treat analyses using the baseline value carried forward were completed for the comparisons between groups . The sample consisted of primarily older (age = 69.4 9.2 years) men . Over one - third of the patients were current smokers, half were diabetic, and the majority of patients received treatment for hypertension and dyslipidemia (table 1). Patients were generally unfit with a peak oxygen uptake of 14.3 2.9 ml kg min. The walking - with - poles group was older than the traditional walking group . Calf muscle oxygenation decreased from 56 17% prior to the treadmill test to 16 18% at peak exercise (71% relative decline from rest to peak exercise). Patients walked 4.45 3.67 minutes before experiencing claudication pain and 4.39 3.99 minutes when the sto2 reached its nadir value . There was no difference between the time elapsed when the sto2 reached its nadir value and pain onset (p = .87). Although all patients reported claudication pain, exercise was not limited by claudication pain for all patients . Therefore, we examined the onset of claudication pain and time to nadir sto2 for those whose exercise was limited by claudication pain only (n = 72). Patients walked 3.35 2.57 minutes before experiencing claudication pain and 3.67 3.49 minutes when the sto2 reached its nadir value . There was no difference between the time elapsed when the sto2 reached its nadir value and pain onset (p = .40). After exercise, the time for the sto2 to return to half of its resting value was 1.3 1.3 min and 2.2 2.1 minutes to return to resting values . The initial claudication time and absolute walking time were not associated with the sto2 values at rest (table 2) but were moderately related to the time elapsed prior to reaching the nadir sto2 value (r = .42, p <.001; r = .58, p = .001, resp . ). There was also a small to moderate relationship between onset of claudication pain and the exercise slope decline in sto2 (r = .28, p = .011). Relationships were similar between initial claudication time and absolute walking time and the time elapsed prior to reaching the nadir sto2 values when the analysis was restricted to those whose exercise was limited by claudication (r = 48, p .001 and r = .47, p <.001). Data comparing groups after exercise training were analyzed using intent - to - treat analysis for all patients . The change in time elapsed prior to reaching nadir sto2 values between baseline and 12 weeks was related to the change in absolute walking time between baseline and 12 weeks (r = .65, p <.001) and initial claudication time at 12 weeks (r = .33, p = .002). Analysis of covariance was completed to compare the groups with age as the covariate since there was a difference in age at baseline (table 3). The change in the time elapsed prior to reaching nadir sto2 values (p = .002) and absolute walking time (p = .002) were greater in the traditional walking group when compared to the walking - with - poles group . There was, however, no difference in the change in initial claudication time between the two groups (p = .38). When the analyses were restricted to those who completed the exercise training only (n = 71), the change in time elapsed prior to reaching nadir sto2 values between baseline and 12 weeks was related to the change in absolute walking time between baseline and 12 weeks (r = .57, p <.001) and initial claudication time at 12 weeks (r = .32, p = .004). Data from a sample patient are depicted in figure 1 . Recovery time was unchanged when baseline and 12-week values were compared (baseline = 2.45 2.17 min, 12 wk = 2.19 1.94 min, p = .81). After 12 weeks of exercise training, within - group changes in initial claudication time, absolute claudication time and time elapsed prior to reaching nadir sto2 values increased (table 4 .) The time elapsed prior to reaching nadir sto2 values increased more in the traditional walking group when compared to the walking - with - poles group (walking group = 4.01 5.13 minutes, walking - with - poles group = .89 3.56 minutes, p = .009). Similarly, absolute walking time increased more in the traditional walking group than in the walking - with - poles group (walking group = 3.97 3.14 minutes, walking - with - poles group = 2.24 2.41 minutes, p = .017). There was no difference between the two groups in initial claudication time (walking group = 1.16 3.64 minutes, walking - with - poles group = .85 3.46 minutes, p = .69). Since there was a significant difference in age between the two groups, age no differences in recovery time were noted between the traditional walking and walking - with - poles groups at the 12-week follow - up time period (traditional walking recovery time = 1.26 1.43 min, walking with poles = 1.41 1.76 min, p = .70). No significant differences were identified in peak oxygen consumption or abi within or between the two groups from baseline to 12 weeks using intent - to - treat analysis or restricting the analysis to those who completed the training only . There are three major findings of this study: (1) the time elapsed before reaching nadir sto2 values is associated with initial claudication time and absolute walking time, (2) there was no difference in the time associated with the nadir of sto2 and the onset of claudication pain, and (3) the time to nadir sto2 of the gastrocnemius muscle was greater after 12 weeks of exercise training in the traditional walking group when compared to the walking with poles group . As bauer notes, during higher work rates and apparent blood flow limitation, muscle oxygen desaturation becomes more rapid in pad subjects . Thus, one would expect that oxygen desaturation would be associated with onset of claudication and walking duration . Our findings reflect these assumptions and were similar to those reported by gardner et al ., that is, shorter time to reach nadir sto2 values were associated with shorter initial claudication time and total walking duration . Second, in gardner's study, relationships between initial claudication time (r = .339), absolute walking time (r = .68) and measured time to minimal exercise sto2 were similar to our relationships between initial claudication time (r = .42), absolute walking time (r = .58) and measured distance to nadir sto2 . These findings suggest that our sample patients were similar to patients in other studies [2, 19]. Exercise - associated decline in sto2 of the gastrocnemius muscle was less after 12 weeks of exercise training when compared to baseline . Wang and colleagues recently demonstrated in 17 subjects with pad who completed a 24-week walking program that an increase in the number of capillaries in contact with type iix and iia calf muscle fibers was related to an improved pain - free walking time (r = .69 and r = .62, p <.05). Investigators hypothesized that the ratio of capillaries to muscle fibers affects oxygen supply thus delaying the mismatch between supply and demand . Both report greater exercise sto2 area under the curve postexercise training when compared to preexercise training . In our study, an increase in the time to nadir sto2 values was associated with delayed initial claudication time and improved absolute walking time . Our findings provide further evidence that walking exercise improves tissue muscle oxygenation in patients with pad . Likewise, our findings support the possibility that the onset of claudication and walking duration are linked by the decline in tissue oxygenation . The onset of claudication and the time elapsed prior to reaching the nadir sto2 values were nearly identical . Thus, the patient's perception of pain is a good indicator of sto2 in the calf muscle . Exercise - induced decline in gastrocnemius muscle sto2 was less in patients assigned to the traditional walking program than in patients assigned to the walking - with - poles exercise training program . We had originally hypothesized that a walking training program with poles for patients would be advantageous because the poles would offload the legs during periods of claudication allowing additional walking exercise . Indeed, oakley et al . Investigated the immediate effects of walking with nordic poles on 20 patients with pad and found that claudication distance increased from 77 m without the poles to 130 m with the poles (p <.0001) and the level of claudication pain also decreased (p = .0002). Our group has also previously reported that patients had decreased calf pain when walking with poles . Oakley et al . Compared muscle histology of 36 patients randomly assigned to 12 weeks of treadmill training, strength training or control . After the 12 weeks, only the treadmill group had histological changes in the muscle . The increase in muscle ischemia with treadmill training was associated with a marked increase in muscle damage . Hiatt suggests that skeletal muscle dysfunction and associated muscle metabolic state can be ameliorated with treadmill exercise training . Tew et al . Provides evidence that even when the exercising muscle is not the calf muscle, tissue oxygenation can improve . Clearly, the patients assigned to the traditional walking group experienced a prolonged exercise time before experiencing claudication pain, prolonged their absolute walking time and increased the amount of time exercising until reaching nadir values . In the walking - with - poles group, there were no changes over time when an intent - to - treat approach was used . However, when the analysis was restricted to those who completed the training program only, there was a difference in absolute walking time and a trend toward increasing time to initial claudication pain onset as well as nadir sto2 . The explanation for these findings may be that leg metabolic changes in the calf muscle occurred to a greater extent in those assigned to the walking - with - poles group or we may not have had enough statistical power to detect changes that were present . Another explanation for this finding may be that the intensity of our training program was based on oxygen uptake associated with heart rate response to exercise . With the addition of the upper body to the exercise regime for patients assigned to the walking - with - poles group thus, the work accomplished by the leg muscles may have been less even though overall exercise intensity was consistent between the two groups of patients . Both groups trained on the treadmill and outdoors . Patients assigned to the walking with poles group were unable to use the handrails on the treadmill for balance; added anxiety may have increased the cardiovascular response thus keeping the overall work accomplished lower in some patients . First, although age was not a contributing factor to the differences between the groups, the walking - with - poles group was older than the traditional walking group . We cannot exclude that the differences in exercise tolerance and willingness to push limits may differ between someone who is 67 years old (mean age for walking group) and someone who is 72 years old (mean age for walking - with - poles group). This five - year age difference may have influenced our outcomes in ways that we did not measure . Next, the sample was primarily men and thus cannot be generalized to women . Additionally, in order to include only those with compressible vessels, we excluded 17 patients who were initially randomized to our study . Lastly, although all of our patients experienced claudication pain, not all patients stopped exercise due to claudication pain . Including patients that did not stop exercise due to claudication pain additional studies examining different walking intensities in patients with peripheral arterial disease should be tested . Further, it may be that use of walking poles early in the rehabilitative effort may augment traditional walking training later by assisting patients to train longer early on . In summary, we conclude that there is a significant decline in sto2 in the gastrocnemius muscle during walking exercise in patients with peripheral arterial disease reflecting muscle tissue ischemia . Contrary to our hypothesis, after 12 weeks of exercise training, absolute walking time and time to reach nadir muscle tissue oxygenation were prolonged in the patients assigned to the traditional walking program more than those assigned to a walking - with - poles exercise training program reflecting greater tissue perfusion and enhancement of arteriovenous oxygen extraction . The precise mechanism for these differences remains unknown but these findings may provide some insight to tissue adaptations to exercise stimulus and may therefore be useful to pad rehabilitation programs.
Access to automated dna sequencing technology has made possible the rapid generation and analysis of gene transcripts expressed in organisms via expressed sequence tags (ests). This information has helped to identify those genes expressed in particular stages of development and in specialized tissues or organs . Novel gene products and target leads for therapeutic intervention can also be gleaned rapidly from ests . A more detailed understanding of the molecular interactions between symbionts, whether pathogenic or mutualistic, is also possible with this approach . For a sequence isolated from interacting symbionts, determining its cellular role (or roles) we refer to this challenge as' the problem': given a sequence x expressed in an interaction between species a and b, did x originate from a or b? Various solutions are readily conceived, each with merits and faults . Here, we show that a comparative lexical analysis of word counts (specifically, hexamer frequencies), previously used to detect library contamination in sequencing projects, provides a powerful computational basis to infer a transcript's species of origin . Experimentally, one can attempt to solve the problem by hybridizing a clone (as probe) to genomic dna (target) from both species and determining to which target the probe hybridizes . However, if a sequence is highly conserved in the two taxa, hybridization stringency conditions can influence the outcome considerably . For high - throughput est sequence analysis, source verification by hybridization is impractical in terms of time and reagents . As an alternative to in vitro hybridization, several computational solutions are possible . Were the genome sequence of both species completely determined, one could simply use sequence similarity searching . However, most plant hosts and their microbial symbionts have little or no genomic sequence data available, which makes this approach very unreliable . Strong similarity to a sequence from one organism does not preclude the possibility that a similar sequence is present in the other species . Exploiting this fact may seem a viable solution to the problem, as it has proven suitable for predicting the presence of introns among exons in genomic dna . However, it really is not practical, because of the need to know the reading frame for translation of a messenger rna into an amino acid . Est data are of notoriously unreliable quality, sometimes having a large proportion of ambiguous bases, and sometimes having single base - pair insertions or deletions, which disrupt a reading frame . Word counting is less prone to these sources of error, and uses information intrinsic to biases in codon usage by counting codon pairs as hexamers in a sliding window, whereas codons are read in non - overlapping, tiled windows . An intuitive approach to the problem that examines sequence composition is to compare the guanine and cytosine (gc) base content of a sequence with other sequences from the species being studied . When two species' genomes have different gc content, this method can be very useful . In a recent investigation, for instance, sequences from the stramenopile plant pathogen phytophthora sojae and its soybean (glycine max) host showed a 20% difference in mean gc content . The origin of a number of sequences could readily be identified this way, but a large proportion could not, because of considerable overlap in the distributions' tails . Counting frequencies of gc is simple word counting, where the word size k is 1/2: only two semi - words, g / c and a / t are counted . An alternative approach to determining the origin of a sequence is suggested by previous work on analysis of word counts, or k - tuple frequencies, which was intended as a means of evaluating a library for contamination when sequencing from a single model organism . The word - counting method provides distinct advantages over other computational methods . Unlike sequence - similarity searching, it does not require that the full protein - coding content of both genomes be known for reasonable inferences to be made . Further, word counting is sensitive to biases in codon usage and gc content commonly observed when comparing taxa, but does not require knowledge of the reading frame for amino - acid translation . That is, the underlying differences between the two organisms that result in base composition or codon usage biases can also be detected by counting words . Unlike gc analysis, lexical analysis establishes a clear threshold above or below which we can infer the species of origin, and a confidence level for an inference can readily be assigned . Dunning's likelihood - ratio test of word dissimilarities also has the appealing property of being non - parametric, having no assumption of normality for the underlying frequency distribution, which makes it statistically powerful . Dunning demonstrated that unreliable results can be obtained from parametric tests, such as, particularly in such cases as lexical analysis . In the experiments detailed below, we first validate the word - counting method on sequences whose origin and function are known, then compare it with ability to diagnose the origin of sequences with distributions of gc content . We examine sequences from pathogenic interactions between species from the genus phytophthora and the plant hosts g. max and medicago truncatula, then apply the word - counting approach to sequences from two microbial mutualists in association with m. truncatula, the arbuscular mycorrhizal zygomycete glomus versiforme, and the nitrogen - fixing bacterium sinorhizobium meliloti . Validation sequence accession numbers, gene names, and comparison results appear in table 1 . The word - counting method was generally quite reliable when tested against sequences of known origin, being wrong in 3 cases out of 50; a phosphate transporter from g. versiforme and two in planta - induced genes from phytophthora infestans were misidentified as plant sequences . This indicates a failure rate of 6% - all false negatives under the null hypothesis that a transcript originates from the plant host . Performance of the method was not influenced by whether the isolated source of a sequence was an mrna or dna molecule, as indicated by the column labeled' mrna?' . Distributions of gc content are approximately normal in two of three cases studied, those of axenic p. sojae cultures (figure 1). For sequences from infected plant cultures, roughly 25% of a total of 927 infected g. max sequences contain less than 50% gc; most of these are likely to be plant transcripts . This is a considerably greater number than for axenic p. sojae cultures, in which fewer than 5% of mycelia and zoospore isolates contain less than 50% gc . Several properties of cumulative distribution functions warrant comment, to help explain similar plots from word dissimilarity comparisons (figures 1b,2a). The median of a distribution occurs where the function reaches a cumulative probability of 0.5 . Medians from all three p. sojae libraries are similar, varying by less than 4% gc (figure 1b). Other moments of the distributions are readily apparent; the variance is inversely related to the slope at the median value of the function . A useful property of cumulative distribution functions is that any point on the y axis gives the integrated area (cumulative probability) under the curve . We use this property to establish experiment - wide false - positive and false - negative rates (figure 2a). In this case, calibration curves from hexamer dissimilarity tests, shown in figure 2b as solid black lines for plant and dashed black lines for stramenopile training sequences are approximately normal . The medians differ considerably, with only about 10% percent overlap in the two distributions' tails about the neutral t - value of zero . Superimposed are comparison curves from p. sojae test sets (figure 2b), which parallel the gc content curves in figure 1b but show slightly less variance . Axenic sequences are clearly more like stramenopiles (b1) than plants (a1) in hexamer composition, with all but a small percentage having positive t values . Plant - like sequences are as abundant in the mixed library as detected by gc content, about 23% . As expected, the two methods agree, having positively correlated values for gc and t (r = 0.852, p <10, v = 2,641). Looking in more detail at the paired dissimilarity values (figure 3), the magnitudes of dissimilarity are also apparent, with longer sequences having larger dissimilarity values . Blastx similarity searches against the protein sequences in nr, a non - redundant library of proteins revealed that none of the 12 plant - like mycelial transcripts significantly resemble known proteins (e> 10). Among the top ten most plant - like transcripts from the infected g. max library, three had no significant matches, four matched putative arabidopsis thaliana proteins, and three matched known g. max proteins: cytochrome p450 (accession af022460, e <10), methylglyoxalase (accession p46417, e <10), and a ripening related protein (accession af127110, e <10). Thus, the majority of the most plant - like transcripts in the infected soybean library strongly resemble characterized plant sequences . Analysis results from all p. sojae and mixed - culture transcripts are available as additional data files, grouped by the library from which transcripts were sequenced . Figure 4 shows that calibration curves from comparing plant and microbial symbiont training sets have good separation and minimal overlap (about 10%) in two of three cases, but not for training set b2, comprised of zygomycetes and chytridiomycetes, which overlaps considerably with plants (figure 4b). When comparing between plants and bacteria, the error rates are = 0.052 and = 0.084, much lower than when comparing plants (a2, medicago) with fungi (b2, zygomycetes and chytridiomycetes). Error rates for comparing stramenopiles and p. infestans ests with plants are as in figure 2 (= 0.088, = 0.032). Also shown in figure 4 all resemble calibration curves from plant sequences, having similar medians and slightly less variance than the plant calibration curves . Comparison curves show that the great majority of test sequences are more plant - like than otherwise, with 20% or less resembling microbial symbionts more closely than plants . A greater proportion of microbial sequences is present in the m. truncatula - g . Versiforme interaction library (20%, figure 4b) than in the p. medicaginis - infected m. truncatula library (5%, figure 4a). However, long's root - hair enriched library (mtrhe) had a greater proportion of putative microbial sequences present (7% and 25%) than any of the libraries isolated from symbiont - associated cultures . The axenic and nodulating root libraries had the smallest portion of putative microbial transcripts (<2%, figure 4c), with the axenic library closely resembling nodulating root libraries . The method of preparing a library can affect the proportion of plant and non - plant sequences, as discussed below . Paired dissimilarity values in figure 5 show in greater detail which sequences are more or less like plant and symbiont . Considerable variation in the degree of dissimilarity to both training sets is clear, largely due to variation in the length of sequences within test sets . D(b) in figure 4, most sequences lie above the identity function, and resemble the plant host more closely than the microbial symbiont . Mycorrhizal test sequences are more difficult to differentiate than sequences from the rhizobacterial or pathogenic associations, as seen by the diminished variation about the identity function in mycorrhizal comparisons (figure 5b), contrasted with comparisons from pathogen - infected and nodulating root libraries (figures 5a and c, respectively). Analysis results from all m. truncatula and mixed - culture transcripts are available as additional data files, grouped by the library from which transcripts were sequenced, and sorted from the least plant - like transcripts to the most plant - like . Clearly, the word - counting approach provides a reliable solution to the problem of source identification with known confidence, and has several significant advantages . The reliability of the method is best justified in terms of the favorable validation test results, and is further corroborated by agreement with an analysis of gc content . In test cases where the correct answer is known a priori, results were correct within error rates expected from overlap in training sets . (recall that = 0.088 for comparisons between plants and stramenopiles, and = 0.052 for comparisons between plants and bacteria .) Unlike gc content, the problem is clearly resolved by word counting with a threshold value of t = 0, and with statistical rigor, because false - positive and false - negative rates for a set of comparisons are readily computed from cumulative distributions of dissimilarity between two training sets . Optimal statistical power (minimal false - negative rate) is ensured when using a likelihood - ratio test statistic, as demonstrated by the pearson - neyman theorem . Rather, it is sufficient that the training set be related to, but not necessarily congeners of, the species from which sequences are being compared . Sequences from several species of the genus phytophthora were correctly distinguished from plant and bacterial sequences, and three genes from agrobacterium tumefaciens were correctly identified as representing a bacterial sequence . However, several caveats warrant prudence . Transcribed sequences that do not encode proteins, but rather catalytic single - stranded rnas such as transfer and ribosomal rnas, should be treated independently because they are more highly conserved across taxa than messenger rnas . Also, filtering or trimming of low - complexity repeat regions, such as poly(a) or poly(t) tracts, is helpful because comparison results can be influenced by the abundance of a single hexamer . Early in our investigations, using one set of training sequences obtained from directionally cloned p. it eventually became clear that, as the p. infestans sequences were all single - pass reads from the 5' end of a clone generated with the t3 primer, few sequences complementary to the 3' end of the mrna sequence were present in the training set . Large amounts of the poly(t) hexamer would be expected when sequencing reverse complements of mrnas obtained from 3' sequences generated with the t7 primer . As a result, any sequence that contained a poly(t) tract tended to resemble the plant sequences . Further, because the error rates for an inference depend on the degree to which calibration curves overlap, the best results are obtained where overlap is minimal . Despite these caveats, word counting presents a viable solution to the problem . The p. sojae - infected g. max library provides a clear example of contrast in both hexamer composition and gc content, resulting in readily diagnosed origins . For clear separation between the two species to appear, the two must differ in composition and a detectable proportion of transcripts from each species must be present in the library . To be detectable, the proportion of transcripts present from a particular species must be greater than the error rate obtained from calibration curves . Though these criteria are true for the infected g. max library (t <0 for <25% of 927 transcripts), they do not appear to be true for the m. truncatula libraries we analyzed (t <0 for 8099% of 8903,017 transcripts). In the p. medicaginis interaction library the p. sojae - infected library was prepared two days after infection, using a susceptible plant host strain, so as to maximize the number of pathogen transcripts present in the host tissue . Medicaginis - infected library was prepared ten days after infection and individual plants varied in their degree of susceptibility (c. vance, unpublished data). Plants were also inoculated in a different manner: ground mycelia were dissolved in sterile water and incubated, and the resulting inoculum was pipetted onto the soil surface, rather than the plant . These differences in how tissues were cultured prior to library preparation could have produced the disparate abundance of plant transcripts, though both libraries were prepared from plant tissues infected with phytophthora . For mycorrhizal root libraries, we might explain the relative lack of symbiont sequences as resulting simply from a relative lack of transcripts in the host tissue . We might therefore expect that most of the transcripts therein originate from the plant host . Confounding this result, the error rates in this comparison are the greatest among all the comparisons we performed, most likely because the evolutionary distance between fungi (zygomycetes and chytridiomycetes) and plants is the least among comparisons . Also, zygomycete protein - coding sequences are rare in genbank, which resulted in a small training set for these fungi, and may have amplified any biases . The high false - negative rate probably led to a failure to detect some symbiont transcripts . In nodulating root libraries, we do not expect to observe an abundance of bacterial transcripts, because bacteria generally do not form polyadenylated mrnas . As the protocols used to extract and purify mrnas from tissue lysate for the libraries cited in this study all relied on the presence of polyadenylation sites the abundance of putative microbial symbiont transcripts among sequences from a pure plant root library is difficult to interpret . The predicted portion of microbial transcripts was greater in the axenic root - hair enriched library than in mixed cultures . Error rates were greatest for comparisons between training sets from plant and pooled zygomycete and chytridiomycete sequences . The 13% false - positive rate does not completely explain why about 15% of root - hair enriched transcripts resemble fungal hexamer composition more closely than plants, and warrants further study . Care had been taken to avoid contaminating plant tissue cultures by culturing seedlings in covered plates . Because of concern that ethylene accumulation in covered plates could improperly stimulate nodulation - related gene expression, seedlings were treated with ag2so4, an inhibitor of the plants' response to ethylene . Inhibition of the ethylene response could have resulted in synthesis of transcripts that are uncharacteristic of plant roots . Analysis of another axenic root - hair enriched library, particularly one provided a carbon source to identify potential contaminants, and not treated with an inhibitor of ethylene response, would be an informative test . The transcripts identified as most and least like plant or symbiont might also be studied in more detail as candidate participants in symbiosis . Symbiotic interactions, whether pathogenic or mutualistic, present novel challenges to both plant hosts and the biologists who study them . Computational approaches, in concert with experimental verification, can help resolve these challenges . To characterize hexamer frequencies in plant hosts and their microbial symbionts, we collected sets of training sequences from public databases and edited them for quality . Training sets were chosen to be representative of, but obtained independently from, taxa participating in symbiotic associations for which a diagnosis of origin would be made . Because the species being compared are represented unevenly in public sequence databases, taxa were chosen so that roughly the same number of genes were analyzed in each training set, rather than simply to maximize the numbers of species or sequences present . Training sets represent protein - coding sequences from three taxonomic groupings: plants (a1, medicago and glycine spp . ), either fungi (b2, zygomycetes and chytridiomycetes) or stramenopiles (b1), including ests from p. infestans, and bacteria (b3, rhizobium, sinorhizobium and bradyrhizobium). We performed pairwise comparisons with two different, taxon - specific training sets (a and b) to infer the origin of a transcript . Training sets were obtained by querying the genbank database using the entrez retrieval tool . A preliminary query by taxon name obtained all available nucleotide sequences from that taxon, then the limits option excluded ests, stss (sequence - tagged sites), gsss (genome survey sequences), working draft sequences, and patented sequences from the query set . Organellar (mitochondrial and chloroplast) dna was also excluded via the limits option . A query term to require that a sequence contain a protein - coding region (cds) was also added, which excluded ribosomal and transfer rna sequences . The results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query . (changing slightly the composition of training sets between those dates did not notably affect the experimental outcome .) Following a previously established protocol, we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . In this protocol, we resampled each training set 40 times by random partitioning into training (for hexamer counts) and test calculation pools . To control for any bias introduced by length variation, a program randomly clipped 300 nucleotide fragments for word counting . As a result, one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate; independent replicates contained different, randomly chosen training sequences and 300 nucleotide fragments . Values of the test statistic from 40 resampled replicates were pooled for calibration purposes . As with the original protocol, we pooled the resulting test statistic distributions, normalized them as cumulative distributions, and then evaluated them for overlap . We call the resulting comparisons' calibration curves', as they are not used directly to make inferences, but rather indirectly, to evaluate the degree of separation in hexamer counts from different taxa . Overlap of calibration curves should be minimal to yield the most statistically powerful results possible . Due to considerable overlap of calibration curves between taxonomically general, inclusive training sets (that is, all eudicots, all fungi and miscellaneous eukaryotes, and all eubacteria, data not shown), we opted to work with specific training sets that included only the most species - specific sequences available, while maintaining approximately equal sample sizes across taxa . The most challenging case was that of the arbuscular mycorrhizal fungi, for which very few protein - coding sequences are available . To increase the amount of data in this training set (b2) without biasing sample sizes, we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences, and compared this training set with a set from a single plant genus, medicago (a2). We chose this option, rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes, because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes, based on 18s ribosomal rna sequence data . That is, the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes, which resemble more closely the ancestral state in modern lineages . Starting with a full set of sequences, we filtered for high - quality sequences by trimming regions having extensive ambiguous bases (n - rich) and poly(a) or poly(t) regions . Thus, we trimmed poly(a) and poly(t) sites to minimize the cases in which a test sequence resembles one training set more closely than the other, simply by virtue of having an abundance of the hexamer aaaaaa or tttttt . Similarly, test results obtained from short or n - rich sequences can be difficult to interpret . We allowed no more than one n per hexamer and trimmed poly(a) or poly(t) tracts longer than 13 nucleotides . To accommodate for possible sequence chimeras, those sequences found to contain an internal poly(a) or poly(t) segment longer than 13 nucleotides were partitioned into two fragments, and the longer of the two fragments was used in analysis, provided its length was at least 300 nucleotides . After trimming, we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn . All blast searches used default parameters and low - complexity filtering with the programs dust or seg . The decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves (data not shown), likely a result of divergent evolutionary rates between protein - coding and non - coding sequences . Chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes . Table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening . To test the validity of word counting as a solution to the problem, we identified a set of 50 gene sequences from plants (m. truncatula and g. max), oomycetes (phytophthora), zygomycetes (glomus versiforme), and bacteria (sinorhizobium meliloti and agrobacterium tumefaciens), for which the function and origin have been characterized experimentally . We chose genes known to play a role in plant - microbe interactions, as well as genes that are found across taxa . We withheld these sequences, and partial transcripts of the same genes, from training sets prior to comparative lexical analysis, and calculated hexamer dissimilarities for each of the three training sets as described below . To diagnose the species of origin for sequences expressed in symbiotic cultures, we collected sequences generated by distinct est sequencing projects from the genbank database . Sequences from pathogenic interactions originated from cultures of a species from the genus phytophthora with its plant host, such as p. sojae and soybean (g. max) isolated from inoculated hypocotyls two days after infection and p. medicaginis and m. truncatula isolated from infected roots 10 days after infection (c. vance, unpublished data). Sequences expressed during mutualistic interactions were obtained from cultures with m. truncatula and mycorrhizal (glomus versiforme; m.j . Harrison, unpublished data) or rhizobacterial (s. meliloti; k. vandenbosch, unpublished data) endosymbionts several days after inoculation . Sequences expressed in pure, axenic cultures from p. sojae mycelia and zoospores and from sterile, uninoculated m. truncatula roots provided a basis for comparison in which no foreign transcripts were expected . To maximize the reliability of diagnostic comparisons, we screened test sequences for high quality as for training sequences, and for low similarity to e. coli, chloroplast and mitochondrial genes, and non - coding rna transcripts (ribosomal and transfer rnas). Independent blastn comparisons identified sequences having very high similarity (e <10) to vector sequences or moderately high similarity (e <10) to non - nuclear or non - coding sequences obtained from genbank . We wrote a perl program (countgc.pl) that calculates the gc base content of a sequence as the portion of guanine and cytosine residues among all unambiguous (non - n) nucleotides in a sequence . The hist method in r, version 1.1.1 aggregated continuous percentages into discrete histogram bins, using bin sizes of 2% difference in gc, with inclusive lower bin boundaries and exclusive upper bounds; the lm method tested for linear correlation of the dissimilarity test statistic t with gc . Used a likelihood - ratio test to determine whether word frequencies from a particular sequence more closely resemble the frequency distribution of control data sets from the taxon being sequenced or a distantly related outgroup . They computed a test statistic t(a, b, x) for each sequence x as the difference of log - likelihood ratio dissimilarity measures, d(a, x) = -2log(a, x), for two data sets, a control set a and an outgroup b, such that t(a, b, x) = d(a, x) - d(b, x). A negative value for t indicates that the sequence more closely resembles words from a; conversely, a positive value indicates a likely contaminant related to b. (dissimilarity is conceptually related to distance . However, dissimilarity does not measure distance because it does not possess the mathematical properties of a distance metric .) Unlike the calculation of calibration curves, in which 300-nucleotide subsequences are randomly resampled, hexamer dissimilarity is measured over the whole length of a test sequence when inferring a transcript's origin . Originally, the investigators used the null hypothesis that no difference exists for dissimilarity measures between the two data sets, or that t(a, b, x) = 0 . Tested two alternative hypotheses: that t <0, being more like a, or t> 0, like b. lexical analysis using pentamers or heptamers yields similar error rates and very highly correlated values for the test result (not shown). Because white et al . Reported the best results were obtained using hexamers, and because a word size of six nucleotides corresponds to the size of a dicodon, we chose to analyze hexamer frequencies . To use longer words requires more training data, because the number of possible words increases exponentially with increasing word size . Use of shorter words may be adequate for some applications and will be investigated in future work . Though we used white's word - counting methods, we did make slight modifications . We simplified one program (called hybridize) to compute individual dissimilarity values, rather than paired differences; a patch that details how to modify the c program is available (see hyb2dis.txt in additional data files). More importantly, we amended the null hypothesis and interpreted calibration curves to test for statistically significant dissimilarity differences . Though the likelihood - ratio test statistic indicates the magnitude of similarity to a or b, we do not know what values for t are significant with known confidence . When testing hypotheses, one can make two types of error: type i, or false positives, and type ii, false negatives . The false - positive rate is denoted and false - negative rate . We determine and from overlap in the calibration curves . Inferring error rates from calibration curves is justified because we know the correct answer and determine the error rate via resampling, as with bootstrap methods to infer error rates or confidence intervals . We are interested in knowing from which of two organisms a sequence originated, and are reasonably confident that it came from either one or the other . Thus, we assume it came from one and test whether we have evidence to refute this assumption . The null hypothesis here is that sequence x is from a. alternatively, it might be from b. evaluating the calibration curve overlap at t = 0 quantifies the associated error rates . The cumulative distribution function (cdf) of taxon b specifies where cdfb intersects 0; the cdf from a specifies as 1-cdfa(0). We can thus resolve the problem with known confidence p: p (t> 0) = . All other computations were performed as described previously . Software used for lexical analysis was obtained via anonymous ftp from the tigr software ftp site . To characterize hexamer frequencies in plant hosts and their microbial symbionts, we collected sets of training sequences from public databases and edited them for quality . Training sets were chosen to be representative of, but obtained independently from, taxa participating in symbiotic associations for which a diagnosis of origin would be made . Because the species being compared are represented unevenly in public sequence databases, taxa were chosen so that roughly the same number of genes were analyzed in each training set, rather than simply to maximize the numbers of species or sequences present . Training sets represent protein - coding sequences from three taxonomic groupings: plants (a1, medicago and glycine spp . ), either fungi (b2, zygomycetes and chytridiomycetes) or stramenopiles (b1), including ests from p. infestans, and bacteria (b3, rhizobium, sinorhizobium and bradyrhizobium). We performed pairwise comparisons with two different, taxon - specific training sets (a and b) to infer the origin of a transcript . Training sets were obtained by querying the genbank database using the entrez retrieval tool . A preliminary query by taxon name obtained all available nucleotide sequences from that taxon, then the limits option excluded ests, stss (sequence - tagged sites), gsss (genome survey sequences), working draft sequences, and patented sequences from the query set . Organellar (mitochondrial and chloroplast) dna was also excluded via the limits option . A query term to require that a sequence contain a protein - coding region (cds) was also added, which excluded ribosomal and transfer rna sequences . The results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query . (changing slightly the composition of training sets between those dates did not notably affect the experimental outcome .) Following a previously established protocol, we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . In this protocol, we resampled each training set 40 times by random partitioning into training (for hexamer counts) and test calculation pools . To control for any bias introduced by length variation, a program randomly clipped 300 nucleotide fragments for word counting . As a result, one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate; independent replicates contained different, randomly chosen training sequences and 300 nucleotide fragments . Values of the test statistic from 40 resampled replicates were pooled for calibration purposes . As with the original protocol, we pooled the resulting test statistic distributions, normalized them as cumulative distributions, and then evaluated them for overlap . We call the resulting comparisons' calibration curves', as they are not used directly to make inferences, but rather indirectly, to evaluate the degree of separation in hexamer counts from different taxa . Overlap of calibration curves should be minimal to yield the most statistically powerful results possible . Due to considerable overlap of calibration curves between taxonomically general, inclusive training sets (that is, all eudicots, all fungi and miscellaneous eukaryotes, and all eubacteria, data not shown), we opted to work with specific training sets that included only the most species - specific sequences available, while maintaining approximately equal sample sizes across taxa . The most challenging case was that of the arbuscular mycorrhizal fungi, for which very few protein - coding sequences are available . To increase the amount of data in this training set (b2) without biasing sample sizes, we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences, and compared this training set with a set from a single plant genus, medicago (a2). We chose this option, rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes, because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes, based on 18s ribosomal rna sequence data . That is, the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes, which resemble more closely the ancestral state in modern lineages . Starting with a full set of sequences, we filtered for high - quality sequences by trimming regions having extensive ambiguous bases (n - rich) and poly(a) or poly(t) regions . Thus, we trimmed poly(a) and poly(t) sites to minimize the cases in which a test sequence resembles one training set more closely than the other, simply by virtue of having an abundance of the hexamer aaaaaa or tttttt . Similarly, test results obtained from short or n - rich sequences can be difficult to interpret . We allowed no more than one n per hexamer and trimmed poly(a) or poly(t) tracts longer than 13 nucleotides . To accommodate for possible sequence chimeras, those sequences found to contain an internal poly(a) or poly(t) segment longer than 13 nucleotides were partitioned into two fragments, and the longer of the two fragments was used in analysis, provided its length was at least 300 nucleotides . After trimming, we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn . All blast searches used default parameters and low - complexity filtering with the programs dust or seg . The decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves (data not shown), likely a result of divergent evolutionary rates between protein - coding and non - coding sequences . Chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes . Table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening . To test the validity of word counting as a solution to the problem, we identified a set of 50 gene sequences from plants (m. truncatula and g. max), oomycetes (phytophthora), zygomycetes (glomus versiforme), and bacteria (sinorhizobium meliloti and agrobacterium tumefaciens), for which the function and origin have been characterized experimentally . We chose genes known to play a role in plant - microbe interactions, as well as genes that are found across taxa . We withheld these sequences, and partial transcripts of the same genes, from training sets prior to comparative lexical analysis, and calculated hexamer dissimilarities for each of the three training sets as described below . To characterize hexamer frequencies in plant hosts and their microbial symbionts, we collected sets of training sequences from public databases and edited them for quality . Training sets were chosen to be representative of, but obtained independently from, taxa participating in symbiotic associations for which a diagnosis of origin would be made . Because the species being compared are represented unevenly in public sequence databases, taxa were chosen so that roughly the same number of genes were analyzed in each training set, rather than simply to maximize the numbers of species or sequences present . Training sets represent protein - coding sequences from three taxonomic groupings: plants (a1, medicago and glycine spp . ), either fungi (b2, zygomycetes and chytridiomycetes) or stramenopiles (b1), including ests from p. infestans, and bacteria (b3, rhizobium, sinorhizobium and bradyrhizobium). We performed pairwise comparisons with two different, taxon - specific training sets (a and b) to infer the origin of a transcript . Training sets were obtained by querying the genbank database using the entrez retrieval tool . A preliminary query by taxon name obtained all available nucleotide sequences from that taxon, then the limits option excluded ests, stss (sequence - tagged sites), gsss (genome survey sequences), working draft sequences, and patented sequences from the query set . Organellar (mitochondrial and chloroplast) dna was also excluded via the limits option . A query term to require that a sequence contain a protein - coding region (cds) was also added, which excluded ribosomal and transfer rna sequences . The results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query . (changing slightly the composition of training sets between those dates did not notably affect the experimental outcome .) Following a previously established protocol, we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . In this protocol, we resampled each training set 40 times by random partitioning into training (for hexamer counts) and test calculation pools . To control for any bias introduced by length variation, a program randomly clipped 300 nucleotide fragments for word counting . As a result, one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate; independent replicates contained different, randomly chosen training sequences and 300 nucleotide fragments . Values of the test statistic from 40 resampled replicates were pooled for calibration purposes . As with the original protocol, we pooled the resulting test statistic distributions, normalized them as cumulative distributions, and then evaluated them for overlap . We call the resulting comparisons' calibration curves', as they are not used directly to make inferences, but rather indirectly, to evaluate the degree of separation in hexamer counts from different taxa . Overlap of calibration curves should be minimal to yield the most statistically powerful results possible . Due to considerable overlap of calibration curves between taxonomically general, inclusive training sets (that is, all eudicots, all fungi and miscellaneous eukaryotes, and all eubacteria, data not shown), we opted to work with specific training sets that included only the most species - specific sequences available, while maintaining approximately equal sample sizes across taxa . The most challenging case was that of the arbuscular mycorrhizal fungi, for which very few protein - coding sequences are available . To increase the amount of data in this training set (b2) without biasing sample sizes, we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences, and compared this training set with a set from a single plant genus, medicago (a2). We chose this option, rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes, because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes, based on 18s ribosomal rna sequence data . That is, the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes, which resemble more closely the ancestral state in modern lineages . Starting with a full set of sequences, we filtered for high - quality sequences by trimming regions having extensive ambiguous bases (n - rich) and poly(a) or poly(t) regions . Thus, we trimmed poly(a) and poly(t) sites to minimize the cases in which a test sequence resembles one training set more closely than the other, simply by virtue of having an abundance of the hexamer aaaaaa or tttttt . Similarly, test results obtained from short or n - rich sequences can be difficult to interpret . We allowed no more than one n per hexamer and trimmed poly(a) or poly(t) tracts longer than 13 nucleotides . To accommodate for possible sequence chimeras, those sequences found to contain an internal poly(a) or poly(t) segment longer than 13 nucleotides were partitioned into two fragments, and the longer of the two fragments was used in analysis, provided its length was at least 300 nucleotides . After trimming, we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn . All blast searches used default parameters and low - complexity filtering with the programs dust or seg . The decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves (data not shown), likely a result of divergent evolutionary rates between protein - coding and non - coding sequences . Chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes . Table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening . To test the validity of word counting as a solution to the problem, we identified a set of 50 gene sequences from plants (m. truncatula and g. max), oomycetes (phytophthora), zygomycetes (glomus versiforme), and bacteria (sinorhizobium meliloti and agrobacterium tumefaciens), for which the function and origin have been characterized experimentally . We chose genes known to play a role in plant - microbe interactions, as well as genes that are found across taxa . We withheld these sequences, and partial transcripts of the same genes, from training sets prior to comparative lexical analysis, and calculated hexamer dissimilarities for each of the three training sets as described below . To diagnose the species of origin for sequences expressed in symbiotic cultures, we collected sequences generated by distinct est sequencing projects from the genbank database . Sequences from pathogenic interactions originated from cultures of a species from the genus phytophthora with its plant host, such as p. sojae and soybean (g. max) isolated from inoculated hypocotyls two days after infection and p. medicaginis and m. truncatula isolated from infected roots 10 days after infection (c. vance, unpublished data). Sequences expressed during mutualistic interactions were obtained from cultures with m. truncatula and mycorrhizal (glomus versiforme; m.j . Harrison, unpublished data) or rhizobacterial (s. meliloti; k. vandenbosch, unpublished data) endosymbionts several days after inoculation . Sequences expressed in pure, axenic cultures from p. sojae mycelia and zoospores and from sterile, uninoculated m. truncatula roots provided a basis for comparison in which no foreign transcripts were expected . To maximize the reliability of diagnostic comparisons, we screened test sequences for high quality as for training sequences, and for low similarity to e. coli, chloroplast and mitochondrial genes, and non - coding rna transcripts (ribosomal and transfer rnas). Independent blastn comparisons identified sequences having very high similarity (e <10) to vector sequences or moderately high similarity (e <10) to non - nuclear or non - coding sequences obtained from genbank . We wrote a perl program (countgc.pl) that calculates the gc base content of a sequence as the portion of guanine and cytosine residues among all unambiguous (non - n) nucleotides in a sequence . The hist method in r, version 1.1.1 aggregated continuous percentages into discrete histogram bins, using bin sizes of 2% difference in gc, with inclusive lower bin boundaries and exclusive upper bounds; the lm method tested for linear correlation of the dissimilarity test statistic t with gc . White et al . Used a likelihood - ratio test to determine whether word frequencies from a particular sequence more closely resemble the frequency distribution of control data sets from the taxon being sequenced or a distantly related outgroup . They computed a test statistic t(a, b, x) for each sequence x as the difference of log - likelihood ratio dissimilarity measures, d(a, x) = -2log(a, x), for two data sets, a control set a and an outgroup b, such that t(a, b, x) = d(a, x) - d(b, x). A negative value for t indicates that the sequence more closely resembles words from a; conversely, a positive value indicates a likely contaminant related to b. (dissimilarity is conceptually related to distance . However, dissimilarity does not measure distance because it does not possess the mathematical properties of a distance metric .) Unlike the calculation of calibration curves, in which 300-nucleotide subsequences are randomly resampled, hexamer dissimilarity is measured over the whole length of a test sequence when inferring a transcript's origin . Originally, the investigators used the null hypothesis that no difference exists for dissimilarity measures between the two data sets, or that t(a, b, x) = 0 . White et al . Tested two alternative hypotheses: that t <0, being more like a, or t> 0, like b. lexical analysis using pentamers or heptamers yields similar error rates and very highly correlated values for the test result (not shown). Because white et al . Reported the best results were obtained using hexamers, and because a word size of six nucleotides corresponds to the size of a dicodon, we chose to analyze hexamer frequencies . To use longer words requires more training data, because the number of possible words increases exponentially with increasing word size . Use of shorter words may be adequate for some applications and will be investigated in future work . Though we used white's word - counting methods, we did make slight modifications . We simplified one program (called hybridize) to compute individual dissimilarity values, rather than paired differences; a patch that details how to modify the c program is available (see hyb2dis.txt in additional data files). More importantly, we amended the null hypothesis and interpreted calibration curves to test for statistically significant dissimilarity differences . Though the likelihood - ratio test statistic indicates the magnitude of similarity to a or b, we do not know what values for t are significant with known confidence . When testing hypotheses, one can make two types of error: type i, or false positives, and type ii, false negatives . The false - positive rate is denoted and false - negative rate . We determine and from overlap in the calibration curves . Inferring error rates from calibration curves is justified because we know the correct answer and determine the error rate via resampling, as with bootstrap methods to infer error rates or confidence intervals . We are interested in knowing from which of two organisms a sequence originated, and are reasonably confident that it came from either one or the other . Thus, we assume it came from one and test whether we have evidence to refute this assumption . The null hypothesis here is that sequence x is from a. alternatively, it might be from b. evaluating the calibration curve overlap at t = 0 quantifies the associated error rates . The cumulative distribution function (cdf) of taxon b specifies where cdfb intersects 0; the cdf from a specifies as 1-cdfa(0). We can thus resolve the problem with known confidence p: p (t> 0) = . All other computations were performed as described previously . Software used for lexical analysis was obtained via anonymous ftp from the tigr software ftp site . The following files are available for download: countgc.pl: perl script used to compute gc content of sequences analyzed . Hyb2dis.txt: patch file that converts white's hybridize program to compute individual dissimilarity values . Training sets (glycinemedicago.txt,rhizobia.txt, stramenopiles.txt, zygochytrid.txt): fasta - formatted text files that contain the sequences used for calibration and comparison . Test sets (psojaeha.txt, psojaemy.txt, psojaezo.txt, mtrhe.txt, dsir.txt, mham.txt, kv0.txt, kv2.txt, kv3.txt): fasta - formatted text files containing transcripts analyzed, edited for quality . Test results (psojaeha.dat, psojaemy.dat, psojaezo.dat, mtrhe-a1b1.dat, mtrhe-a2b2.dat, dsir.dat, mham.dat, kv0.dat, kv2.dat, kv3.dat): text files that contain transcript analysis results, sorted from least to most plant - like . Fasta - formatted text files containing transcripts analyzed, edited for quality . Text files that contain transcript analysis results, sorted from least to most plant - like . We thank callum bell, mark gijzen, maria harrison, tom kepler, deb samac, and bruno sobral for valued discussions and feedback . Comments from b. m. tyler and an anonymous reviewer on an earlier version of this work greatly enhanced its presentation . Distribution of gc content in pure and mixed - culture libraries . (a) probability densities for histogram bin sizes of 0.02 (2%) in base content . (a) calculation of statistical parameters from cdfs a and b. overlap in the upper tail of cdfa with cdfb and the lower tail of cdfb with cdfa are likely regions for error . We find the false - positive rate where 1 - cdfa intersects 0 [cdfa(0)= 1 -], and the false - negative rate where cdfb crosses 0 . Also shown are the medians () for each distribution, where cdf() = 0.5 ., solid black line) and stramenopile plus p. infestans est (b1, dashed black line) training sequences . Superimposed distributions of test results show dissimilarity differences for infected g. max (green) and axenic p. sojae mycelial and zoospore sequences (blue and cyan, respectively). Each point corresponds to an expressed tag from either (a) infected g. max or (b) axenic p. sojae mycelial or (c) zoospore sequences, compared with plant (a1) and stramenopile plus p. infestans est training sequences (b1). The identity function indicates equal dissimilarity to both training sets, t = d(a) - d(b)= 0 . Calibration curves compare plant training sets (a1 and a2, solid black lines) with one of three microbial symbiont training sets (broken black lines): (a) stramenopile and p. infestans est sequences (b1); (b) pooled zygomycete and chytridiomycete coding sequences (b2); and (c) sequences from the genera rhizobium, sinorhizobium and bradyrhizobium (b3). Cumulative distributions of test results from m. truncatula axenic and microbial symbiont mixed cultures appear in each panel (colored lines). Each point indicates the dissimilarity of a test sequence compared with a plant training set (a1 or a2) and one of three microbial symbiont training sets: (a) stramenopile and p. infestans est sequences (b1); (b) pooled zygomycete and chytridiomycete coding sequences (b2); and (c) sequences from the genera rhizobium, sinorhizobium and bradyrhizobium (b3). Sequences from m. truncatula axenic (green) and microbial symbiont mixed culture libraries are represented in each panel . Number of sequences (n) and nucleotides (nt), as raw, trimmed (removed n - rich regions, poly(a) and poly(t) sites), and screened sequences (removed ribosomal, chloroplast, and mitochondrial dna and remaining sequences shorter than 300 nucleotides). Number of est sequences (n) and nucleotides (nt) as raw, trimmed (limited lengths of n - rich regions, poly(a) and poly(t) sites), and screened (removed ribosomal, chloroplast, and mitochondrial dna, and remaining sequences shorter than 300 nt) sequences.
The transition - metal - catalyzed amination of aryl halides has become one of the most powerful methods to construct arylamines . The development of methods to couple aryl halides with amines has been studied extensively in the past decade . Palladium - catalyzed reactions occur with a broad scope of both coupling partners, high functional group tolerance, and good selectivity for monoarylation of the amine nucleophile . Both the palladium metal and the ligands used for this reaction are costly, and the ligand is difficult to recycle . For these types of reasons, much interest has been paid recently to develop reactions catalyzed by first - row metal complexes that are typically catalyzed by precious metals . For example, copper complexes have been studied extensively for the coupling of aryl iodides, and to a lesser extent aryl bromides, with nitrogen nucleophiles . Yet, the scope of such coupling with electron - rich bromoarenes, ortho - substituted bromoarenes, unactivated aryl chlorides, and heteroaryl halides is limited . Many of the first - row metals are more electropositive than those of the second row and form electron - rich low - valent species . Indeed, nickel complexes have been shown to catalyze the amination of aryl chlorides and phenol derivatives (scheme 1). Such nickel - catalyzed amination reactions could be more practical than palladium - catalyzed amination reactions on large scale because the metal is much less expensive, and much simpler ligands on the nickel center are needed for the catalyst to react with aryl chlorides . For example, hidai showed over 40 years ago that ni(pph3)4 adds phenyl chloride at room temperature to form (pph3)2ni(ph)(cl), whereas pd(pph3)4 does not react with aryl chlorides . However, the scope of the nickel - catalyzed amination of aryl halides has been limited to the coupling of secondary alkylamines and arylamines . Primary alkylamines, one of the most significant classes of amines for such cross coupling, have been shown to couple only with activated aryl chlorides . In addition to the limitations on the scope of the coupling of aryl halides with amines, the mechanism of the coupling of aryl halides with amines catalyzed by nickel complexes has not been studied . Many papers cite the potential of ni(i) intermediates, either as part of a one - electron redox event or as part of a mechanism involving ni(i) and ni(iii) intermediates . In contrast, the mechanism for the palladium - catalyzed amination of aryl electrophiles has been studied in detail, including the kinetic behavior of the catalytic reaction and stoichiometric reactions of isolated intermediates . Such studies have not been conducted on nickel - catalyzed couplings to form c n bonds, but are particularly important if one is to determine how to create catalysts of nickel that are as long - lived as those of palladium and that react with similarly broad scope . Here, we present the first nickel - catalyzed amination of unactivated aryl chlorides with primary aliphatic amines . The reaction occurs with aryl and heteroaryl chlorides and bromides catalyzed by a single - component binap - ligated nickel(0) precursor . Kinetic studies have been conducted on the reaction, and studies on the relative rates of the reaction with various isolated nickel complexes have been conducted . These studies rule out a catalytic cycle occurring through a ni(i) halide intermediate and are consistent with a ni(0)/ni(ii) catalytic cycle for the amination of aryl chlorides and aryl bromides, which react with different ni(0) species . We initiated our studies of nickel - catalyzed amination of aryl chlorides with primary aliphatic amines by studying the reaction between 3-chloroanisole and octylamine . We investigated nickel complexes generated in situ from ni(cod)2 and various phosphine ligands as catalysts . These reactions were conducted with naobu as base in toluene at 50 c . Reactions catalyzed by the combination of ni(cod)2 and monophosphines, such as pph3 and pcy3, afforded only trace amounts of the desired amine product (entries 14). Similar results (<5% conversion and <2% yield) were obtained for reactions catalyzed by the combination of ni(cod)2 (1 mol%) and dppe, dppp, or dppb (1 or 2 mol%) (entries 510). However, reactions catalyzed by the combination of ni(cod)2 and dppf or binap gave significant amounts of product (1667%, entries 1114). In particular, the reaction catalyzed by the single component catalyst (binap)ni(-nc - ph) afforded the product in 92% yield (entry 15). An explanation for the high activity of this catalyst will be revealed during the presentation of our mechanistic studies . Conditions: 3-chloroanisole (0.400 mmol), octylamine (0.600 mmol), naobu (0.600 mmol), 24 h. conversion and yield were determined by gc analysis using dodecane as internal standard . [ni] = (binap)ni(-nc - ph). With an active catalyst in hand and reliable conditions identified for this ni - catalyzed amination, we studied the scope of aryl chlorides that undergo these amination reactions, and the results are summarized in table 2 . In general, a range of electron - rich (2a2e), electron - neutral (2h), and electron - deficient (2i2l) aryl chlorides coupled with octylamine in high yields (7696%) under the developed conditions with 1 mol% of nickel complex 1 . For very electron - rich aryl chlorides, 4 mol% of the catalyst was required to obtain modest to good yields (2f and 2 g). For these two reactions, aryl chlorides were not fully converted, and only trace amounts (<5%) of biaryl compounds were detected by gc analysis . Mono - ortho - substituted aryl chlorides reacted in high yields (2a, 2c2e, 2h, and 2j). However, di - ortho - substituted aryl chlorides, such as 2-chloro-1,3-dimethylbenzene, did not couple with octylamine under the developed conditions . Conditions: arcl (0.400 mmol), octylamine (0.600 mmol), (binap)ni(-nc - ph) (4.0 mol, 1 mol%), naobu (0.600 mmol), toluene (1 ml); temperature, 50 c; reaction time, 24 h. (binap)ni(-nc - ph) (16.0 mol, 4 mol%). The scope of primary amines was studied by conducting the amination reactions of 3-chloroanisole catalyzed by 2 mol% complex 1 . The results of this study are summarized in table 3 . In general, a variety of primary aliphatic amines underwent this amination process in good to excellent isolated yields (7596%). Conditions: 3-chloroanisole (0.400 mmol), primary amine (0.600 mmol), (binap)ni(-nc - ph) (8.0 mol, 2 mol%), naobu (0.600 mmol), toluene (1 ml); temperature, 50 c; reaction time, 24 h. (binap)ni(-nc - ph) (16.0 mol, 4 mol%). The steric bulk of the alkyl group of the primary amines significantly influenced the yields of these reactions . The reactions of an amine containing a primary alkyl group (2 m) afforded the product in higher yield than the reactions of amines containing a secondary alkyl group (2n and 2o), although the primary amines containing a secondary alkyl group still coupled with aryl chlorides in good yield . However, the amination of 3-chloroanisole with primary amines containing a tertiary alkyl group, for example, tert - butylamime and amylamine, gave only trace amounts of the desired products . Amines containing aryl (2p), heteroaryl (2q and 2r), allyl (2s), and cyano (2 t) groups reacted in high yields (9096%). Primary amines bearing 1,3-dioxolane (2u and 2v) and morpholine (2w) moieties also reacted in high yields (8388%). The reaction of n - isopropylethylenediamine, a substrate containing both a primary and a secondary amine, occurred selectively with the primary amine, leaving the secondary amine intact (2x). Nitrogen - containing heterocycles containing amino substituents are of particular importance for pharmaceutical applications . However, the c n coupling reactions between nitrogen - containing heteroaryl electrophiles and primary amines are challenging, presumably because of the coordinating properties of both pyridines and primary amines . Nickel catalysts have been reported for the amination of heteroaryl chlorides, but the amine nucleophiles are limited to secondary amines . Cross - coupling reactions between nitrogen - containing heteroaryl chlorides and primary amines catalyzed by 2 mol% of complex 1 are summarized in table 4 . The scope of heteroaryl chlorides that underwent this amination reaction encompassed a range of 2-pyridyl (3a3f), 3-pyridyl (3g3l), and 4-pyridyl (3m3p) chlorides containing electron - donating groups (me and ome) and electron - withdrawing groups (cn and cf3). In general, these reactions afforded the corresponding heteroaryl alkylamine in good to excellent isolated yields (5796%, average yield: 81%). This amination also occurred in high yields with 2-chloroquinoline (3q) and 2-chloropyrazine (3r). The scope of primary amines that underwent reactions with chloropyridines was similar to that of the reactions of 3- chloroanisole described in table 3 . Conditions: 3-chloroanisole (0.400 mmol), primary amine (0.600 mmol), (binap)ni(-nc - ph) (8.0 mol, 2 mol%), naobu (0.600 mmol), toluene (1 ml); temperature, 50 c; reaction time, 24 h. (binap)ni(-nc - ph) (16.0 mol, 4 mol%). Compared to the well - established palladium - catalyzed amination of aryl bromides, the nickel - catalyzed amination of aryl bromides has been less studied, and conditions for coupling of aryl bromides with amines by nickel catalysts have not been identified . Only two examples of nickel - catalyzed amination of aryl bromides have been published, and these two reactions occurred in poor yields (eqs 1 and 2). Having identified conditions for the nickel - catalyzed amination of aryl chlorides, we tested these conditions for the coupling of aryl bromides with primary amines . The amination of aryl bromides catalyzed by benzonitrile complex 1 in toluene with naobu base occurred in good yields in some cases, but the scope of the reaction was limited . Bromobenzene and 4-bromobenzotrifluoride reacted with octylamine in high gc - yields (98% and 94%, repectively) under conditions listed in table 2 with 3 mol% of complex 1 (eq 3). However, 3-bromoanisole coupled with 2-(1,3-dioxolan-2-yl)ethan-1-amine in only 55% gc - yield, whereas the reaction of the same amine with 3-chloroanisole afforded the desired product in 87% isolated yield (eq 4). This limited scope for the amination of aryl bromides may stem from the faster thermal decomposition of arylnickel(ii) bromide species to nickel(i)3 bromide, relative to the rate of decomposition of arylnickel(ii) chloride species . The catalyst precursor employed in this study is a single - component nickel(0) complex (binap)ni(-nc - ph) (1). Previously, we reported the synthesis of this nickel complex by substitution of the 1,5-cyclooctadiene (cod) in ni(cod)2 by binap, and benzonitrile . To avoid using the thermally labile, air- and moisture - sensitive ni(cod)2, we developed a new synthesis (scheme 2) of this complex starting from the air - stable nickel precursor (dme)nicl2 . Reaction of (dme)nicl2 with 1 equiv of binap in thf at 50 c afforded (binap)nicl2, which was then reduced by zinc metal in the presence of cod . P nmr analysis of this reaction indicated a clean formation of (binap)ni(cod). The byproduct zncl2 was removed by washing the reaction mixture with degassed aqueous naoh . At this stage, the residue was suspended in toluene and then allowed to react with benzonitrile . Arylnickel halide complexes are important intermediates in nickel - catalyzed cross - coupling reactions . In general, arylnickel halides ligated by monophosphines are synthesized by oxidative addition reactions of aryl halides to ni(pr3)x (r = alkyl or aryl), but arylnickel halides ligated by bisphosphines are prepared by ligand substitution reactions between monophosphine - ligated arylnickel halide complexes with bisphosphines . The direct oxidative addition of aryl halides to bisphosphine - ligated ni(0) has rarely been investigated, mainly due to the lack of bisphosphine - ligated ni(0) precursors that can react with aryl halides at ambient temperature . Recently, we reported the synthesis of binap - ligated arylnickel halide complexes by reactions of aryl halides with (binap)ni(cod) in the presence of catalytic amounts of benzonitrile, and we identified (binap)ni(-nc - ph) as the active ni(0) species . In our communication on the nickel - catalyzed asymmetric -arylation of ketones, we reported the single reaction of electron - deficient 4-chlorobenzotrifluoride with this newly defined ni(0) precursor 1 and no isolation of the products . Here we show the results of the reactions of complex 1 with electronically varied aryl chlorides . Electron - neutral and electron - deficient aryl chlorides reacted smoothly with ni(0) complex 1 in thf at room temperature to afford the arylnickel(ii) chloride complexes 47 in 7689% isolated yields (eq 5). These complexes are stable in the solid state at ambient temperature for at least six months without noticeable decomposition . However, upon standing in thf solution at room temperature for about 48 h, they fully decomposed to [(binap)ni(-cl)]2 (8) with concomitant release of biaryls, as detected by gc - ms analysis . However, the reaction of electron - rich 4-chloroanisole with complex 1 in thf did not form the arylnickel chloride complex resulting from simple oxidative addition, as determined by p nmr spectroscopy . Instead, p nmr signals corresponding to two mutually trans phosphine resonances (doublets at 24.2 and 14.4 ppm with jpp = 352 hz) were observed . 56single - crystal x - ray diffraction of the isolated material (figure 1) showed that this product is complex 9 (eq 7). Complex 9 contains a coordinated monophosphine ligand ph2p(c6h4 - 4-ome) and a six - membered nickelacyclic structure . Nickelacyclic complex 9 contains a small dihedral angle between the two naphthyl groups of 63, and the nickel center consists of a distorted square planar geometry . Complex 9 is formed by p c bond cleavage of the binap backbone after the initial formation of binap - ligated arylnickel chloride complex (binap)nicl(c6h4 - 4-ome). C bond cleavage of the binap backbone in a binap - ligated phenylpalladium bromide complex was reported by our group previously . All the ellipsoids are drawn at the 50% probability and all the hydrogen atoms are omitted for clarity . Complex 9 was tested as a catalyst for the amination of 4-chloroanisole with octylamine under the conditions described in table 2 . Only trace (<5% by gc analysis) amounts of the amination product were formed . Thus, the formation of catalytically inactive complex 9 by the reaction of 4-chloroanisole and nickel precursor 1 explained the high catalyst loading required for the amination of electron - rich 4-chloroanisole . The oxidation state of the nickel intermediates in the amination of aryl halides with amines has been ambiguous, and pathways involving both ni(0)/ni(ii) and ni(i)/ni(iii) couples have been proposed for these reactions . With isolated, binap - ligated ni(0), ni(i) halide, and arni(ii) halide complexes in hand, we were able to assess precisely the competence of these complexes to be intermediates in the amination reactions . To do so, we conducted the reaction of the isolated (binap)nicl(c6h4 - 4-cf3) (7) with octylamine and naobu in the presence of 1 equiv of binap to trap a ni(0) product (a, scheme 3). This reaction afforded n - octyl-4-(trifluoromethyl)aniline in quantitative yield in less than 5 min at room temperature, as indicated by f nmr spectroscopic analysis . In addition, we conducted the amination reaction of 4-chlorobenzotrifluoride with octylamine in the presence of naobu catalyzed by 2 mol% of (binap)nicl(c6h4 - 4-cf3) at room temperature (b, scheme 3). Full conversion of 4-chlorobenzotrifluoride was achieved in 2 h, and this reaction afforded n - octyl-4-(trifluoromethyl)aniline in 91% isolated yield . The same reaction catalyzed by 2 mol% of [(binap)ni--cl]2 did not afford any amination product (c, scheme 3). These results show clearly that the binap - ligated arylnickel(ii) chloride compound is kinetically and chemically competent to be an intermediate in the catalytic process and the binap - ligated nickel(i) halide is not . To identify the resting state of the catalyst in this nickel - catalyzed amination reaction, we monitored by p{h} nmr spectroscopy the reaction of 4-chlorobenzotrifluoride with octylamine in the presence of naobu catalyzed by 2 mol% of (binap)ni(-nc - ph) (1) at room temperature . During the first 50% conversion of 4-chlorobenzotrifluoride, however, during the second 50% conversion, a new species corresponding to a p{h} nmr resonance at 31.7 ppm gradually accumulated . This new species is (binap)2ni, as determined by independently preparing this complex by reduction of (dme)nicl2 with zn metal in the presence of 2 equiv of binap in thf and comparing the p nmr chemical shift . To test the catalytic activity of (binap)2ni for this amination process, we conducted the reaction of 4-chlorobenzotrifluoride with octylamine catalyzed by 5 mol% of (binap)2ni in the presence of naobu at room temperature . This reaction did not afford detectable amounts of the amination product (a, scheme 4). This result is consistent with the early report that (binap)2ni does not undergo oxidative addition with aryl chlorides or bromides at ambient temperatures . The same reaction catalyzed by 5 mol% (binap)2ni in the presence of a catalytic amount of phcn (30 mol%) afforded the amination product in 84% yield in 20 h (b, scheme 4). For comparison, we conducted the same reaction catalyzed by only 2 mol% (binap)ni(-nc - ph). This reaction afforded the amination product in almost quantitative yield in 2 h at room temperature (c, scheme 4). Thus, (binap)ni(-nc - ph) is a more active catalyst than the combination of (binap)2ni and phcn . To assess the origin of the effect of benzonitrile on the rate of the catalytic reaction, we performed the reaction of (binap)2ni with benzonitrile and the reaction of (binap)ni(-nc - ph) with binap . The reaction of (binap)2ni with 10 equiv of benzonitrile did not form detectable amounts of (binap)ni(-nc - ph), as determined by p nmr spectroscopy (d, scheme 4). The reaction of (binap)ni(-nc - ph) with 1 equiv of binap afforded (binap)2ni quantitatively at ambient temperature (e, scheme 3). Based on these stoichiometric reactions and the catalytic reactions in scheme 3, we conclude that the formation of (binap)2ni is reversible in the presence of benzonitrile, but the equilibrium lies largely on the side of (binap)2ni and free nitrile . The formation of bis(amine)-ligated palladium complexes was reported as one possible pathway for deactivation of the catalyst in the palladium - catalyzed amination of aryl halides . Formation of analogous bis(amine) complexes of nickel was proposed to explain the much lower reactivity of primary aliphatic amines than that of cyclic secondary aliphatic amines and anilines in the nickel - catalyzed amination reaction . However, these bis(amine)-ligated nickel complexes have never been prepared or characterized . In the nickel - catalyzed amination of aryl chlorides with primary amines catalyzed by (binap)ni(-nc - ph), we observed the formation of (binap)2ni . The stoichiometry of this conversion indicates that there are nickel complexes lacking binap ligand in the reaction mixture . Thus, we studied the ligand substitution reactions between primary aliphatic amines and binap - ligated ni(0) and binap - ligated ni(ii) complexes . The reaction of (binap)ni(-nc - ph) with 50 equiv of n - butylamine in toluene did not form any new nickel species, and free binap was not detected by p nmr spectroscopy (a, scheme 5). This result is consistent with the affinity of the electron - rich ni(0) center for softer, -accepting ligands . However, the reaction of (binap)nicl(c6h4 - 4-cn) with 50 equiv of n - butylamine afforded free binap and trans-(n - butylamine)2nicl(c6h4 - 4-cn) (10) (b, scheme 5). Complex 10 was isolated in 76% yield . With the bis(amine)-ligated complex 10 in hand, we tested its potential to serve as an intermediate in the amination reaction . We conducted the reaction of 10 with 2 equiv of naobu in the presence or in the absence of 1 equiv of binap (c, scheme 5). This result indicates that the formation of bis(amine)-ligated nickel complexes is one possible pathway for decomposition of the nickel catalyst . After identifying the resting state of the nickel catalyst and possible paths for catalyst deactivation, we sought to identify the rate - limiting step for this nickel - catalyzed amination process . To do so, we measured the dependence of the initial rates of the amination reaction on the concentrations of each reagent under conditions in which the other reagents were present in large excess . We chose the reaction of 4-chlorobenzotrifluoride with octylamine in the presence of naobu for the kinetic studies because this reaction occurred at ambient temperature, and we were able to monitor the reaction conveniently by both p and f nmr spectroscopy . Furthermore, no background nucleophilic aromatic substitution reaction was observed for this aryl chloride and octylamine in the presence of naobu . For the initial rates to be a valid method for kinetic analysis of this reaction, all reagents must be soluble in the reaction medium, and no induction period can be present . The single - component catalyst precursor 1 is only marginally soluble in the reaction medium . Thus, we used (binap)nicl(c6h4 - 4-cf3) (7) as the source of nickel for our kinetic experiments because it dissolves in seconds once mixed with other reagents . Benzonitrile was added to the reaction mixture to trap the binap - ligated ni(0) species to form (binap)ni(-nc - ph), which had been identified as the catalyst resting state . In the absence of benzonitrile, the irreversible formation of (binap)2ni occurs quickly . Benzonitrile (10 equiv relative to complex 7) was added so that catalyst decomposition was not observed by p nmr spectroscopy over the time of the reaction . The kinetic behavior was assessed for the coupling of 4-chlorobenzotrifluoride with octylamine catalyzed by the combination of complex 7 in the presence of phcn and naobu at room temperature (eq 8). The initial rates were measured by f nmr spectroscopy using 4-ocf3-anisole as the internal standard . The rate of the amination reaction was determined by measuring the formation of n - octyl-4-(trifluoromethyl)aniline with respect to time . The plots of concentrations of n - octyl-4-(trifluoromethyl)aniline vs time at various concentrations of each reagent are shown in figures s1s5 in the supporting information . The plots of the initial rates of the reaction vs the concentrations of the catalyst, aryl chloride, and benzonitrile are shown in figure 2.8 the kinetic analysis showed that this nickel - catalyzed amination reaction of aryl chlorides is first order in the nickel catalyst, first order in aryl chloride, and inverse first order in phcn . The remaining reagents, naobu and amine, did not affect the initial rate of the reaction . These data indicate that oxidative addition of the aryl chloride to (binap)ni(0), formed by reversible dissociation of phcn from the catalyst resting state (binap)ni(-nc - ph), is the turnover - limiting step in the amination reaction . Plots of initial rates vs concentration of catalyst, aryl chloride, and benzonitrile for the amination of 4-chlorobenzotrifluoride (0.101.0 m) with n - octylamine (0.30 m) catalyzed by (binap)nicl(c6h4 - 4-cf3) (0.00600.018 m) in the presence of benzonitrile (0.0900.180 m) and naobu (0.30 m) at room temperature . The mechanism of palladium - catalyzed cross - coupling reactions with aryl halides as electrophiles has been extensively studied . These studies reveal that the halide identity has a dramatic effect on the mechanism for the oxidative addition of aryl halides to pd(0) species . The additions of the less reactive aryl chlorides occurs through lower - coordinated pd(0) intermediate than the additions of more reactive aryl iodides . However, such mechanistic data has not been gained for the nickel - catalyzed amination of aryl halides . For the nickel - catalyzed enantioselective -arylation of ketones, we found that aryl chlorides react in much higher yields and with much higher enantioselectivities than aryl bromides, and we attributed this dramatic effect of halide on the reaction yield and enantioselectivity to the faster decomposition of the arylnickel(ii) bromide intermediate relative to the arylnickel(ii) chloride intermediate to their corresponding ni(i) halide complexes . Because the ni(0) complex 1 ligated by binap and phcn catalyzes the amination of both aryl chlorides and aryl bromides, we can compare directly the kinetics and mechanism of the amination reactions of aryl chlorides and aryl bromides catalyzed by this nickel system . To do so, we conducted kinetic experiments on the amination of aryl bromides that were similar to those we conducted on the amination of aryl chlorides . We monitored by p nmr spectroscopy the reaction of 4-bromobenzotrifluoride with octylamine catalyzed by (binap)ni(-nc - ph) in the presence of naobu at room temperature . As was the case in the reactions of chloroarenes, (binap)ni(-nc - ph) was identified as the resting state of the catalyst in the reaction of 4-bromobenzotrifluoride with octylamine . Kinetic studies were conducted on the reaction of 4-bromobenzotrifluoride with octylamine catalyzed by (binap)nibr(c6h4 - 4-cf3) (11) in the presence of added phcn (eq 8). The initial rates were measured for various concentrations of each reagent while keeping the other reagents in excess . The plots of concentrations of n - octyl-4-(trifluoromethyl)aniline vs time at various concentrations of each reagent are shown in figures s7s11 . The plots of the initial rates of the reaction vs the concentrations of the catalyst, aryl chloride, and benzonitrile are shown in figure 3 . Plots of initial rates vs concentration of catalyst, aryl bromide, and benzonitrile for the amination of 4-bromobenzotrifluoride (0.100.80 m) with n - octylamine (0.30 m) catalyzed by (binap)nicl(c6h4 - 4-cf3) (0.00600.018 m) in the presence of benzonitrile (0.0900.180 m) and naobu (0.30 m) at room temperature . Like the amination of 4-chlorobenzotrifluoride, the amination of 4-bromobenzotrifluoride with octylamine is first order in the nickel catalyst, first order in aryl halide, zero order in naobu, and zero order in amine . In contrast to the reaction of 4-chlorobenzotrifluoride, which is inverse first order in phcn, the amination of 4-bromobenzotrifluoride is zero order in phcn . This order in phcn, in combination with the first order in aryl bromide, indicates that the bromoarene reacts directly with (binap)ni(-nc - ph) during the turnover - limiting step of the amination of the bromoarene without dissociation of phcn . The different reactivities of aryl halides with binap - ligated nickel complexes of different coordination numbers parallels a trend in oxidative addition to pd(0) complexes noted in the introduction to this section . Based on the stoichiometric reactions of the isolated binap - ligated arylnickel halides with primary amines in the presence of base and the conducted kinetic studies, we propose a catalytic cycle depicted in scheme 6 for the nickel - catalyzed amination of aryl chlorides and aryl bromides reported in this paper . The observed first order rate - dependence on the concentrations of both the catalyst and aryl halides indicates that the oxidative addition of aryl halides to binap - ligated ni(0) species to form (binap)niar(x) (x = cl or br) is the turnover - limiting step for both amination processes . This binap - ligated arylnickel(ii) halide complex subsequently reacts with primary amine and naobu to form an arylnickel amido complex, followed by reductive elimination of the n - alkyl aniline product in the presence of phcn to regenerate (binap)ni(-nc - ph), which was confirmed as the catalyst resting state . Interestingly, different rate - dependences on the concentration of phcn were observed for this nickel - catalyzed amination of aryl chlorides and aryl bromides . The reaction of aryl chlorides is inverse first order in phcn, whereas the reaction of aryl bromides is zero order in phcn . These reaction orders indicate that aryl chlorides and aryl bromides react with different nickel(0) species . The aryl chlorides react after reversible dissociation of phcn from (binap)ni(-nc - ph) to form the lower - coordinate nickel(0) species (binap)ni(0) and ultimately the binap - ligated arylnickel(ii) chloride complex . However, the aryl bromides react directly with (binap)ni(-nc - ph) to form binap - ligated arylnickel(ii) bromide complex and the nitrile dissociates during or immediately after the oxidative addition process . One could imagine the zero - order dependence on added phcn could indicate that dissociation of this ligand is rate limiting, but the first - order dependence of the reaction on the aryl bromide is inconsistent with this proposal . In addition to the steps within the catalytic cycle that form the arylamine product, there are several pathways that generate catalytically inactive nickel species . First, the reaction of (binap)niar(cl) with amine rnh2 forms the catalytically inactive (rnh2)2niar(cl) and free binap . The released free binap in the reaction mixture traps the (binap)ni(0) fragment generated by dissociation of phcn from (binap)ni(-nc - ph) or reductive elimination of n - alkyl aniline from the binap - ligated arylnickel(ii) amido complex to form (binap)2ni . (binap)2ni does not undergo oxidative addition of the aryl halide and does not catalyze the amination reaction . Second, thermal decomposition of (binap)niar(x) (ar = electron - neutral and electron - rich aryl groups) affords the catalytically inactive [(binap)ni(-x)]2 with concomitant formation of biaryl . C bond cleavage of the binap backbone is a potential pathway for the deactivation of the nickel catalyst . Indeed, this complex was observed to accumulate (approximately 6.4 mm of the 16 mm concentration of initial catalyst) during the reaction of 4-chloroanisole with octylamine . To gain information on the relative rates of the amination of aryl chlorides and aryl bromides and the effect of temperature and concentration of phcn on the selectivity of the amination reaction for an aryl chloride versus an aryl bromide, we conducted a series of reactions with an aryl halide containing both chloride and bromide and a mixture of aryl halides in the presence of various amounts of phcn at room temperature or 50 c . The amination of 1-bromo-4-chlorobenzene with octylamine catalyzed by 3 mol% of (binap)ni(-nc - ph) at 50 c occurred selectively at the c br bond, and the product of the reaction at the c cl bond was not detected by gc - ms analysis . (a, scheme 7). However, side - by - side reactions of chlorobenzene and bromobenzene under the same conditions occurred in the same gc yield (98%) (b, scheme 7). The amination of equal amounts of 1-bromo-4-butylbenzene and 4-chlorotoluene with octylamine catalyzed by 3 mol% of (binap)ni(-nc - ph) was conducted at both room temperature and 50 c (c, scheme 7). At both temperatures, these reactions occurred in similar yields, but with different selectivity . At room temperature, 1-bromo-4-butylbenzene was converted selectively over 4-chlorotoluene, and a product ratio of 3:1 was obtained . However, at 50 c, 1-bromo-4-butylbenzene and 4-chlorotoluene converted equally . Br bond in 1-bromo-4-chlorobenzene (a, scheme 7), the decreased selectivity for amination of the c br bond over the c cl bond in different haloarenes might stem from irreversible or nearly irreversible binding of the haloarene to ni(0) to form an arene -complex prior to cleavage of the carbon halogen bond . This preferential intramolecular oxidative addition has recently been observed for the ni - catalyzed kumada cross - coupling reactions that are part of chain - growth polymerizations . To evaluate the effect of phcn on the selectivity between the amination of an aryl chloride and an aryl bromide, we conducted the amination of equal amounts of 1-bromo-4-butylbenzene and 4-chlorotoluene with octylamine catalyzed by 3 mol% of (binap)ni(-nc - ph) in the presence of 0.9 equiv of phcn (relative to octylamine) (d, scheme 6). Because our kinetic studies revealed different dependences of the reaction rate on phcn for this nickel - catalyzed amination (inverse first order and zero order on phcn for the reaction of aryl chloride and aryl bromide, respectively), we considered that an increased selectivity for the conversion of aryl bromide over aryl chloride would be observed for reactions conducted with higher concentrations of phcn than for reactions conducted with lower concentrations of phcn . Consistent with this prediction, the selectivity was higher (30:1 for the reaction at room temperature and 9:1 for the reaction at 50 c) for the amination of 1-bromo-4-butylbenzene versus the amination of 4-chlorotoluene for reactions run with higher concentrations (from 0.004 to 0.18 m) of benzonitrile at both room temperature and 50 c for 24 h. these data show that the difference in ni(0) species that adds the different aryl halides can be used to beneficially affect selectivity . Several first - row metal complexes catalyze cross coupling reactions, and many catalyze the coupling of certain classes of chloroarenes . However, no first row metal complex has been shown to catalyze the coupling of primary alkylamines with unactivated chloroarenes . Copper complexes couple bromoarenes and iodoarenes with primary amines, but they do not catalyze the coupling of chloroarenes with primary amines . Previously, the combination of ni(cod)2 and dppf was shown to couple an electron - poor chloroarene with a primary amine, but this catalyst did not couple unactivated chloroarenes with primary amines . A set of preliminary studies we conducted have shown that the complexes (dppf)ni(cod) or (dppf)2ni generated in situ by the reaction of ni(cod)2 and dppf do not add aryl chlorides at ambient temperature, and the reaction of chloroarenes with (dppf)ni(cod) or (dppf)2ni at elevated temperatures forms (dppf)ni(i)cl and biaryl . This formation of (dppf)2ni(0) and (dppf)ni(i)cl at the elevated temperatures required for oxidative addition of chloroarene to occur, likely prevented productive coupling reactions with unactivated chloroarenes . These observations further illustrate the importance of the nitrile - ligated precursor with the general formula l2ni(-nc - ph). Nickel complexes of n - heterocyclic carbenes have been shown to couple aryl chlorides and sulfonates with secondary amines and with arylamines, but not primary alkylamines . Mechanistic studies on the reactions of secondary amines catalyzed by the complexes of n - heterocyclic carbenes are too limited to determine the origin of their lack of reactivity with primary amines, but recent studies have shown that the arylnickel halide complexes containing hindered n - heterocyclic carbenes are not formed by the reaction of (imes)2ni(0) with chlorobenzene, bromobenzene, and iodobenzene . Instead, ni(i) species are formed, and this formation of ni(i) complexes could either constitute the pathway for catalyst decomposition or a mechanism for reaction with this catalyst involving odd - electron nickel intermediates . We have shown that nickel complexes containing common aryl bisphosphine ligands can catalyze the coupling of aryl chlorides and bromides with primary amines . In particular, we have shown that the mixed ni(0) species (binap)ni(-nc - ph) containing one bisphosphine and one side - bound nitrile is a particularly active catalyst for the coupling of chloroarenes with primary amines and is more active than the related (binap)2ni catalyst . A range of electronically varied aryl halides and nitrogen - containing heteroaryl chlorides, including pyridine, quinoline, and isoquinoline derivatives, couple with a variety of aliphatic primary amines in high isolated yields . Several aspects of the current system lead to the ability of this first - row metal system to catalyze this class of amination reaction . In general, we attribute the high yields for these amination reactions to the facile oxidative addition of aryl chlorides and heteroaryl chlorides to the (binap)ni(-nc - ph) precatalyst at ambient temperatures . Detailed studies on the mechanism of the amination reaction the following conclusions about the mechanism of this coupling process have been drawn from these studies: (1) the oxidative addition of aryl chlorides to (binap)ni(-nc - ph), together with the stoichiometric reactions of (binap)niar(cl) with primary aliphatic amines in the presence of base, support a reaction pathway involving a ni(0)/ni(ii) couple in the catalytic cycle . (2) the oxidative addition of aryl chlorides and heteroaryl chlorides to the single - component nickel precursor (binap)ni(-nc - ph) at ambient temperatures prevents the formation of inactive ni species . (3) ni(i) intermediates, which have been observed in oxidative addition studies to phosphine - ligated ni(0) complexes, appear to result from decomposition of the ni(ii) product of oxidative addition as opposed to forming from one - electron pathways for oxidative addition to ni(0). (4) the reaction of (binap)ni(-nc - ph) with electron - rich aryl chlorides, such as 4-chloroanisole, leads to the p c bond cleavage of the backbone of binap and forms the nickel complex 9 containing two monophosphine ligands . The formation of the catalytically inactive complex 9 explains the high catalyst loading required for the amination of electron - rich aryl chlorides . (5) mechanistic and kinetic studies revealed that the turnover - limiting step of these nickel - catalyzed amination reactions of aryl chlorides is oxidative addition of the aryl chloride to (binap)ni(0), which is formed by dissociation of phcn from the catalyst resting state (binap)ni(-nc - ph). (6) mechanistic and kinetic studies imply that the turnover - limiting step of the reactions of aryl bromides is also oxidative addition, but that aryl bromides react with a different nickel species, of composition (binap)ni(-nc - ph) containing a bound nitrile . (7) the formation of catalytically inactive bis(amine)-ligated ni(ii) species (rnh2)2niar(cl), ni(i) species [(binap)ni(-cl)]2, and bis - binap - ligated ni(0) species (binap)2ni are the major pathways for catalyst inactivation . Further studies to improve the lifetime of the ni - bisphosphine catalysts, to determine the mechanism by which the phosphine - ligated ni(i) species form as a means to prevent this detrimental path, and to determine if ni(i) complexes are generated during oxidative addition of haloarenes will be the subject of future work.
Children diagnosed with jia were referred by pediatricians to our immuno - ophthalmology unit for ophthalmic examination . Children 10 years old or younger presenting to our clinic with intraocular inflammation were referred for pediatric consult to verify a possible jia association . Included in this study is a cohort of 172 children with jia who were examined with the slit lamp at least on three consecutive visits and followed for a minimum of 3 years and a maximum of 15 years after the original referral visit . At the time of initial examination all were diagnosed by pediatricians as having jia either prior to their initial ophthalmic examination (152 patients) or after pediatric consult was sought following the initial detection of intraocular inflammation (20 patients). All 172 children fulfilled the criteria for the diagnosis of jia: children with arthritis of at least 3 months duration, less than 16 years old at the time of first arthritic manifestation and without any other detectable cause for the arthritis . History of possible systemic disease and ocular manifestations were carefully reviewed with the patients and parents . All patients underwent an ocular examination which included assessment of the visual acuity using the snellen charts or the illiterate e charts and/or familiar pictures for the young verbal children . In a few of the very young and pre - verbal children, the pattern of fixation for near and distance and the elicitation of optokinetic nystagmus were used to assess their visual functions (benezra and rose 1990). Slit lamp biomicroscopy, thorough fundus examination and assessment of refractive errors in both eyes were performed in all cases . The intraocular pressure (iop) using the goldman applanation tonometer was obtained in cooperative children . In a few of the very young children with early ocular complications the iop assessment, fundus examination and refraction data ocular movement abnormalities, the presence or absence of strabismus and the binocular functions were also routinely assessed . Ocular medical treatment consisted on instillations of corticosteroids and cycloplegic eye drops when the intraocular inflammation was confined to the anterior segment . The frequency of eye drops instillations in these cases was arbitrarily modulated for every affected eye according to the intensity of inflammatory reaction in the aqueous humor as assessed at the slit lamp . Systemic corticosteroids (up to 1.0 mg / kg / day) and/or methotrexate (up to 25 mg once a week) were prescribed if the intermediate and posterior segments demonstrated active inflammatory process or at the advice of the treating rheumatologist for the control of severe arthritic manifestations . Complete blood count (cbc), sedimentation rate (esr), c reactive protein (crp), urine culture, kidney and liver function tests were performed in all cases . According to the clinical observations and the results of these routine preliminary examinations, a tailored individual more arduous battery of tests the type of intraocular inflammation (uveitis) was classified according to the anatomical site of the major inflammatory manifestations following the intraocular inflammation society guidelines (forrester et al 1998; benezra et al 2000). The intraocular inflammation was further subdivided to whether the disease was strictly ocular or it was associated with a systemic disease . Systemic disease associations were determined according to established sets of criteria (bloch - michel and nussenblatt 1987; weiner and benezra 1991; dollfus 1998; forrester et al 1998; benezra et al 2000; benezra et al 2005). History of possible systemic disease and ocular manifestations were carefully reviewed with the patients and parents . All patients underwent an ocular examination which included assessment of the visual acuity using the snellen charts or the illiterate e charts and/or familiar pictures for the young verbal children . In a few of the very young and pre - verbal children, the pattern of fixation for near and distance and the elicitation of optokinetic nystagmus were used to assess their visual functions (benezra and rose 1990). Slit lamp biomicroscopy, thorough fundus examination and assessment of refractive errors in both eyes were performed in all cases . The intraocular pressure (iop) using the goldman applanation tonometer was obtained in cooperative children . In a few of the very young children with early ocular complications the iop assessment, fundus examination and refraction data ocular movement abnormalities, the presence or absence of strabismus and the binocular functions were also routinely assessed . Ocular medical treatment consisted on instillations of corticosteroids and cycloplegic eye drops when the intraocular inflammation was confined to the anterior segment . The frequency of eye drops instillations in these cases was arbitrarily modulated for every affected eye according to the intensity of inflammatory reaction in the aqueous humor as assessed at the slit lamp . Systemic corticosteroids (up to 1.0 mg / kg / day) and/or methotrexate (up to 25 mg once a week) were prescribed if the intermediate and posterior segments demonstrated active inflammatory process or at the advice of the treating rheumatologist for the control of severe arthritic manifestations . Complete blood count (cbc), sedimentation rate (esr), c reactive protein (crp), urine culture, kidney and liver function tests were performed in all cases . According to the clinical observations and the results of these routine preliminary examinations, a tailored individual more arduous battery of tests the type of intraocular inflammation (uveitis) was classified according to the anatomical site of the major inflammatory manifestations following the intraocular inflammation society guidelines (forrester et al 1998; benezra et al 2000). The intraocular inflammation was further subdivided to whether the disease was strictly ocular or it was associated with a systemic disease . Systemic disease associations were determined according to established sets of criteria (bloch - michel and nussenblatt 1987; weiner and benezra 1991; dollfus 1998; forrester et al 1998; benezra et al 2000; benezra et al 2005). Of the 172 children with jia, 152 were referred by pediatricians for ocular examination after the diagnosis of jia was ascertained . In 20 children, intraocular inflammation was the presenting symptom while jia disease was established later by a pediatrician . Of the 152 children with the presenting symptom of arthritis, 18 (11.8%) had the systemic type of jia, 87 (57.2%) had the pauciarticular type and 47 (30.9%) had the polyarticular type . Of the 152 children presenting with all types of jia arthritic manifestations, in 14 children (9.2%), the intraocular inflammation (uveitis) was diagnosed during the first ophthalmic examination (12/14 had clinical evidence for the pauciarticular type of jia). All these 14 children had a recent history (3 to 9 months) of arthritic disease . Ten additional children developed uveitis less than three years of the initial ocular examination and, in 7 children the uveitis was detected 3 to 7 years later (table 1). Among the 20 children presenting initially with uveitis, 19 developed clinical manifestations compatible with the pauciarticular type of jia . A systemic type of jia was confirmed two months after the diagnosis of uveitis in one out of these 20 children (table 2). Thus, out of a total of 172 children with jia, 51 children had an associated intraocular inflammation (uveitis). Thirty nine of these 51 children (76.5%) had the pauciarticular type of jia . In 32 of these 39 children (82%), the uveitis was either the presenting symptom or it was detected during the initial ophthalmic examination . On initial examination, the intraocular inflammation processes were confined to the anterior segment in 45 of the 51 (88.2%) affected children . In 5 of the affected children (9.8%), a few cells were also observed in the anterior vitreous and in one child (2%), a marked vitritis with peripheral retinal vasculitis was observed in one eye . On further follow up however, only in 4 out of the 51 patients (7.8%), the inflammatory processes remained strictly confined to the anterior segment . Thus, with time, a tendency towards the involvement of the intermediate and posterior segments was observed . On last ophthalmic examination (3 to 15 years after initial diagnosis), in 22 patients (43.1%) the inflammatory signs were detected in all eye segments (pan uveitis) and in 24 children (47%), the inflammatory reaction involved the anterior and intermediate segments . When the intraocular inflammation was initially diagnosed, it was uniocular in 29 of the 51 children (56.9%). On further follow up, the intraocular inflammation remained uniocular only in 8 of the 51 patients (15.7%) while in 43 children (84.3%) there was involvement of both eyes . When both eyes were affected, the intensity of intraocular inflammation was unequal in the majority of patients . The eye with more severe inflammation showed invariably a poorer visual acuity and more prominent secondary ocular complications . The ocular complications observed in the 94 affected eyes of the 51 children are illustrated in table 3 . More severe and early complications were observed in girls manifesting the intraocular inflammatory processes when 4 years of age or younger . In 46 of the 94 eyes with uveitis (48%), a band - shaped keratopathy developed . In most, the keratopathy was mild and did not interfere with vision . In 5 eyes (5.3%), the keratopathy was dense, encroached on the visual axis and affected the visual acuity . An increased iop above 24mmhg and possible development of glaucoma was detected in 24 of the 94, (25%) affected eyes . Medical treatment consisting of beta blockers and carbonic anhydrase inhibitor drops along with a decrease in the instillation frequency of corticosteroid eye drops induced a decrease of iop below 20 mmhg in all cases . Lens opacity interfering with the visual acuity (cataract) was recorded in 56 of the 94 affected eyes (59.6%). In eyes developing severe lens changes (dense cataract) accompanied by band keratopathy, marked impairment of the visual functions interfering with the daily activity and/or inducing amblyopia it was assessed that the lens opacity and/or the ensuing amblyopia were the main cause for the decrease of vision to a level lower than 0.05 (6/120 or 20/400) (table 4). Twenty nine of these eyes underwent lensectomy combined with anterior vitrectomy while in one case, the parents declined surgical intervention . An intraocular lens (iol) was implanted in 19 eyes (pseudophakia) while 10 eyes remained aphakic . Glasses or contact lenses were prescribed for the correction of aphakia in these 10 eyes . The reported incidences of this association vary markedly with lower incidences and less severe ocular disease being reported more recently (sherry et al 1991; oren et al 2001; sim et al 2006; saurenmann et al 2007). A close look at these published differences reveals that the severity of ocular affection in the various studies is probably associated with population selection and referral trends in specific medical environments . It appears that the severity of ocular complications is higher when the study authors are ophthalmologists . This tendency is increased further when the papers originate from tertiary clinics specializing in uveitis and surgery of its complications . Noteworthy is the fact that 20 of the 51 children with jia and uveitis in our cohort presented with severe intraocular inflammation . The diagnosis of jia was made only after these children were referred specifically for pediatric examination to assess the possible presence of jia . In most of these 20 children thus, a study regarding the incidence of uveitis in jia carried out by pediatricians will not include these children biasing the incidence as well as the severity of ocular inflammation . Concomitant with other reports (sim et al 2006; grassi et al 2007; henligenhaus et al 2007), we observed in our present study that the highest associated incidence of arthritis and ocular manifestations is found in the group of children with the pauciarticular type . We have also noted that, in many cases, the articular and ocular clinical disease severity do not parallel . In many children, we observed severe ocular exacerbations during quiescent periods of arthritic manifestations and vice versa . An interesting aspect of our study is the observation that during the initial phase of the ocular disease, the inflammation is strictly restricted to the anterior segment . Later during the follow up however, the intermediate and posterior segments become involved in the intraocular inflammatory processes . These observations may result from a spill over of the chronically stimulated inflammatory cells from the anterior segment to the posterior structures dragging them to react following the release of inflammatory cytokines . The major points of clinical interest which can be derived from our study are: thirty nine of the 51 children with uveitis in our study group (76.5%) had the pauciarticular type of jia.the majority of children with pauciarticular jia developing intraocular inflammation harbor signs of ocular disease either before or shortly after the development of arthritic manifestations.most children with polyarticular or systemic jia types do not present signs of intraocular inflammation during the first three years following the initial arthritic manifestations.the potential for a child with jia to develop de novo ocular inflammation, at any time during the ophthalmic follow up, is low . The majority of children within all jia types who devolop an associated uveitis harbor ocular signs on initial examination . Therefore, frequent ophthalmic visits for all children with jia seems unjustified.nearly all children presenting with ocular disease and developing later jia characteristics, had a mild arthritis involving only one or two joints . Thirty nine of the 51 children with uveitis in our study group (76.5%) had the pauciarticular type of jia . The majority of children with pauciarticular jia developing intraocular inflammation harbor signs of ocular disease either before or shortly after the development of arthritic manifestations . Most children with polyarticular or systemic jia types do not present signs of intraocular inflammation during the first three years following the initial arthritic manifestations . The potential for a child with jia to develop de novo ocular inflammation, at any time during the ophthalmic follow up, is low . The majority of children within all jia types who devolop an associated uveitis harbor ocular signs on initial examination nearly all children presenting with ocular disease and developing later jia characteristics, had a mild arthritis involving only one or two joints . From our present study observations, it can be concluded that rigorous and very frequent ophthalmic exams for jia children are not obligatory if during the first ophthalmic examination ocular inflammatory signs are not detected . Furthermore, from our experience, a rigorous time frame for regular follow up visits for these children as suggested (rosenberg and oen 1986) seems unnecessary and, in our opinion, may be counterproductive for the following reasons: (a) uveitis may be detected during the ophthalmic examination only if the inflammatory signs are already present, (b) there are no predictive signs for the potential development of intraocular inflammation in these children, and (c) an intraocular inflammation may develop 24 or 48 hours after the regular periodic visit . In these cases, parents may be reluctant to seek again ophthalmic consultation (even if they suspect a problem) until serious complications develop . Therefore, education of the parents about the ocular disease, its potential evident signs and symptoms should be the main line of conduct for follow up instead of regular frequent ophthalmic visits . A drop of vision affecting the child s daily behavior can be noticed by parents . Also, parents can be taught to perform a visual acuity test at home when playing with the child . These measures in our experience are more effective, practical, easily carried out and have the highest possible cost / benefit ratio . In this study we confirm, as published earlier (benezra and cohen 2000) and also reported by others (wolf et al 1987; akduman et al 1997; woreta et al 2007) that, severe complications with loss of vision are still to be feared in children with jia who are developing uveitis . Most severe ocular complications have been observed in children 4 years of age or younger who presented first with ocular disease or had an ocular involvement detected during the initial ophthalmic examination . If not treated early, loss of vision may ensue in these cases even if successful control of the intraocular inflammation is achieved but proper anti amblyopic measures are not applied early . Last but not least, in the management of jra associated ocular inflammation, surgery should be considered despite the potential for complications . In properly selected cases, early surgery may even be considered in children with severe visual impairment in one eye due to band keratopathy, seclusion of the pupil by inflammatory membranes and/or dense cataract . In these cases, adequate surgical procedure of the cataract combined with intraocular lens implantation is to be considered and can result in good restoration of vision (benezra and cohen 2000; khotaniemi and penttila 2006).
Increasing cytosolic calcium ion (ca) concentration is essential for the contraction of smooth muscle cells . The increase results from the influx of ca through the plasma membrane and release of ca from intracellular stores, mainly the sarcoplasmic reticulum (sr) [14]. Phenylephrine (pe) or angiotensin ii (ang ii) induces receptor - coupled g protein - induced phosphoinositide 3-kinase (pi3k) and phospholipase c (plc) activation, resulting in ca - dependent vasoconstriction in smooth muscle cells [510]. Pi3k activity facilitates the production of 3-phosphorylated phosphoinositides such as phosphatidylinositol 3-phosphate (pi(3)p), phosphatidylinositol (3,4)-bisphosphate (pi(3,4)p2), and phosphatidylinositol (3,4,5)-triphosphate (pi(3,4,5)p3) [11, 12]. Among these, pi(3,4,5)p3 stimulates the l - type ca channel (12), a voltage - dependent ca channel that plays an important role in the regulation of vascular tone [5, 12, 13]. Activated plc is an effector in the stimulation of ca release from the endoplasmic reticulum (er) or the sr [1416]. Plc hydrolyzes phosphatidylinositol 4,5-bisphosphate (pip2) into diacylglycerol (dag) and inositol (3, 4, 5)-triphosphate (ip3). The latter is released as a soluble structure into the cytosol, where it binds to ip3 receptors in the sr [15, 17]. This binding process increases the cytosolic ca concentration and smooth muscle constriction [15, 16]. The herb cinnamomi ramulus (cr) has traditionally been used in asia and europe to treat maladies involving blood circulation and inflammation . In one study, an aqueous extract of cr ameliorated sucrose - induced blood pressure elevation in spontaneously hypertensive rats . Recently, we reported that cr ethanol extract (cre) reduces vascular contraction through the inhibition of voltage - dependent ca channels . The present study explored the suggestion that the vasodilatory effect of cre is related to ca - dependent mechanisms in rat aorta . All animals were provided with food and water ad libitum and allowed to adapt to the experimental conditions (temperature, 21 2c; humidity, 5060%) for 1 week . Rabbit polyclonal antibodies against phosphorylated plc (pplc), -actin, and anti - rabbit secondary antibody were purchased from santa cruz biotechnology (santa cruz, calif, usa). Pi3k / p85 antibody was purchased from cell signaling technology (beverly, mass, usa). Pe, ang ii, verapamil, nifedipine, fpl64176, 2, 4, 6-trimethyl - n-(meta-3-trifluoromethyl - phenyl)-benzenesulfonamide (m-3m3fbs), u73122 and caffeine were purchased from sigma - aldrich (st . Louis, mo, usa). Cr (twigs of cinnamomum cassia blume) collected in china in november 2009 was purchased from humanherb (gyeongsan, korea). A voucher specimen (cre08) has been deposited in the college of oriental medicine, dongguk university . Dried cr (100 g) was extracted with 500 ml of 70% ethanol by heating at 75c for 3 h. the extract was filtered through whatman filter paper (whatman international, maidstone, uk) to remove the insoluble materials . After filtration, the extracts were concentrated by rotary evaporation using a model vv2000 apparatus (heidolph, walpersdorfer, germany) at a temperature of 75c and then dried using a model fd8508s freeze dryer (ilshin, busan, korea). The ec50 value of 0.1 mg / ml cre was used in all experiments . In a previous research, cinnamaldehyde and coumarin also, cinnamaldehyde was known as major active compound of cr for vasodilation, antitumor, and antifungal activity . All procedures were performed according to protocols approved by the institutional animal care and use committee of dongguk university . Briefly, rats were sacrificed and their thoracic aortas were immediately excised and immersed in ice - cold krebs solution (115.0 mm nacl, 4.7 mm kcl, 2.5 mm cacl2, 1.2 mm mgcl2, 25.0 mm nahco3, 1.2 mm kh2po4, and 10.0 mm dextrose). The aortas were cleaned of all adherent connective tissue and cut into 3 mm long ring segments . Endothelium was removed from the internal surface of each segment by gentle rubbing with forceps . One triangle was anchored to a stationary support and the other was connected to a ft03 isometric force transducer (grass, quincy, mass, usa). Each vessel ring was incubated in a water - jacketed organ bath (10 ml) that was maintained at 37c and aerated with a mixture of 95% o2 and 5% co2 . Each ring was stretched passively by imposing the optimal resting tension of approximately 2.0 g, which was maintained throughout the experiment . Each endothelium - free aortic ring was allowed to equilibrate in the organ bath for at least 50 min before the experiment involving the contractile response to 5 m ang ii, 1 m pe, 10 m fpl64176, 5 g / ml m-3m3fbs, or 30 mm caffeine . Endothelium - free rings were used because preliminary experiments (data not shown) established that cre relaxes vascular constriction in an endothelium - independent manner . The denudation of endothelium was assessed by treating the rings with 1 m acetylcholine . Isometric tension was recorded using a powerlab/8sp computerized data acquisition system (adinstruments, castle hill, nsw, australia). The influence of cre on extracellular ca influx was studied in ca - free krebs solution . After equilibration of the ring in ca - free krebs solution containing 60 mm kcl, cumulative doses of cacl2 were added (0.3, 0.6, 1, 1.5, 2.5, 5, and 10 mm, in order) with preincubation of cre in organ bath . The cacl2 dose - dependent maximum constriction of the aortic ring with 60 mm kcl in ca - free krebs solution was expressed as 100% . To determine the influence of cre on ca influx through the l - type ca channel, aortic rings were pretreated with nifedipine or verapamil before pe contraction, and were preincubated with cre before contraction by fpl64176 . To investigate the inhibitory effect of cre on intracellular ca release by pe in ca - free conditions, and by caffeine in normal krebs solution, the transient contraction of cre preincubated aortic rings was measured . To further investigate the relationship with the plc pathway, aortic rings were constricted with m-3m3fbs, and were preincubated with u73122 prior to contraction by pe . A previously described protocol was used for preparation of protein extract with some modifications . Briefly, endothelium - free aortic rings were contracted with 1 m pe or 5 g / ml m-3m3fbs, and then treated with cre for 30 min . The aortic rings were quick frozen by immersion in acetone containing 10% trichloroacetic acid (tca) and 10 mm dithiothreitol (dtt) precooled to 80c . When used, recovered samples were homogenized in buffer containing 320 mm sucrose, 50 mm tris, 1 mm edta, 1% triton x-100, 1 mm dtt, and the following protease inhibitors: leupeptin (10 g / ml), trypsin (10 g / ml), aprotinin (2 g / ml), or phenylmethylsulphonyl fluoride (100 g / ml). The protein samples were electrophoresed and the resolved proteins were transferred to a nitrocellulose membrane . The membrane was incubated with primary antibodies and then treated with horseradish peroxidase - conjugated anti - rabbit igg as a secondary antibody . All bands were detected using an enhanced chemiluminescence system (amersham biosciences, buckinghamshire, uk). Each set of experiments was done at least three times and results are presented as the mean sd . The statistical significance of differences between mean values was assessed with student's t - test or anova . Ang ii increases the intracellular ca concentration in vascular smooth muscle cells through a sequence of events following activation of ang ii type 1 receptor and l - type calcium channels [6, 7]. Pe- or ang ii - induced contraction was significantly dilated by 53.6 7.8% and 66.0 5.4%, respectively, as compared to maximal tension (figure 1), indicating that that cre - mediated vasodilation may be related to decreased intracellular ca concentration . To determine the influence of cre on ca influx, the change of contraction was measured by adding cacl2 in an accumulative manner (0.3, 0.6, 1, 1.5, 2.5, 5, and 10 mm, in order) before and after cre pretreatment in ca - free krebs solution containing 60 mm kcl . The vasoconstriction rate at the aforementioned cacl2 concentrations were 7.7 8.1%, 23 16%, 44 16.7%, 59 17%, 72 14%, 88.4 9.4%, and 100%, respectively, (the latter represents the maximum contraction value of the aortic ring, achieved at 10 mm cacl2). These contractions were significantly reduced by 1.9 2.2%, 5.8 4.1%, 8.3 6.3%, 11.4 4.4%, 22.3 1.9%, 24.8 6.2%, and 29.7 3.8% (same respective cacl2 concentrations) with cre - pretreatment (figure 2). To discern the effect of cre on the l - type calcium channel, the influence of the l - type calcium channel blocker nifedipine (100 m) or verapamil (1 m), and the l - type calcium channel activator fpl64176 (10 m) on vasorelaxation of cre against pe - induced contraction of aortic rings was measured . Pretreatment of aortic rings with nifedipine or verapamil significantly inhibited the relaxant effect of cre (figure 3(a)). Previous studies have shown that fpl64176 increases extracellular ca entry, thereby enhancing the cytosolic ca concentration [23, 24]. Presently, fpl64176 induced contraction, which plateaued at 3.75 0.22 g in 30 min, was inhibited by 2.4 0.1 g with preincubation of cre (figure 3(b)). To assess whether cre is involved in ca release - mediated vasoconstriction from intracellular stores, the transient contraction by pe or caffeine was examined in cre preincubated aortic rings . Preincubation reduced the magnitude of contraction by pe from 0.32 0.6 g to 0.1 0.6 g (figure 4(a)). The transient contraction induced by 30 mm caffeine was also reduced by cre pretreatment (figure 4(b)). To evaluate whether the relaxant effect of cre was involved in the plc pathway, the plc pathway inhibitor u73122 and activator m-3m3fbs were used . U73122 pretreatment significantly inhibited the relaxant effect of cre on pe - induced contraction from 56.9 2.7% to 8.5 1.3% (figure 5(a)). M-3m3fbs (5 g / ml)-induced contraction was relaxed significantly with cre treatment (figure 5(b)). Pi3k is directly activated by g - protein for generation of pip3, which eventually stimulates l - type calcium channels in vascular myocytes [5, 12]., ip3 stimulates intracellular ca release from the sr for vasoconstriction [15, 17]. Presently, pe and m-3m3fbs increased the expression levels of pi3k (4.5 0.38 and 2.0 0.32, resp .) And pplc (4.1 0.30 and 2.5 0.14, resp . ), which were significantly decreased by cre . Pe - induced pi3k expression was decreased by 2.8 0.70 at 50 g / ml and by 0.2 0.6 at 100 g / ml . Pplc expression was also decreased by 2.0 0.30 at 50 g / ml and by 1.8 0.1 at 100 g / ml (figure 6(a)). M-3m3fbs - induced pi3k expression was downregulated by 0.6 0.1 at 50 g / ml and by 0.5 0.2 at 100 g / ml, and pplc expression was decreased by 1.4 0.1 at 50 g / ml and by 0.3 0.1 at 100 g / ml (figure 6(b)). Its bark and twig are known as cinnamomi cortex (cc) and cinnamomi ramulus (cr), respectively . Cc inhibits helicobacter pylori and ameliorates sucrose - induced blood pressure elevation in spontaneously hypertensive rats . Furthermore, cre exerts an endothelium - independent vasodilatory response through inhibition of voltage - dependent ca channels . The present study investigated the vasodilatory effect of cre resulting from the inhibition of both ca influx and release in rat aorta . Pe or ang ii stimulate plc isoforms to generate ip3 through the activation of g proteins, causing release of activator ca from sr [9, 10, 1416]. Presently, cre markedly and similarly relaxed aortic rings that were precontracted with pe or angii . These results suggest that cre - mediated vasodilation may be involved in the regulation of ca mobilization . To assess this, firstly, whether cre actually inhibits extracellular ca influx or not, we measured the tension of aortic rings by accumulative addition of cacl2 in ca - free krebs solution containing 60 mm kcl . Preincubation with cre significantly reduced rat aortic contraction by addition of cacl2,, indicating inhibition of ca influx . Nifedipine (100 m) and verapamil (1 m) pretreatment inhibited the vasodilative effect of cre on pe - induced constricted aortic rings from 54.9 4.1% to 15.3 2.1% and 13.9 5.2%, respectively . In addition, preincubation with cre reduced the contraction of aortic ring by 10 m fpl64176 . These results support the suggestion that the vasodilative effect of cre is related to the inhibition of l - type calcium channel in the cell membrane . The sr is the major source of ca release into the cytosol [1, 16, 17]. This ca release is induced by the ip3 second messenger, which is generated by plc activation . Ca release from the sr is considered to be the initial mechanism in agonists such as pe- and ang ii - induced vasoconstriction [6, 17]. Pe - induced transient constriction is dependent on ca release from the sr through the ip3 signal pathway in ca - free krebs solution; however, caffeine is dependent on ca - induced ca release from the sr [27, 28]. To demonstrate the effects of cre on ca release from the sr, transient contractions induced by pe in ca - free krebs solution and induced by caffeine in normal krebs solution were investigated . Cre significantly reduced the magnitudes of transient contraction by pe and caffeine, suggesting cre inhibits ca release from the sr by blocking the ip3-induced ca release and ca - induced ca release mechanisms . Pretreatment with the plc inhibitor u73122 significantly reduced the vasorelaxation of cre on pe - induced vasoconstriction, and cre relaxed m-3m3fbs - induced vasoconstriction . Additionally, we analyzed the expression levels of the intracellular signaling regulator proteins pi3k and plc . Pi3k generates various 3-phosphorylated phosphoinositides through activation by g - proteins, especially pi(3,4,5)p3 stimulates the l - type ca channel that plays an important role in the regulation of vascular tone [8, 11, 12]. On the other hand, plc formats the two potent second messengers ip3 and dag especially, ip3 induces the activation of ip3 receptor on the sr membrane, opening a calcium channel, resulting in the release of ca into the cytosol [14, 17]. Presently, pe- or m-3m3fbs - induced phosphorylation of plc and upregulation of pi3k / p85 protein expression were inhibited by cre (figure 6). The collective data supports the idea that cre dilates vascular contraction through the inhibition of both ca influx via the l - type ca channel and ip3-induced ca release from the sr . In conclusion, the data supports the vasorelaxation of cre through the inhibition of ca influx and ca release . Therefore, cre may be useful as a drug for the treatment and prevention of high blood pressure associated with ca - dependent contraction of smooth muscle.
After an information campaign in horse journals and at meetings, 278 samples of maple tree leaves, sprouts, and seeds collected by horse owners in 2014 were sent to utrecht university (uu) and rikiltwageningen ur (figs 1, 2, 3). The samples were taken from the ground, packed in plastic, and immediately sent to the laboratory where they were stored at 20c within 24 hours of collection . They were classified by an experienced botanist at the species level (acer pseudoplatanus, acer platanoides, or acer campestre) on the basis of the samples and accompanying photographs sent by owners . Sprouts were only used in this study upon positive classification by means of accompanying leaves, seeds or both . The minimum amount of material was 50 g for seeds and sprouts and 100 g for leaves . Owners were asked to complete a questionnaire with information about date of collection, address of the pasture, species of maple tree (in their opinion), signalment (name, breed and age) of the horse(s), and presence of disease (am) in horses that were grazed in the pastures where the material was collected . If the pasture had contained horses with am, details about the disease (including date of onset and the final outcome), laboratory results and name of the veterinarian were provided . Inclusion criteria to confirm am were recumbency, dark colored urine, high serum activity of creatine kinase (ck, either too high to measure in practice, or> 100,000 iu / l in a laboratory) and postmortem examination, which was performed in 4 cases . Exclusion criterion for am was lack of ck analysis unless postmortem examination was performed . The geographic location of the pastures where the acer seeds, sprouts and leaves were sampled . A, b, c leaves of acer pseudoplatanus, acer campestre and acer platanoides . Hypoglycin a was measured using a newly validated method based on liquid chromatography coupled to tandem mass spectrometry (lc ms / ms).1 seed samples (50 g, as received) were ground in a retsch mill, and leaf samples (50100 g) were cryogenically milled into a powder, using liquid nitrogen . One gram of homogenized sample material was extracted with 10 ml of milliq water (mechanical shaker, 1 h). After centrifugation (5000 g, 10c, 10 min), 10 l of the clear extract was injected into an lc ms / ms system, acquiring 2 transitions (m / z 142.174.1 and 142.196.0). Hypoglycin a was identified based on matching retention time and ion ratio of the 2 transitions, against an analytical reference standard (shypoglycin a2) dissolved in milliq water . (residuals <20%) between the concentration of hypoglycin a and the msresponse was obtained in the range 0.012 mg / l, corresponding to 0.120 mg / kg in nondiluted extract and 102000 mg / kg for 100fold diluted extracts . The accuracy of the method was determined as the recovery obtained after spiking known amounts of hypoglycin a to blank leaf (1 mg / kg) and seed (10 mg / kg) samples in 5fold dilutions . The corresponding repeatabilities (relative standard deviation, rsd) were 11% and 7%, respectively . The rsd obtained for repetitive analysis (n = 5) of a homogenized seed sample containing hypoglycin a at a concentration of 194 mg / kg was 8% . Recoveries obtained for hypoglycin a in spiked seed samples (10 mg / kg) concurrently analyzed with the study samples were consistent with those of the validation and in the range of 7395% . Samples with incomplete questionnaire data on relevant facts, such as address and obtainable data about the disease if present, were excluded from further statistical analysis (9 samples). If multiple samples from 1 pasture were submitted, which happened often, the mean result of the analyses per sample type (sprout, seed or leaf) were counted as 1 result in further statistical analysis . Samples that could not be unambiguously assigned to an acer species (6), samples that were not fresh (17), and samples that contained material other than seeds, sprouts and leaves (10) also were excluded from the study . This approach resulted in 143 usable samples, 30 from the spring and 113 from the autumn of 2014 . The samples were grouped by whether they came from pastures grazed by healthy horses or horses with am (nonam versus am pastures). Mean concentrations of hypoglycin a were compared for the type of sample (seed versus sprout versus leaf), season (spring versus autumn) and disease status for the pasture (nonam versus am). Statistical analysis was performed using univariate generalized linear models with multiple comparisons, corrected with a posthoc bonferroni test.3 control for normal distribution was performed . After an information campaign in horse journals and at meetings, 278 samples of maple tree leaves, sprouts, and seeds collected by horse owners in 2014 were sent to utrecht university (uu) and rikiltwageningen ur (figs 1, 2, 3). The samples were taken from the ground, packed in plastic, and immediately sent to the laboratory where they were stored at 20c within 24 hours of collection . They were classified by an experienced botanist at the species level (acer pseudoplatanus, acer platanoides, or acer campestre) on the basis of the samples and accompanying photographs sent by owners . Sprouts were only used in this study upon positive classification by means of accompanying leaves, seeds or both . The minimum amount of material was 50 g for seeds and sprouts and 100 g for leaves . Owners were asked to complete a questionnaire with information about date of collection, address of the pasture, species of maple tree (in their opinion), signalment (name, breed and age) of the horse(s), and presence of disease (am) in horses that were grazed in the pastures where the material was collected . If the pasture had contained horses with am, details about the disease (including date of onset and the final outcome), laboratory results and name of the veterinarian were provided . Inclusion criteria to confirm am were recumbency, dark colored urine, high serum activity of creatine kinase (ck, either too high to measure in practice, or> 100,000 iu / l in a laboratory) and postmortem examination, which was performed in 4 cases . Exclusion criterion for am was lack of ck analysis unless postmortem examination was performed . The geographic location of the pastures where the acer seeds, sprouts and leaves were sampled . A, b, c leaves of acer pseudoplatanus, acer campestre and acer platanoides . Hypoglycin a was measured using a newly validated method based on liquid chromatography coupled to tandem mass spectrometry (lc ms / ms).1 seed samples (50 g, as received) were ground in a retsch mill, and leaf samples (50100 g) were cryogenically milled into a powder, using liquid nitrogen . One gram of homogenized sample material was extracted with 10 ml of milliq water (mechanical shaker, 1 h). After centrifugation (5000 g, 10c, 10 min), 10 l of the clear extract was injected into an lc ms / ms system, acquiring 2 transitions (m / z 142.174.1 and 142.196.0). Hypoglycin a was identified based on matching retention time and ion ratio of the 2 transitions, against an analytical reference standard (shypoglycin a2) dissolved in milliq water . Quantification was performed by multilevel calibration . A consistent linear relationship (residuals <20%) between the concentration of hypoglycin a and the msresponse was obtained in the range 0.012 mg / l, corresponding to 0.120 mg / kg in nondiluted extract and 102000 mg / kg for 100fold diluted extracts . The accuracy of the method was determined as the recovery obtained after spiking known amounts of hypoglycin a to blank leaf (1 mg / kg) and seed (10 mg / kg) samples in 5fold dilutions . The corresponding repeatabilities (relative standard deviation, rsd) were 11% and 7%, respectively . The rsd obtained for repetitive analysis (n = 5) of a homogenized seed sample containing hypoglycin a at a concentration of 194 mg / kg was 8% . Recoveries obtained for hypoglycin a in spiked seed samples (10 mg / kg) concurrently analyzed with the study samples were consistent with those of the validation and in the range of 7395% . Samples with incomplete questionnaire data on relevant facts, such as address and obtainable data about the disease if present, were excluded from further statistical analysis (9 samples). If multiple samples from 1 pasture were submitted, which happened often, the mean result of the analyses per sample type (sprout, seed or leaf) were counted as 1 result in further statistical analysis . Samples that could not be unambiguously assigned to an acer species (6), samples that were not fresh (17), and samples that contained material other than seeds, sprouts and leaves (10) also were excluded from the study . This approach resulted in 143 usable samples, 30 from the spring and 113 from the autumn of 2014 . The samples were grouped by whether they came from pastures grazed by healthy horses or horses with am (nonam versus am pastures). Mean concentrations of hypoglycin a were compared for the type of sample (seed versus sprout versus leaf), season (spring versus autumn) and disease status for the pasture (nonam versus am). Statistical analysis was performed using univariate generalized linear models with multiple comparisons, corrected with a posthoc bonferroni test.3 control for normal distribution was performed . A p value of .05 was set as statistically significant . In total, 143 of the collected samples (93 seed samples, 14 sprout samples, and 36 leave samples) from a total of 81 locations were included (fig 1). None of the acer platanoides (n = 11) and acer campestre (n = 13) samples (29 samples of seeds, 11 of leaves and 1 of sprouts) contained detectable concentrations of hypoglycin a. in contrast, most acer pseudoplatanus leaves, seeds, and sprouts contained the toxin . In 2 samples of seeds and 6 samples of leaves, no hypoglycin a was found (ie, concentration below limit of quantification [1 mg / kg for leaves and 10 mg / kg for seeds]). The factor season was not statistically significant (p = .841) and was excluded from further statistical analysis . The mean sd concentrations of hypoglycin a in the samples are presented in table 1 . Hypoglycin a concentrations differed significantly among the sample types (seeds and sprouts, p <.001; seeds and leaves, p <.001; sprouts and leaves, p = .000). Concentrations were significantly higher in seeds from the am group than in seeds from the nonam group (seeds, 856 677 versus 456 358 mg / kg; p = .039). In sprouts, (1365 795 mg / kg versus 1097 310, p = .493) and leaves (31 49 versus 37 34 mg / kg, p = .819) this was not the case and when values for all seeds, sprouts, and leaves were combined the difference between nonam and am was not significant either (p = .127); (table 1). The concentration (mg / kg) of hypoglycin a in samples of acer pseudoplatanus taken from pastures or paddocks grazed by healthy horses and horses with atypical myopathy (am) concentration significantly different between pastures used by healthy and am horses (p = 0.039). There were 16 horses (providing 29 samples) with am of which 11 died and 5 survived . There was no relationship between the concentration of hypoglycin a in the samples and outcome of the disease (p = .250)., acer negundo (box elder tree) and acer pseudoplatanus (sycamore maple) have been associated with am.15, 17 the species acer campestre (hedge maple) and acer platanoides (norway maple) did not contain detectable hypoglycin a concentrations, and thus it is probably safe, in terms of am, to grow these species in or around pastures and paddocks . In 1973, semiquantitative measurements (notdetected, trace, weak, moderate, and strong) also indicated that these tree species were free of hypoglycin a.18 the most common maple tree in the netherlands is acer pseudoplatanus . We found that the leaves, sprouts, and seeds contained measurable concentrations of hypoglycin a. although hypoglycin a concentrations were significantly lower in seeds collected from pastures grazed by healthy horses than in samples from pastures grazed by am horses, concentrations were too variable to allow predictions to be made as to whether or not the presence of an individual tree would be a threat to horses grazing in its vicinity . Our results are consistent with a study that found hypoglycin a concentrations in seeds to be highly variable among trees on the same or different farms.17 hypoglycin a concentrations in our study were similar to those previously reported.17 in the previous study, a mean concentration of 690 mg / kg was found with variation between 40 and 2810 mg / kg and in our study a mean of 856 mg / kg with variation between 25 and 3683 mg / kg was found . The question arises as to why not all horses grazing pastures containing or surrounded by acer pseudoplatanus develop am . The most likely explanation might relate to differences in the amount of seeds, sprouts, and leaves ingested, other components of the diet, and the sensitivity of the individual horse . The availability of seeds, leaves, and sprouts largely is determined by the season and weather, with changes in wind and temperature loosening the attachment of leaves and seeds.6, 10, 15, 19 indeed, inclement weather or high winds have been reported to precede clinical signs of am.6, 12, 15 strong winds can disperse leaves and seeds over a long distance, perhaps depositing them in pastures that do not contain acer trees . All of these factors could explain why acer trees are not always found in the paddock or pastures grazed by horses with am.8 our results suggest that, per kilogram, sprouts are most dangerous to horses, followed by seeds, with leaves being potentially least harmful . Data show that there are more cases of am in autumn than in spring.8 because sprouts are more common in spring and seeds are more common in autumn, it would appear that horses eat more seeds than sprouts either because more seeds are available or because they prefer seeds to sprouts . However, whether horses eat seeds or sprouts is to a large extent determined by the availability of other feedstuff.20 in general, spring pasture contains more and better grass than autumn pasture, and thus horses may have less reason to eat other feedstuff.20 the finding that the concentration of hypoglycin a is high in sprouts suggests that horse owners should be alert to the presence of acer sprouts in pastures . Owners should be advised to mow areas of pasture with maple sprouts and to remove the mown material . A factor that needs further investigation is tree stress.20, 21 tree stress or abiotic stress may increase the hypoglycin a concentration in seeds . Several owners who sent in samples did not return the questionnaire or did not complete it fully . Moreover, if there was doubt (after consultation with the owner's veterinarian) about whether a horse suffered from am or not, data for samples collected from that horse's paddock or pasture were not used . Results of the samples of 1 type (seed, sprout, or leaf) originating from 1 pasture were pooled and the mean concentration of these was treated as 1 sample . Because toxin concentrations are highly variable among trees on the same farm, it would have been better to have taken more samples of all materials . The seeds were fresh frozen and not desiccated before freezing and changes in toxin concentrations as a result of artificial freezing could have occurred in the spring samples in the same fashion as occurs naturally in autumn . Ideally all cases would have been confirmed by analyzing blood or urine for madd or toxin or toxin metabolites . Because doing so was not always possible, the described inclusion criteria were used to make the diagnosis . In conclusion, it is important to know the tree species when giving advice regarding the toxicity of maple trees and the potential risk for horses, because some acer species contain hypoglycin a and others do not . Unfortunately, the amount of hypoglycin a in seeds is so variable that a reliable prediction of the occurrence of am cannot be made for individual farms . Therefore, it is not worthwhile to measure hypoglycin concentrations in individual seed samples in order to predict am risk . However, in all cases in which no hypoglycin a was found, no clinical signs of am were observed . Thus, the most important recommendation is that owners should prevent horses from eating seeds, sprouts, leaves, or any combination of these from acer pseudoplatanus (sycamore maple), whereas the seeds of acer campestre (hedge maple) and acer platanoides (norway maple) appear to be safe for horses . Steps to prevent the ingestion of toxin include moving the horse to a safer pasture, decreasing the size of the pasture (away from the trees), blowing away seeds and leaves, or mowing and removing sprouts.
Cell migration requires the rearrangement of adhesions and cytoskeleton in response to extracellular stimuli to form a leading edge that allows directed locomotion . In most cell types, migration - related adhesive structures are composed of integrin heterodimers and intracellular associated proteins clustered in plaques termed focal complexes and their more mature variant, focal adhesions . In addition to these adhesive structures, dcs also assemble distinctive adhesions named podosomes that are characteristic of cells of the myeloid lineage including macrophages and osteoclasts [4, 5]. The molecular composition of podosomes is similar to that of focal complexes and focal adhesions . However, they have a unique organization with actin filaments bundled in discrete foci forming a conical core containing distinctive elements such as gelsolin, cortactin, the actin - nucleating factor arp 2/3 complex, and the scaffolding protein wasp surrounded by a ring of integrins and integrin - associated proteins [4, 5]. Wasp is an essential component of podosomes, where it activates the arp2/3 complex inducing actin polymerization . Wip has also been detected in the core of podosomes in endothelial cells expressing egfp - wip, and although there is evidence indicating that wip plays a major role in regulating the activity of wasp family proteins and the dynamics of actin filaments [8, 9], it remains unknown how wip participates in the assembly of podosomes . To study the role of wip in podosome formation, the distribution of f - actin and vinculin was examined in wild - type and wip dcs by confocal microscopy . As expected, podosomes were grouped in clusters in wild - type dcs (figures 1a and 1c). Those few podosomes formed in wip dcs displayed an abnormal structure having less discrete actin cores and disorganized vinculin rings around f - actin (figure 1d). Large focal contacts containing vinculin comprised the most striking adhesion structures in wip dcs (figures 1b and 1e), replacing podosomes as the major points of contact with the substratum . The critical involvement of wip in dc podosome formation is also supported by the presence of endogenous wip in the core of podosomes in wild - type cells (figure 1f). Our data suggest that the key role of wip in podosome formation is in the organization of f - actin and the clustering of integrins and associated proteins . The absence of wip had a profound effect on the organization of f - actin within dcs . While the bulk of f - actin in wild - type dcs was associated with the core of podosomes (table 1, see figure s1a in the supplemental data available online), in wip dcs it distributed among various anomalous structures including actin cables (table 1, figure s1b), large fused actin aggregates, commonly with a circular shape (table 1, figure s1c), and extensive membrane ruffles (table 1, figure s1d). Clustering of podosomal integrins and the integrin - associated proteins paxillin and talin was also affected . In wild - type dcs, 2-integrins formed rings around the f - actin cores of podosomes (figure s2a) as previously described . In contrast, wip dcs failed to cluster 2-integrins into rings, and instead they formed small integrin - containing focal contacts scattered through the basal surface and around the periphery of the cell (figure s2b) or large plaques associated with anomalous actin - rich aggregates (figure s2c). Facs analysis showed that wild - type and wip dcs expressed equivalent levels of 1- and 2-integrins (figure s3), indicating that wip does not affect integrin expression . Paxillin and talin, which normally array around the podosome core in wild - type dcs (figures s2d and s2 g), in wip dcs localized to focal adhesions (figures s2e and s2h) containing 1 integrins (data not shown) or formed amorphous clusters colocalizing with the anomalous actin aggregates (figures s2f and s2i). The shift of adhesion composition and structure from podosomes (short half - life in the range of 30 s to 5 min) to large focal contacts (long half life in the range of 30 to 60 min) in wip dcs had a negative impact on adhesion turnover (figure 1 g). As previously described, interference reflection microscopy (irm) analyses showed podosomes in wild - type dcs rapidly assembled and disassembled, giving a high turnover index (figure 1 g). In wip dcs, although multiple transient and unstable lamellar protrusions were developed at the cell periphery, the major adhesion sites in contact with the substratum were highly stable with a significantly lower turnover index compared to wild - type (figure 1 g). Interestingly, we found a decrease in podosome turnover in wild - type dcs plated on fibronectin (fn) and icam-1, indicating that integrin ligands promote podosome stability (figure 1 g). This stability of adhesion on integrin ligands correlates with an increase in the percentage of wild - type dcs with well - defined podosomes and small focal contacts (figure 1a) and accumulation of podosomal f - actin, integrins, and associated proteins (data not shown). In contrast, no significant differences were detected in turnover index of adhesions between wip dcs plated on different substrata (figure 1 g). The presence of integrin ligands failed to induce major changes in the composition of adhesions of wip dcs (figure 1b) except for an increase in the size of the focal adhesions . Taken together, our data indicate that wip plays a key role in regulating dc adhesion structure and dynamics on biologically relevant substrata, processes crucial for the migratory capacity of cells . Stabilization of the leading edge in the direction of dc movement is provided by podosomes [13, 14]. Migrating wild - type dcs clearly polarized, forming a patent leading edge, with the rear of the cell retracting at a similar rate, allowing net translocation of the cell body . In contrast, wip dcs failed to develop a major leading front and instead formed multiple simultaneous and unstable lateral lamellae and ruffles (figures 2a2e; movies s1 and s2). Cortactin is an arp2/3 activator that plays a major role in the formation of podosomes [15, 16], establishment of cell polarization, and also generation of cellular cortical protrusions . It has been proposed that cortactin synergises with the wasp family of proteins to induce actin polymerization, and it is thought that the actin filament meshwork triggered by the n - wasp / cortactin pair may have a different organization from actin meshworks triggered by n - wasp or cortactin alone . Importantly, wip has been shown to interact with cortactin and induce cortactin - mediated actin polymerization . Our results show that in the absence of wip, cortactin is displaced from podosome cores where it normally colocalizes with f - actin (figure 2f) and is instead associated with random lamellar protrusions and ruffles over the cell periphery (figure 2 g). In addition, we found there was a higher proportion of cortactin associated with the cytoskeletal fraction (triton insoluble) in adherent wip dcs compared to wild - type dcs (figure 2h). Taken together, these results show that in the absence of wip, association of cortactin with the cytoskeleton is deregulated and associated with the formation of random cortical protrusions and loss of dc polarity . This suggests that the wasp / wip complex may couple with cortactin to induce a type of meshwork of actin filaments and associated proteins that allow the clustering of 2-integrins during the assembly of podosomes and dc polarization . In the absence of wip, cortactin cannot be recruited to form a complex with wasp, discrete actin foci in podosomes fail to form, and instead, cortactin codistributes with actin filaments at the cell margin, resulting in the random protrusions and reduced motility (data not shown) observed in wip dcs . We have previously shown that wasp dcs display equivalent anomalies in 2-integrin clustering and focal contact formation to that observed in wip dcs . Also we find here that like wasp dcs, wip dcs fail to polarize and form and stabilize consistent leading edges . Recently, it has been proposed that wip contributes to the stabilization of wasp expression and the regulation of wasp - mediated actin rearrangement [8, 22]. We find here that wip dcs express low levels of wasp at the protein level (figure 3a), as recently described for wip t - lymphocytes . Levels of wip expression were not affected by the lack of wasp in wasp dcs (figure 3b). Since mrna levels of wasp in wip dcs were comparable to those of wild - type dcs (figure 3c) the levels of expression of the wip binding partners cortactin, nck, and crkl was not affected in wip dcs (figure s4). It has been predicted that wip may prevent the exposure of wasp family proteins to proteases, because the 25-residue wasp binding motif from wip wraps around the wip binding domain of n - wasp . Wasp, but not n - wasp, is a substrate for the protease calpain in platelets, and we have recently found that calpain also cleaves wasp in migrating dcs . Our present results show that wip prevents wasp cleavage by calpain in dcs since inhibition of this protease in wip dcs resulted in major recovery of wasp expression (figures 3d and 3e). This suggests that wip may play a crucial role in regulating the accessibility of wasp for calpain cleavage, which takes place during dissolution of podosomes . A role for wip in the maintenance of wasp levels is also suggested by recent findings showing that in was patients, mutations that prevent wasp binding to wip result in diminished expression of wasp [24, 25]. We suggest that the low levels of wasp in these was patients may be the result of extensive calpain - mediated cleavage because of failure of wasp - wip interaction . Surprisingly, the recovery of wasp levels in wip dcs by treatment with calpain inhibitors was not sufficient to induce podosome formation, and large focal contacts remained the major adhesion structures (figure 4a). In addition, forced expression of egfp - wasp in wip dcs treated with calpain inhibitors alone or in conjunction with proteasome inhibitors failed to restore podosome formation, and egfp - wasp remained diffusely distributed in the cytoplasm or associated with anomalous f - actin aggregates that could contain vinculin forming abnormal plaques colocalizing with f - actin (figure 4b, top and middle). This contrasts with the observed recovery of podosome formation and dynamics in wasp dcs (where wip levels are normal) after the expression of egfp - wasp (figure 4b, bottom) [11, 13]. We excluded the possibility that wip binding is essential for the intrinsic actin polymerizing activity of wasp because specific wasp mutants (wasp a134 t and wasp r138p) that interrupt wip binding [24, 27] showed an actin polymerizing activity equivalent to wild - type wasp (figure 4d). These results indicate that wip not only protects wasp from calpain cleavage but also facilitates the localization of wasp to sites of actin polymerization and the organization of integrins, vinculin, and other integrin - associated proteins in circular clusters . The mechanisms by which wip regulates wasp activity to allow podosome formation are yet to be fully elucidated . It is possible that wasp / wip interactions may directly facilitate the recruitment of wasp to cell - surface receptors . Alternatively, wip may facilitate the bridging of wasp to these receptors by mediating the interaction with other adaptor proteins or may regulate the function of regulatory proteins such as kinases, known to affect wasp activity [30, 31]. Our data show that wip by itself is not sufficient to promote podosome assembly because wasp dcs, which express normal levels of wip, also lack podosomes and assemble large focal contacts and disorganized actin clumps instead . It is tempting to speculate that the cellular responses (of dc) to external soluble ligands such as chemoattractants would recruit wasp / wip complexes coupled to integrin - associated proteins to trigger the assembly of podosomes at appropriate sites in the developing leading edge of the cell . These complexes would provide a cytoskeletal platform that regulates the accurate clustering and activation of 2-integrins, facilitating their function . Taken together, our results suggest that wasp and wip work as a functional unit in a cellular context as has been previously proposed [8, 9] to regulate podosome formation and dynamics during the establishment of cell polarity as seen in the chemotactic response to extracellular stimuli . In summary, we have shown that in dcs wip is essential for podosome formation and cell polarity by preventing the extensive degradation of wasp by calpain and by facilitating the recruitment of wasp to discrete foci to form the core of podosomes . The tight coupling between wasp and wip is therefore a key contributor to normal podosome dynamics and likely of major importance to the cell trafficking behavior of dcs.
Ventriculoperitoneal (vp) shunting is commonly utilized as a diversion procedure for patients with hydrocephalus . Multiple techniques for initial placement of the catheters have been described, including conventional minilaparotomy, peel - away introducer sheaths, as well as full laparoscopic approaches . Vp shunts fail for a variety of reasons, including failure secondary to disconnection of abdominal tubing with migration of the tubing, most commonly further into the peritoneal cavity; this occurs in 2.5% to 4% of shunts placed according to current published series . Initial shunt failure rates continue to be high, with a reported failure rate of 31% at 6 months and an infection rate of 7% . Common presenting features of shunt failure include headache, mental status changes, and vomiting . Laparoscopy avoids laparotomy and may provide improved confirmation of shunt functionality and placement at the time of revision . A 31-year - old obese female was initially treated for pseudotumor cerebri with placement of a lumbar peritoneal shunt in 2003 . After 5 failed revisions for nonfunction, a ventriculoperitoneal shunt was placed in september 2007 . The patient subsequently presented 11 days postoperatively with headaches, dizziness, and formation of a seroma in her right upper quadrant . Ct scan revealed the vp catheter tubing coiled in the subcutaneous tissue of the right upper quadrant . Four 5-mm titanium clips were placed on the catheter in an attempt to prevent repeated migration of the tubing . One month later, the patient presented with recurrence of headaches, nausea, and right upper quadrant abdominal pain . Ct scan was again obtained, revealing that the shunt tubing had migrated back into the subcutaneous tissue (figure 1). A laparoscopic approach was again utilitzed for retrieval and attempted improved fixation of the vp shunt . Adhesions were taken down at the original entry site of the shunt, revealing a 2-cm fascial defect in the abdominal wall with the shunt tubing coiled within it (figure 2). The tubing was brought down through the defect out of the subcutaneous space and into the peritoneal cavity (figure 3). One port was upsized to 10 mm, and the tubing was externalized through the port and secured to a 3-cm x 10-cm piece of ptfe mesh with 30 prolene sutures (figure 4). Csf flow was confirmed, and the tubing attached to the mesh was reintroduced into the peritoneal cavity . The mesh was secured to the abdominal wall by using spiral tacks and 0 gore - tex sutures (figure 5). The separate 2-cm fascial defect was repaired with a 10-cm x 10-cm piece of ptfe mesh placed as an onlay with the vp shunt exiting from its inferior border . It was secured with spiral tacks and 0 gore - tex sutures for transabdominal fixation (figure 6). The patient was seen in follow - up 7 months postprocedure and reported no recurrence of her symptoms . Traditionally, laparotomy was necessary for both peritoneal shunt placement as well as subsequent shunt revisions . The advent of laparoscopy has afforded a minimally invasive approach that has resulted in decreased patient morbidity related to shunt placement . Retrieval and repositioning of a displaced vp shunt in the peritoneal cavity using laparoscopic techniques has been previously described . In the present case, our subsequent laparoscopic approach utilized ptfe mesh, well described in the use of laparoscopic ventral hernia repair, to fixate the tubing to the abdominal wall to prevent further tubing migration . This approach also allowed us to simultaneously repair the fascial defect associated with the shunt entry site into the peritoneal cavity, allowing us to reap the benefits of decreased recurrence and decreased patient morbidity associated with this technique . The technique described was well tolerated without complication and resulted in improvement of clinical symptoms.
Ventilator - associated pneumonia (vap) is defined as pneumonia occurring more than 48 hours after patients have been intubated and received mechanical ventilation . Vap is the most common nosocomial infection in the intensive care unit (icu) with an incidence ranging from 8% to 28% in intubated mechanically ventilated patients . Ventilator - associated pneumonia complicates the course of patients receiving mechanical ventilation inspite of major advances in techniques for its diagnosis and treatment . In the absence of a gold standard, vap is assumed to be diagnosed more accurately by bronchoscopic sampling and microbiological cultures of the lower respiratory tract . Bronchoscopy, being invasive, is not uncommonly associated with complications, especially in patients on high respiratory supports . This has paved the way for less invasive tests such as endotracheal aspirates (eta) and quantitative eta cultures with a threshold of 10 to 10 bacteria per milliliter of exudates that is considered as optimal for the microbiological confirmation of vap. [46] the american thoracic society (ats) guidelines recommend that quantitative cultures can be performed on eta or samples collected either bronchoscopically or nonbronchosopically . More importantly, recent small trials have repeatedly shown that there is no advantage of bronchoscopic cultures over quantitative eta cultures when mortality was considered as the end - point further strengthening the case for quantitative eta as a diagnostic tool. [811] detection of causative organisms and their antibiotic susceptibility is crucial for diagnosis of vap in order to initiate the appropriate antibiotic treatment thereby reducing the adverse effects of inadequate antibiotic treatment on the patient prognosis . Hence, the present study is undertaken to isolate, identify and quantitate bacteria and to perform the antibiotic susceptibility testing from the endotracheal aspirates of the clinically suspected patients of vap . The prospective study was carried out during the period from january 2010 to december 2010 in department of microbiology and medical intensive care unit (micu) on 54 patients with clinical suspicion of ventilator - associated pneumonia . The approval of the institutional review board was obtained during the planning phase of the study and each patient (or his / her caregivers) gave informed consent prior to participation in the study . All the patients 18 years and above who were under mechanical ventilation for more than 48 hours and clinically suspected of having contracted vap were included in this study . The diagnosis of vap was established using clinical pulmonary infection score (cpis), which was evaluated on a daily basis until the patient was on ventilator support . Clinically diagnosed ventilator - associated pneumonia were observed and clinical parameters were recorded from their medical records and bedside charts . Endotracheal aspirate (1 ml) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu . The eta was collected using a 22-inch ramson's 12 f suction catheter with a mucus extractor, which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm . Chest vibration or percussion for 10 min was used to increase the retrieved volume (1 ml) in case the patient produced very little secretions . Only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing . The aspirate specimens showing presence of <10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study . Samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min . 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10, 10, 10, 10, 10, 10 . The samples were then plated on sheep blood agar (sba), chocolate agar (ca), and macconkey agar (ma) by using 4 mm nichrome wire loop (hi - media, mumbai, india), which holds 0.01 ml of solution . All plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator . Growth of any organism below the threshold was assumed to be due to colonization or contamination . Organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method: erythromycin (e) (15 g), clindamycin (cd) (10 g), cotrimoxazole (co) (25 g), cephalexin (cp) (5 g), linezolid (lz) (30 g), doxycycline (do) (30 g), ciprofloxacin (cip) (5g), ceftazidime (caz) (30 g), amoxyclav (amc) (30 g), vancomycin (va) (30 g), amikacin (ak) (30 g), imipenem (i) (10 g), cefotaxime (ce) (30 g), piperacillin - tazobactam (pt) (100 g/10 g) (hi - media laboratories, mumbai). Escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains . Isolates showing reduced susceptibility to either ceftazidime (30 g) or cefotaxime (30 g) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . As per clsi 2011 guidelines, when using the new interpretive criteria, routine esbl testing is no longer necessary before reporting results (ie, it is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins to resistant). Isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases (mbl) enzymes by imipenem - edta combined disc method . All the patients 18 years and above who were under mechanical ventilation for more than 48 hours and clinically suspected of having contracted vap were included in this study . The diagnosis of vap was established using clinical pulmonary infection score (cpis), which was evaluated on a daily basis until the patient was on ventilator support . Clinically diagnosed ventilator - associated pneumonia were observed and clinical parameters were recorded from their medical records and bedside charts . Endotracheal aspirate (1 ml) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu . The eta was collected using a 22-inch ramson's 12 f suction catheter with a mucus extractor, which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm . Chest vibration or percussion for 10 min was used to increase the retrieved volume (1 ml) in case the patient produced very little secretions . Only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing . The aspirate specimens showing presence of <10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study . Samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min . 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10, 10, 10, 10, 10, 10 . The samples were then plated on sheep blood agar (sba), chocolate agar (ca), and macconkey agar (ma) by using 4 mm nichrome wire loop (hi - media, mumbai, india), which holds 0.01 ml of solution . All plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator . Growth of any organism below the threshold was assumed to be due to colonization or contamination . Organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method: erythromycin (e) (15 g), clindamycin (cd) (10 g), cotrimoxazole (co) (25 g), cephalexin (cp) (5 g), linezolid (lz) (30 g), doxycycline (do) (30 g), ciprofloxacin (cip) (5g), ceftazidime (caz) (30 g), amoxyclav (amc) (30 g), vancomycin (va) (30 g), amikacin (ak) (30 g), imipenem (i) (10 g), cefotaxime (ce) (30 g), piperacillin - tazobactam (pt) (100 g/10 g) (hi - media laboratories, mumbai). Escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains . Isolates showing reduced susceptibility to either ceftazidime (30 g) or cefotaxime (30 g) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . As per clsi 2011 guidelines, when using the new interpretive criteria, routine esbl testing is no longer necessary before reporting results (ie, it is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins to resistant). Isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases (mbl) enzymes by imipenem - edta combined disc method . Endotracheal aspirate (1 ml) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu . The eta was collected using a 22-inch ramson's 12 f suction catheter with a mucus extractor, which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm . Chest vibration or percussion for 10 min was used to increase the retrieved volume (1 ml) in case the patient produced very little secretions . Only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing . The aspirate specimens showing presence of <10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study . Samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min . 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10, 10, 10, 10, 10, 10 . The samples were then plated on sheep blood agar (sba), chocolate agar (ca), and macconkey agar (ma) by using 4 mm nichrome wire loop (hi - media, mumbai, india), which holds 0.01 ml of solution . All plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator . Growth of any organism below the threshold was assumed to be due to colonization or contamination . Organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method: erythromycin (e) (15 g), clindamycin (cd) (10 g), cotrimoxazole (co) (25 g), cephalexin (cp) (5 g), linezolid (lz) (30 g), doxycycline (do) (30 g), ciprofloxacin (cip) (5g), ceftazidime (caz) (30 g), amoxyclav (amc) (30 g), vancomycin (va) (30 g), amikacin (ak) (30 g), imipenem (i) (10 g), cefotaxime (ce) (30 g), piperacillin - tazobactam (pt) (100 g/10 g) (hi - media laboratories, mumbai). Escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains . Isolates showing reduced susceptibility to either ceftazidime (30 g) or cefotaxime (30 g) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . As per clsi 2011 guidelines, when using the new interpretive criteria, routine esbl testing is no longer necessary before reporting results (ie, it is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins to resistant). Isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases (mbl) enzymes by imipenem - edta combined disc method . This prospective study was done in the period from january 2010 to december 2010 in our hospital icu . The meansd age of patients was 46.418.45 years (range 18 to 86 years), having a predominance of male population . Bimodal distribution of age was observed with the first peak between 18 - 28 years of age group and second peak at around 59 years of age(28.89%). Distribution of total number of cases was done depending on the duration of mechanical ventilation into early - onset vap developing within 4 days of intubation and late - onset vap developing after 4 days of intubation . The percentage of patients with early onset vap was 39.62% and late onset vap was 60.38% . In our study, acinetobacter baumannii caused predominantly bilateral and right sided pneumonia whereas pseudomonas aeruginosa caused more of bilateral bronchopneumonia [table 1]. Chest x - ray finding in relation to the organism isolated of 53 patients diagnosed as vap based on a cpis score of more than six, 51 (96.23%) patients had monomicrobial infection and 2 (3.77%) had polymicrobial infection . Among the 27 isolates of acinetobacter baumannii, 1 (3.70%) was resistant to all group of antibiotics tested in the study, including carbapenems . Other 25 (92.59%) isolates of acinetobacter baumannii were resistant to cotrimoxazole, ciprofloxacin and amikacin, 24 (88.89%) to imipenem, 23 (85.18%) to ceftazidime, 11 (40.74%) to doxycycline, 10 (37.04%) to piperacillin - tazobactam . All the 27 (100%) isolates of acinetobacter baumannii were multidrug resistant (mdr) i.e. Resistant to three or more class of antibiotics . Pseudomonas aeruginosa was resistant to gentamicin (100%), aztreonam (88.23%), ciprofloxacin and amikacin (82.35%), imipenem (47.06%), ceftazidime (35.29%) and piperacillin - tazobactam (23.53%). Figure 1 shows that late - onset vap was more common than early - onset vap . Among the early onset vap common organisms isolated were acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae, citrobacter freundii and staphylococcus aureus while in case of late onset vap common organisms were acinetobacter baumannii, pseudomonas aeruginosa and methicillin - resistant staphylococcus aureus . Organisms causing vap (early and late onset vap) eight (47.06%) of the 17 pseudomonas aeruginosa isolates were mbl producers . Among 27 isolates of acinetobacter baumannii, 12 (44.44%) were ampc producers while 14 (51.85%) were mbl producing strains . The co - existence of ampc and mbl was reported in 9 (33.33%) isolates of acinetobacter baumannii and 2 (11.76%) isolates of pseudomonas aeruginosa [figure 2]. Different enzymes produced by the isolated strains in the present study it was found that the mortality rate was 45.28%.rate of mortality was less in early - onset vap (23.80%) as compared to late onset vap group (59.37%) and the difference was found to be statistically significant (x1=6.473; df=1; p=0.011). Out of 53 cases of ventilator - associated pneumonia, 16 (30.19%) were female and 37 (69.81%) were male . Aetiological agents widely differ according to the population of the patients in the intensive care unit, duration of hospital stay and prior antimicrobial therapy . The increase of acinetobacter baumannii (49.09%) infections is due to its great resistance to the environment which enables it to spread, its limited virulence and its extraordinary ability to develop resistance to all the antimicrobials and spread by aerosols . One of them was methicillin - resistant staphylococcus aureus (mrsa), but mrsa is generally associated with late onset vap . This may be due the fact that this case showed mixed infection with predominant organism being citrobacter freundii (1.8 10 cfu / ml) while mrsa was showing 2.1 10 cfu / ml in quantitative cultures . Secondly, the patient had history of prior antibiotic therapy which increases risk of mrsa . Found x - ray pattern in acinetobacter baumannii vap is non - specific . In half of the cases it causes lung infiltration and a diffuse bilateral pattern some enzymes of pseudomonas aeruginosa have invasive properties, causing thrombosis of pulmonary vessels and pulmonary infarction; this produces nodular bilateral lesions, predominantly in inferior lobes, and it is such a characteristic image that it should make the presence of pseudomonas aeruginosa suspected . The antibiotic resistance pattern of nonfermenters was almost the same in both early- and late - onset vap . Many of the early - onset vap cases had the risk factors such as prior antibiotic therapy and current hospitalization for five days or more for infection with mdr pathogens . That could be the reason for almost similar susceptibility pattern of the isolates from early- and late - onset vap . Even the american thoracic society guidelines support the same reasoning by suggesting that patients with early - onset vap who have received prior antibiotics or who had the prior hospitalization within the past 90 days are at greater risk for colonization and infection with mdr pathogens and should be treated similarly to patients with late - onset vap . Ampc beta - lactamases hydrolyze cephamycins and are not inhibited by commercially available beta - lactamase inhibitors . Bacteria, mostly klebsiella pneumoniae and e. coli, producing plasmid - mediated ampc beta - lactamases have been responsible for nosocomial outbreaks of infection and colonization . Twelve (44.44%) out of 27 isolates of acinetobacter baumannii have shown production of ampc beta lactamase enzyme, and no other enterobacteriaceae showed production of ampc beta lactamase . Presently, there is concern about the acquisition of plasmid - mediated metallo - beta lactamases active against carbapenems, penicillins and cephalosporins . In this study, 14 (51.85%) isolates of acinetobacter baumannii and 8 (47.06%) isolates of pseudomonas aeruginosa were plasmid - mediated metallo - beta lactamases enzyme producing strains detected by imipenem - edta combined disc method which corresponds to a study were metallo - beta lactamases were produced by 21.74% of acinetobacter spp . And 50% of pseudomonas aeruginosa . Early - onset vap in our study was found to be 39.62% while in various study it was found to be around 40% . The low incidence of early onset vap in our study may be due to antibiotic use before admission to the icu . Studies have shown that previous antibiotic use decreases early - onset vap but markedly increases multidrug - resistant (mdr) pathogens, which is also reflected in our study . In the present study, it was found that the mortality rate was 45.28% which was in accordance with previous studies such as those by gupta a et al . The bacteriological approach for the management of vap avoids the problem of overtreatment by separating colonizers from infecting pathogens . This study showed that quantitative culture of eta is a useful test for early diagnosis of vap . The antibiotic susceptibility pattern of these isolates will also help the clinicians to choose the appropriate antimicrobial agents for prophylactic as well as treatment purposes.
Alpha1-adrenergic receptors (1-ars) are a heterogeneous family of g - protein - coupled receptors that present in most human and animal tissues, and there are considerable variations in the expression levels of 1-ar subtypes in various tissues of different species [14]. 1-ars play important roles in regulating physiological and pathological responses mediated by catecholamines, particularly in the cardiovascular and urinary systems . In addition to mediating catecholamine - induced vasoconstriction, the 1-ars in vascular wall have been shown to promote proliferation and hypertrophy of arterial smooth muscle cells and adventitial fibroblasts [5, 6]. In the urinary system, the demonstration of 1-ar expression in human prostate, bladder muscle, and smooth muscles has led to the treatment of bladder outlet obstruction and ureteral stones, via blocking these receptors [7, 8]. Of the three 1-ar subtypes (1a, 1b, and 1d), the 1d - ar has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [911]. However, recent experiments performed in 1d - ar knockout models suggest that this 1-ar subtype plays an important role in the overall regulation of blood pressure . Additionally, armenia et al . Reported that 1a- and 1d - ars are the major functional subtypes of renal 1-ars in both normal and streptozotocin - induced diabetic sprague - dawley rats . Using in situ hybridization, kurooka et al . Identified the gene expression of all three 1-ar subtypes in human kidney cortex they found that intense 1-ar mrna staining was apparent especially in the smooth muscle of arterial walls, whereas weak staining of each of the 1-ar mrnas was observed in the glomeruli and renal tubules . More recently, the presence and distribution of subtypes in human renal pelvis and calyces were evaluated, and 1d - ar was most dense in both followed by 1a and 1b . However, the expression and distribution patterns of 1d - ar in normal and diabetic rat kidneys remain unknown . In the present study, we examined mrna levels of three 1-ar subtypes in the kidney cortex of normal zucker lean (zl) and zucker diabetic (zd) rats by microarray and real - time pcr analyses . Confocal immunofluorescence microscopy was performed to evaluate the distribution of 1d - ar in the kidney of zucker rats . Furthermore, we investigated whether activation of peroxisome proliferator - activated receptor- (ppar) or ppar, known renoprotective intervention in animal models of type 2 diabetes, would affect renal expression of 1-ar subtypes . Six - week - old male zucker lean (zl) and zucker diabetic fatty (zd) rats were purchased from charles river laboratories (wilmington, ma, usa). Rats were housed in a temperature - controlled room with a 12: 12-hour light - dark cycle and free access to purina 5008 rat chow and water . Blood glucose was monitored using the accu - chek glucometer by tail - vein blood sampling . In a previous study in which we characterized the time course of blood glucose in this model, we showed that blood glucose of the zd rats began to increase at week 8, reached a peak at week 12, and remained at this higher level thereafter . The rats were housed in the animal care facility at the morehouse school of medicine that is aaalac accredited . All animal protocols were approved by the institutional animal care and use committee and were in accordance with the requirements stated in the national institutes of health guide for the care and use laboratory animals . For fenofibrate treatment, 12-week - old zucker rats were divided into 3 experimental groups: vehicle- (0.5% carboxymethylcellulose ig) treated zl, vehicle - treated zd, or fenofibrate- (150 mg / kg / day ig) treated zd (f - zd) rats for 10 weeks . For rosiglitazone treatment, 12-week - old zd animals were treated with rosiglitazone (10 mg / kg / day in drinking water) or vehicles for 8 weeks . Glomeruli were isolated from the kidney cortex of zl and zd rats at the age of 7, 12, and 2022 weeks, respectively, by a modified procedure as described previously . Briefly, the rats were anesthetized and the kidneys were rapidly removed and placed in hanks' balanced salt solution (hbss) at ph 7.4 . The renal cortex was dissected and cut into small pieces with a surgical blade . Glomeruli were isolated by passing the tissues successively through calibrated sieves (pore size: 200, 125, and 65 m) and rinsed with hbss . Isolated glomeruli, collected on the 65 m sieve, were resuspended in hbss . Total rna was prepared from isolated glomeruli or kidney cortex by using ultrapure trizol reagent according to the manufacturer's instructions (gibco - brl, grand island, ny). The quality of the rna samples was assessed using the agilent 2100 bioanalyzer (g2938a). Seven hundred and fifty nanograms of total rna per sample were used for crna synthesis and amplification . Cyanine-3-(cy3-) labeled crna was purified and hybridized to agilent whole rat genome 44k oligo microarray chips (p / n g2519f-14870, agilent technologies) according to the manufacturer's instructions . The processed microarrays were scanned with the agilent g2565ba dna microarray scanner (p / n g2505-a). The scanned images were analyzed with agilent feature software (version 9.5.1.1) using default parameters . The resulting text files were loaded into the agilent genespring gx software (version 7.3) for further analysis . Significantly differentially expressed -adrenergic receptor genes among zl, zd, and f - zd groups were identified by a threshold of 2 fold change and p 0.05 . Reverse transcription was performed on equal amounts of total rna by using random hexanucleotide primers to produce a cdna library for each sample . Real - time pcr reactions were run on an icycler iq real - time pcr detection system by using taqman universal pcr master mix (applied biosystems, p / n 4304437). 1a-, 1b-, and 1d - ar and -actin gene - specific taqman probe and primer sets were obtained from applied biosystems as assays - on - demand (aod) gene expression products . The aod identification numbers were rn00567876 for 1a - ar, rn01471343 for 1b - ar, rn00577931 for 1d - ar, and 4331182 for rat -actin each sample was run in triplicate, and the comparative threshold cycle (ct) method was used to quantify fold increase (2) compared with controls . For immunofluorescent staining, 5 m thick cryostat sections of oct - embedded kidney samples were used . To study the localization of 1d - ar in the rat kidney, the sections were incubated with a mixture of two antibodies overnight: rabbit anti-1d - ar antibody (1: 100, sigma - aldrich, st . Louis, mo), mouse anti--smooth muscle actin (-sma, 1: 100, santa cruz biotechnology, dallas, tx), or goat anti - kidney injury molecule-1 (kim-1, 1: 100, r&d systems, minneapolis, mn). As a negative control, the sections were exposed to nonimmune igg (in replacement of primary antibodies) with the same secondary antibodies, and no specific staining occurred . Comparisons among multiple groups were performed by one - way anova and newman - keuls post hoc test . We performed microarray analysis to assess gene expression levels of -adrenergic receptor subtypes in the kidney cortex of 22-week - old normal zl (blood glucose: 108 8 mg / dl) and diabetic zd (blood glucose: 425 35 mg / dl) rats . The expression profile of each experimental group was determined in three animals per group . Table 1 shows gene expression levels of 1- and 2-adrenergic receptor subtypes detected in rat kidney tissue . The rank order of expression levels of the three 1-ar mrnas in normal rat kidney cortex was 1b> 1d> 1a . Among these genes compared to normal zl controls, renal cortical 1d - ar gene was increased by 553% in 22-week - old zd rats (table 1). To verify the relative transcript levels of 1a-, 1b-, and 1d - ar subtypes derived from the microarray experiment, we performed quantitative real - time pcr (qpcr) assay . The relative expression levels of 1a-, 1b-, and 1d - ar mrnas normalized against -actin in rat kidney cortex are shown in figure 1 . In consistency with the microarray results, qpcr analysis showed that renal cortical 1d mrna was increased by 16-fold in 22-week - old zd rats compared to zl controls . We have previously shown that ppar activation protects against kidney injury in zucker diabetic fatty rats [15, 17]. Here, we further examined the effect of ppar activation on the expression level of 1-ar mrnas in the diabetic kidneys . Gene microarray analysis revealed that ppar activation inhibits the upregulation of 1d gene in the diabetic kidneys . Chronic administration of fenofibrate, a ppar agonist, resulted in a decrease in 1d - ar mrna by 64% compared to vehicle - treated zd animals (table 1). Qpcr analysis confirmed a reduction of 1d - ar mrna in the kidney of f - zd rats (figure 1). In contrast, both microarray and qpcr analyses indicated that mrna expression of 1a- and 1b - ar subtypes was not affected by fenofibrate in the diabetic kidneys . Additionally, the effect of ppar activation on gene expression of 1-ar subtypes was examined in the zd rats after rosiglitazone treatment . Compared to vehicle - treated zd rats, renal cortical 1d - ar mrna was significantly lower when rosiglitazone was administered for 8 weeks (figure 2). Similar to ppar activation, 1a- and 1b - ar mrna expression in the diabetic rats was not affected by rosiglitazone treatment . In a previous study, we showed that blood glucose levels were not different between normal zl and diabetic zd rats at the age of 7 weeks . Blood glucose of the zd rats began to increase at week 8, reached a peak at week 12, and remained at this higher level thereafter . To analyze the temporal pattern of renal expression of 1d - ar receptors, renal cortical and glomerular 1d - ar mrna levels were compared in the zucker rats at the ages of 7, 12, and 2022 weeks . As shown in figure 3, zd rats at week 7 had slightly lower 1d - ar mrna level in the glomeruli compared to their zl littermates, whereas renal cortical 1d - ar mrnas were not different between the two groups . At the age of 12, both renal glomerular (2.4-fold) and cortical (1.7-fold) 1d - ar mrna levels were significantly higher in the zd animals, which were further increased by 3.4-fold and 12.9-fold, respectively, at the age of 2022 weeks . We performed immunofluorescence staining to correlate 1d - ar gene expression results with its protein level and distribution in the kidney of zucker animals . As expected, 1d - ar protein was clearly detected in the renal arteries and arterioles in both normal and diabetic animals (figure 4). The intense staining was primarily in the smooth muscle of renal arterial walls as evidenced by its colocalization with -sma . In normal zl rats, weak 1d - ar staining was also detected in the glomeruli, whereas there was no obvious staining in the renal tubules (figure 4). In consistency with the mrna expression results, immunofluorescence staining identified increased 1d - ar signal in the glomeruli of diabetic rats, which was partially colocalized with -sma (figure 4). Intense tubulointerstitial staining of 1d - ar was apparent in the diabetic kidneys . As shown in figure 5, 1d - ar was expressed in both tubular epithelial cells and activated interstitial fibroblasts, which was positive for -sma staining . To further characterize 1d - ar expression in renal tubules of diabetic animals, we performed double staining for 1d - ar and kim-1, a sensitive tubular injury marker . Dual labeling revealed a spatial relationship between kim-1 and 1d - ar in the diabetic kidneys . As shown in figure 6, virtually all dilated tubules expressing 1d - ar were also kim-1-positive, suggesting that 1d - ar was expressed in the injured dedifferentiated proximal tubules . In these tubules, 1d - ar expression was predominantly cytoplasmic, whereas kim-1 staining was prominent at the apical membrane . In this study, the expression and distribution of 1d - ar mrna and protein were determined by the gene microarray, qpcr, and confocal immunofluorescence analyses . Although mrna expression of all three 1-ar subtypes (1b> 1d> 1a) was detected in rat kidney cortex, only 1d - ar gene was massively upregulated in the diabetic animals . Moreover, diabetes - related increase in 1d - ar mrna was inhibited when the zd rats were treated with fenofibrate or rosiglitazone . Immunostaining for 1d - ar confirmed that intense 1d - ar staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys . Weak 1d - ar protein staining was detected in the glomeruli of normal zl controls, but there was no obvious staining in the normal tubular epithelium . In consistency with the gene expression results, 1d - ar protein was significantly increased in the glomeruli and proximal tubules of diabetic animals . The expression of 1-ar subtype mrnas has previously been studied in various animal and human organs, and the predominant subtype mrna expressed differs among species and organs . For example, kurooka et al . Reported that 1a - ar gene was detected more than 1b - ar or 1d - ar in human kidney cortex . In contrast, karabacak et al . Recently evaluated 1-ar subtype protein expression in human renal pelvis and calyx tissues and found that 1d - ar was most dense in both followed by 1a- and 1b - ar subtypes, respectively, where the rate of 1b - ar was significantly lower than the other two . In the rat kidney, it was reported that the 1b - ar is predominant when detected by a radioligand binding assay and rnase protection assay . In consistency with these findings, we confirmed the expression of all three 1-ar subtype mrnas (1b> 1d> 1a) in rat kidney cortex by microarray and qpcr analyses . Of the three 1-ar subtypes, the 1d - ar has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [911]. However, recent experiments performed in 1d - ar knockout models suggest that this 1-ar subtype plays an important role in the overall regulation of blood pressure . Moreover, armenia et al . Reported that 1a- and 1d - ars are the major functional subtypes of renal 1-ars in both normal and streptozotocin - induced sprague - dawley rats . Interestingly, among the three 1-ar subtypes detected in rat kidney cortex, we found that 1d - ar mrna was markedly increased by 16-fold in the diabetic kidneys . Additionally, diabetes - associated upregulation of 1d - ar mrna expression was inhibited when the diabetic animals were treated with ppar agonists, known renoprotective interventions . Therefore, previous evidences and present results strongly suggest that 1d - ar may play an important role in renal physiology and/or pathophysiology . Diabetic nephropathy is characterized by a series of ultrastructural changes, including glomerular and tubular hypertrophy, mesangial expansion, glomerulosclerosis, and tubulointerstitial fibrosis . We have previously demonstrated a progressive loss of renal function in the diabetic animals as evidenced by an increase in urinary protein to creatinine ratio in the zd rats between the ages of 7 and 20 weeks . In the current study, we further observed a gradual increase in cortical and glomerular 1d - ar mrna expression during disease progression in the diabetic animals . We speculate that increased 1d - ar may play a role in the development of diabetic renal hypertrophy and fibrosis . In fact, a role for 1d - ar in vascular hypertrophy and remodeling has been suggested by a recent report that a decrease in the lumen area and an increase in the wall thickness of arteries in hypoxic pulmonary hypertension were strongly inhibited in 1d - ar knockout mice . In this study, we evaluated the expression and distribution of 1d - ar protein in the kidneys of zl and zd rats by immunofluorescence staining . As expected, intense 1d - ar staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys . Compared to low expression level of 1d - ar protein in the glomeruli of normal zl controls, zd rats demonstrate a significant increase in 1d - ar signal . Moreover, zd kidneys displayed strong tubulointerstitial 1d - ar signal within fibrotic areas . A colocalization of 1d - ar with -sma indicates that 1d - ar expressing interstitial cells are myofibroblasts . Future work in the field could be necessary to establish the exact role of 1d - ar in activation and proliferation of renal interstitial fibroblasts . Another interesting novelfinding in our set of data reported here is the 1d - ar expression in tubular epithelial cells of diabetic kidneys . Although there was no obvious staining in the normal tubules, 1d - ar staining was apparent in the dilated tubules in the fibrotic areas . To further characterize 1d - ar expression in renal tubules of diabetic animals, the spatial relationship between tubular 1d - ar and kim-1, a tubular injury marker, the selective increased expression by dedifferentiated epithelial cells and activated interstitial fibroblasts supports the potential importance of 1d - ar in renal tubulointerstitial injury . Moreover, recent studies on the pulmonary circulation suggest that catecholamines may participate in the excessive muscularization and fibrosis of the arteries through erk1/2 signaling pathway in hypoxic pulmonary hypertension [5, 2022]. Therefore, further studies are warranted to evaluate the functional consequences of 1d - ar induction in diabetic kidney injury . In summary, the current study highlights mrna expression of the three 1-ar subtypes in rat kidney cortex . An increase in renal expression of 1d - ar mrna and protein was associated with glomerular and tubulointerstitial injury in diabetic nephropathy . Chronic treatment with ppar agonists prevents the increase in 1d - ar mrna in the diabetic kidneys . These findings may provide new insights into the prevention and early management of diabetic kidney disease.
While the occurrence of dental caries has decreased during the last decades, researchers had focused on non - carious lesions, including erosion . Dental erosion is an acid - induced tooth loss not involving microorganisms caused by external and/or internal acids . Recent studies indicate a meaningful increase in dental erosion prevalence, especially in dentin, due to modifications in diet, lifestyle and socioeconomic status . Early signs of erosive tooth loss have been found in children and young people . Considering that the modification in population habits and the decrease of acid exposure are very tough, alternatives to reduce the progression of tooth erosive loss have been investigated . Naf is one of the most tested fluoride salt, whose mechanism of action against erosion is based on the deposition of caf2-like layer on the surface promoting an additional barrier that inhibits the contact of the acid with the tooth . However, the anti - erosive effect of naf on dentin is limited since its effect is only seen when the demineralized organic matrix (dom) is preserved . Nevertheless, some loss of the dom by enzymatic activity is expected in the clinical situation, especially in patients with eating disorders . Therefore, the use of other fluoride salts is likely to supply the lack of action of conventional fluoride (naf) on dentin, and consequently, it could be more effective against erosion . Accordingly, titanium tetrafluoride (tif4) has been widely studied against tooth erosive demineralization since 1997, demonstrating the enamel erosion - inhibiting effect . On the other hand, generally, tif4 has a similar effect as naf on the prevention of dentin erosion when it is applied as varnish, or a better effect than naf when applied as high f concentrated solution . Therefore, the protective effect of tif4 on dentin erosion is still in debate . Considering that the application of professional fluoride, such as varnish, is not often done, patients at high risk of erosion would benefit from other alternatives to increase the frequency of fluoride exposure . Accordingly, the daily application of mouthrinses with low concentration of f, as those containing sncl2/amf / naf marketed in europe (erosion protection), has shown some protective effect against enamel and dentin erosion in vitro and in situ . Therefore, the use of low concentrated tif4 mouthrinse by the patient could be a good alternative; however, there is a clinical limitation due to the low ph of the solution, which might cause some side effects in oral cavity, since it has cytotoxic effect on fibroblasts . This study hypothesized that the formulation of an experimental solution containing both naf and tif4 would increase the ph, allowing its use in the clinical situation without losing its protective effect against dentin erosion compared to tif4 alone and the commercial solution erosion protection . The null hypothesis is that there is no significant difference in the protective effect against dentin erosive loss among the tested fluoride mouthrinses . Ninety dentin samples, which were stored in 0.1% thymol/0.9% nacl solution during the preparation phase, were cut from bovine dental roots . The root was separated from the crown using a water - cooled diamond saw and a cutting machine (isomet low speed saw, buehler, lake bluff, il, usa), and embedded in pre-30 self - polymerized acrylic resin in cylindrical shape to facilitate the handling . Thereafter, they were serially flattened with water - cooled abrasive discs (320, 600, and 1200 grades of al2o3 papers; buehler, lake bluff, il, usa), and finally polished with felt paper wetted with a diamond solution (1 m thickness of particles; buehler, lake bluff, il, usa) on a rotating polishing machine (arotec sa ind . After polishing, the samples were cleaned in an ultrasonic device with deionized water for 2 min . The reference areas on the polished dentin surface were marked with two parallel lines made with a scalpel blade, 1.0 mm apart . Small drilling was also done on the outer area of the dentin surface to allow the correct position of the sample in the profilometric system . Prior to the experiment, the baseline profile was measured and two layers of nail varnish (colorama, com ., so paulo, sp, brazil) were applied on 2/3 of the control surface (sound surfaces), leaving only 1/3 central of the exposed dentin (1.0 mm x 5 mm). Dentin samples were randomly allocated to each of the six treatment groups (n=15): 1) commercial sncl2/naf / amf solution (800 ppm sn, 500 ppm f, ph 4.5, erosion protection, gaba int . Ag, basel, switzerland, positive control); 2) experimental 0.0815% tif4 solution (315 ppm ti, 500 ppm f, ph 2.5); 3) experimental 0.105% naf solution (500 ppm f, ph 4.5 adjusted with phosphoric acid); 4) experimental 0.042% naf+0.049% tif4 solution (naf- 190 ppm f, tif4 190 ppm ti and 300 ppm f, ph 4.4); 5) experimental 0.063% naf+0.036% tif4 solution (naf 285 ppm f, tif4 140 ppm ti and 220 ppm f; ph 4.5); 6) no treatment (untreated, negative control). All solutions had approximately 500 ppm f based on the calculation obtained from the salts concentrations diluted in deionised water, and their ph was measured using a ph electrode . The experimental fluoride solutions were prepared using the analytical grade reagents from sigma - aldrich (st . The fluoride treatments were performed twice a day (immediately after the first and the last erosive challenges of the day; v=0.5 ml / sample) for 1 min, during 7 days of erosive challenges . Erosive challenges took place by immersion in a freshly opened bottle of soft drink (sprite zero, coca - cola company spal, porto real, rj, brazil, ph 2.6, 30 ml / sample) four times a day for 90 s each, at 25c . Then, the samples were rinsed with deionized water (5 s) and exposed to artificial saliva (ph 6.8, 30 ml / samples, 25c) for 2h between the erosive challenges and overnight . The artificial saliva (v=500 ml) consisted of 0.001 g ascorbic acid, 0.015 g glucose, 0.290 g nacl, 0.085 g cacl2, 0.080 g nh4cl, 0.635 g kcl, 0.080 g nascn, 0.165 g kh2po4, 0.100 g carbamide and 0.170 g na2po4, and it was daily renewed . Dentin erosive loss (m) was quantitatively determined by a contact profilometer (mahr perthometer, gttingen, lower saxony, germany) before (baseline) and after 7 days of experiment . For the profilometric measurement, the nail varnish was carefully removed using a scalpel and acetone solution (1:1 water). Five profile measurements were performed at exactly the same sites as the baseline measurement, at intervals of 0.5 mm . To achieve this outcome, the dentin samples presented the identification marks (small drillings made with drill 1/4) and were inserted into a metal device, allowing the stylus to be accurately repositioned at each measurement . Baseline and final profiles were done and compared using the software mahrsurf cxr20 (mahr, gttingen, lower saxony, germany). The scans were superposed and the average depth of the under curve area was calculated (m). For a better understanding of the treatments effect, the prevention fraction (%) of each treatment the software graphpad instat version 2.0 for windows (graphpad software, la jolla, ca, usa) was used for the statistical analysis . The assumptions of equality of variances and normal distribution of data were checked using the bartlett and kolmogorov - smirnov tests, respectively . Once the homogeneity was not achieved, the data from dentin loss (m) were analyzed using kruskal - wallis followed by dunn s test . Ninety dentin samples, which were stored in 0.1% thymol/0.9% nacl solution during the preparation phase, were cut from bovine dental roots . The root was separated from the crown using a water - cooled diamond saw and a cutting machine (isomet low speed saw, buehler, lake bluff, il, usa), and embedded in pre-30 self - polymerized acrylic resin in cylindrical shape to facilitate the handling . Thereafter, they were serially flattened with water - cooled abrasive discs (320, 600, and 1200 grades of al2o3 papers; buehler, lake bluff, il, usa), and finally polished with felt paper wetted with a diamond solution (1 m thickness of particles; buehler, lake bluff, il, usa) on a rotating polishing machine (arotec sa ind . After polishing, the samples were cleaned in an ultrasonic device with deionized water for 2 min . The reference areas on the polished dentin surface were marked with two parallel lines made with a scalpel blade, 1.0 mm apart . Small drilling was also done on the outer area of the dentin surface to allow the correct position of the sample in the profilometric system . Prior to the experiment, the baseline profile was measured and two layers of nail varnish (colorama, com ., so paulo, sp, brazil) were applied on 2/3 of the control surface (sound surfaces), leaving only 1/3 central of the exposed dentin (1.0 mm x 5 mm). Dentin samples were randomly allocated to each of the six treatment groups (n=15): 1) commercial sncl2/naf / amf solution (800 ppm sn, 500 ppm f, ph 4.5, erosion protection, gaba int . Ag, basel, switzerland, positive control); 2) experimental 0.0815% tif4 solution (315 ppm ti, 500 ppm f, ph 2.5); 3) experimental 0.105% naf solution (500 ppm f, ph 4.5 adjusted with phosphoric acid); 4) experimental 0.042% naf+0.049% tif4 solution (naf- 190 ppm f, tif4 190 ppm ti and 300 ppm f, ph 4.4); 5) experimental 0.063% naf+0.036% tif4 solution (naf 285 ppm f, tif4 140 ppm ti and 220 ppm f; ph 4.5); 6) no treatment (untreated, negative control). All solutions had approximately 500 ppm f based on the calculation obtained from the salts concentrations diluted in deionised water, and their ph was measured using a ph electrode . The experimental fluoride solutions were prepared using the analytical grade reagents from sigma - aldrich (st . The fluoride treatments were performed twice a day (immediately after the first and the last erosive challenges of the day; v=0.5 ml / sample) for 1 min, during 7 days of erosive challenges . Erosive challenges took place by immersion in a freshly opened bottle of soft drink (sprite zero, coca - cola company spal, porto real, rj, brazil, ph 2.6, 30 ml / sample) four times a day for 90 s each, at 25c . Then, the samples were rinsed with deionized water (5 s) and exposed to artificial saliva (ph 6.8, 30 ml / samples, 25c) for 2h between the erosive challenges and overnight . The artificial saliva (v=500 ml) consisted of 0.001 g ascorbic acid, 0.015 g glucose, 0.290 g nacl, 0.085 g cacl2, 0.080 g nh4cl, 0.635 g kcl, 0.080 g nascn, 0.165 g kh2po4, 0.100 g carbamide and 0.170 g na2po4, and it was daily renewed . Dentin erosive loss (m) was quantitatively determined by a contact profilometer (mahr perthometer, gttingen, lower saxony, germany) before (baseline) and after 7 days of experiment . For the profilometric measurement, the nail varnish was carefully removed using a scalpel and acetone solution (1:1 water). Five profile measurements were performed at exactly the same sites as the baseline measurement, at intervals of 0.5 mm . To achieve this outcome, the dentin samples presented the identification marks (small drillings made with drill 1/4) and were inserted into a metal device, allowing the stylus to be accurately repositioned at each measurement . Baseline and final profiles were done and compared using the software mahrsurf cxr20 (mahr, gttingen, lower saxony, germany). The scans were superposed and the average depth of the under curve area was calculated (m). For a better understanding of the treatments effect, the prevention fraction (%) of each treatment the software graphpad instat version 2.0 for windows (graphpad software, la jolla, ca, usa) was used for the statistical analysis . The assumptions of equality of variances and normal distribution of data were checked using the bartlett and kolmogorov - smirnov tests, respectively . Once the homogeneity was not achieved, the data from dentin loss (m) were analyzed using kruskal - wallis followed by dunn s test . All experimental mouthrinses promoted significantly lower dentin erosive loss when compared to the negative control (p<0.0001), except naf solution (prevention fraction of 13.3%; p>0.05). The best anti - erosive effect was found for experimental solutions containing 0.0815% tif4 (prevention fraction of 100%) and 0.042% naf+0.049% tif4 (58.3%). Sncl2/naf / amf (erosion protection, 52%) and 0.063% naf+0.036% tif4 (40%) did not significantly differ from 0.042% naf+0.049% tif4 and naf alone, but both were less effective than tif4 alone . The median values (minimum - maximum) of dentin erosive loss for each group table 1median (minimum - maximum) of the dentin erosive loss for different groupssolutionsmedian (min; max)erosion protection(positive control)0.86 (0.67; 1.85) tif4 (0.0815%)-0.19 (-0.45; -0.05) naf (0.105%)1.56 (1.01; 2.38) naf+tif4 (0.042%+0.049%)0.75 (0.21; 1.59) naf+tif4 (0.063%+0.036%)1.08 (0.59; 1.66) negative control1.80 (1.23; 4.94) * negative value means increase of the surface (deposition)different letters show significant differences among the groups; min = minimum; max = maximum (p<0.0001) * negative value means increase of the surface (deposition) different letters show significant differences among the groups; min = minimum; max = maximum (p<0.0001) considering the increase of dental erosion s prevalence, the attention has been focused on the development of preventive approaches to reduce the progression of this dental condition . The present study investigated the protective effect of the daily application of solution containing tif4/naf . The null hypothesis tested in this study was rejected because the tested fluoride mouthrinses had a significantly different effect among them against dentin erosive loss . The solution containing pure tif4 showed the best protective effect, differing from all other groups except from a specific combination of tif4 and naf . This new approach would benefit patients with high risk of erosion presenting gingival recession due to periodontal disease, brushing habits (abrasion) or / and occlusal disorders (abfraction). The dentin, in these cases, may be likely exposed to extrinsic acid sources from the diet, and therefore, susceptible to the development of erosion . The present study aimed to simulate the home - care application of low - concentrated fluoride (500 ppm f) solution, after two meals (morning and evening), in periods in which the patient could perform a rinse after the daily hygiene habit . The idea behind the combination of two fluorides, tif4 and naf, into an experimental mouthrinse is based on the fact that pure tif4 has low ph, impairing its clinical use . The addition of naf to tif4 solution was able to increase its ph to a suitable value to be applied in vivo and to be compared with commercial products . In this study, tif4 alone reduced in 100% dentin loss, which might be due to the deposition of acid - resistant surface layer rich in caf2, titanium dioxide and hydrated titanium phosphate (unpublished data). The protocol of tif4 application tested in this study has not been applied in previous studies, since most of them tested high concentrated tif4 solution applied at once . The addition of naf into tif4 solution decreased the protective effect, considering the percentage of prevention fraction, due to a likely lower precipitation of ti and f salts . However, one of the combinations (0.042% naf+0.049% tif4) was still statistically similar to pure tif4 solution . The present results in respect to the daily application of fluoride mouthrinses are more promising than those found for a unique application of a product with high concentration of fluoride, as varnish, against dentin erosion and erosion - abrasion . The findings suggest the importance of a frequent low concentrated fluoride exposure rather than a unique application of a high concentrated fluoride product . A recent study was conducted in enamel showing similar results however, only one of the combinations (0.042% naf+0.049% tif4) was effective in reducing enamel erosive loss (41% preventive fraction), while the other one did not differ from the negative control . Generally, the tested fluoride mouthrinses had better impact on dentin compared to enamel, which might be explained by the differences in the composition between the dental tissues . In case of dentin, the effect of the combinations of tif4 and naf was similar to those provided by a commercial fluoride solution (positive control), which has been widely used in europe for prevention of tooth erosion . The preventive fraction found by the application of erosion protection in the present study was similar to a previous in situ study performed by other research group . Based on this finding, we can speculate that the acid - resistance of ti and f precipitates found for our experimental solutions are similar to tin and fluoride precipitates produced by the application of erosion protection on dentin . On the other hand, naf solution presented the worst performance, not differing from the negative control . It is widely known that naf is ineffective to protect against tooth erosion especially in case of dentin, in which its effect depends on the presence of the dom . In this study, the dom was not removed, but it would be interesting to test the effect of the experimental fluoride solutions on dentin without dom . Further studies should also test the effect of the experimental fluoride solutions on both dentin erosion and brushing abrasion to check the stability of the protective effect faced by two different challenges (chemical and mechanical). Another point to consider is that erosion in dentin is very complex due to the role of dom in the progression of erosive loss . Therefore, erosive loss is difficult to be quantified, since the quality of the remaining organic layer may interfere with the profilometric measurement . Therefore, to generate reliable data, the profiles must be measured with the samples immersed 100% in water or without dom . We have decided to perform the analysis with dom under 100% humidity, since in previous study we have not found differences in the comparison between tif4 and naf varnishes in the profile analysis of dentin with or without dom . Future studies, including in situ and in vivo models, must be performed to confirm the findings, since saliva can be able to buffer the ph of the fluoride solutions, which might lead to different results . Under the conditions of the present study, we can conclude that the daily application of an experimental mouthrinse containing a specific combination of tif4 and naf has the ability to reduce dentin erosion in vitro, and may be a good alternative for high - risk populations.
Allergic diseases are among the most common chronic diseases throughout the world and the prevalence of atopic diseases in childhood has significantly increased during the past several decades [2, 3]. Although there is a general consensus on the importance of a genetic predisposition for atopic diseases, only changes in environmental factors can explain this increase [46]. There is a strong association between sensitisation and symptoms of allergic diseases although this association is not absolute . The allergy march refers to the natural history of sensitisation to allergens and symptoms of eczema, asthma, and rhinitis, which is characterized by a typical sequence of sensitisation and manifestations of symptoms that appear during a certain age period [7, 8]. Food allergy and atopic eczema during the first years of life have been considered risk factors for subsequent asthma and rhinitis caused by indoor and outdoor inhalant allergens [5, 9, 10]. However, allergy - like symptoms in childhood like wheezing and eczema need not have an atopic background [11, 12]. Wheezing episodes in young children are often transient and associated with viral respiratory infections [13, 14]. Thus, better predictors of disease development and diagnostic markers for allergic disease are needed to give proper treatment and valuable advice, especially concerning environmental control to parents of young children . The present study was carried out in order to evaluate the diagnostic performance of phadiatop infant, an in vitro test designed to detect allergen - specific ige antibodies known to be relevant in the development of ige - sensitisation in early childhood . The study was conducted retrospectively in consecutively included children, below the age of five years and admitted to bkl voksentoppen, rikshospitalet, oslo, during a period of twelve months . The children were referred from paediatricians and other paediatric departments at hospitals in norway and thus selected for more severe allergic symptoms . Frozen - serum samples from 122 children were analysed and clinical data documented in the patients' records were compiled for the study . The study conformed to the principles of helsinki's declaration and was approved by the local ethics committee . Skin prick test (spt) was performed according to standard procedure at the hospital . Glycerinated allergen extract (soluprick alk - abello, denmark and a standard lancet with 1 mm tip and a shoulder (alk - abello, denmark) was used . Histamine chloride 10 mg / ml was used as positive - control and as negative - control glycerol . The reaction was recorded as 3 + when the reaction was equal to the histamine control, 2 + when half the histamine control, and 4 + when double the histamine control . The panel included egg white, cow's milk, cod fish, peanut, hazel nut, wheat, house dust mite, cat dander, birch, timothy, cladosporium, and latex . Total and allergen - specific ige antibodies were determined in immunocap 100 (phadia ab, sweden) at the hospital laboratory when appropriate according to clinical findings . Serum samples were sent to phadia ab, uppsala, sweden, for determination of specific ige antibodies using immunocap phadiatop, fx5 (a food mixture of milk, egg white, fish, peanut, wheat, and soy bean), and immunocap phadiatop infant using immunocap100 (phadia ab, uppsala, sweden). Phadiatop infant is designed to detect allergen - specific ige antibodies to food and inhalant allergens, relevant in the development of ige - mediated disease in young children . The following allergens are included in the test: hen's egg, cow's milk, peanut, shrimp, cat epithelium and dander, dog dander, house dust mite, common silver birch, timothy, ragweed, and wall pellitory . The laboratory analyses were performed in a blinded manner and the results of phadiatop and fx5 were sent back to the investigating physician . Atopy was defined as a constitutional disposition to produce ige antibodies to common allergens whether the patients had clinical symptoms or not, as judged by the investigator . The study was designed to make the diagnosis with best possible available tools (case history, spt, and allergen - specific ige results) to evaluate the clinical performance of phadiatop infant . A preliminary diagnosis whether a child was atopic or not was assessed by the investigating physician in retrospect, taking into account clinical history and diagnostic procedures, which includes available spt and allergen - specific ige results, noted in the patient records . In the final diagnosis of the atopic state, the laboratory results from phadiatop and fx5 were also taken into consideration in order to get comparable data of allergen - specific ige sensitisation on all children . This final diagnosis was used as the reference for calculation of the diagnostic performance of phadiatop infant . Quantitative variables are described in appropriate measures of localisation and dispersion, quantitatively variables presented by counts and percentages . The diagnostic performance of phadiatop infant was evaluated by calculating sensitivity, specificity, negative and positive predictive values (npv, ppv), respectively, with 95% confidence intervals . The subgroups divided by age, <2 years and 2 years were compared with regard to the diagnostic performance of phadiatop infant using descriptive statistics . The demographic characteristics of the children classified according to the final diagnosis of atopy / nonatopy or inconclusive diagnosis are shown in table 1 . Thirty - eight (31%) subjects in the study population were girls and 84 (69%) were boys with a median age of 2.7 years . Of the 122 children, 86 (70%) were atopic, 26 girls and 60 boys, which is a commonly found gender distribution among atopic children at that age . Only 18% of the children presented with wheezing as a single symptom and the majority of these children were nonatopic, 55% below 2 years and 40% above this age, respectively . Eczema as a single symptom was the most prevalent symptom among the children below two years of age (49%) and 63% were classified as atopic . In the older age group of children, eczema and/or wheezing in combination with other allergic symptoms dominated (41%) and other allergy - like symptoms such as rhinitis, rhinoconjunctivitis, anaphylaxis, and gastrointestinal symptoms were registered in 31 (26%) children, out of whom 22 (71%) were older than 2 years (data not shown). Eighty - three of the children (68%) reported at least one first degree relative, with about the same proportion for the atopic as for the nonatopic children, 71% and 61%, respectively (data not shown). The diagnostic performance characteristics of phadiatop infant in this study population with a prevalence of 70% are presented in table 2 . The sensitivity calculated for the whole group of children was 98% (95% ci: 92100%) and the specificity 89% (95% ci: 7497%). The ppv and npv values were 95% (95% ci: 8999%) and 94% (95% ci: 8099%), respectively . The diagnostic performance of the test was found to be similar when the children were separated in the two age groups, below or above two years, but due to small numbers of children in the separated age groups, the calculated values are not presented . Symptoms of allergic disease in young children are generally unspecific and the diagnosis without objective tests could be an arbitrary process . The paediatric section of the european academy of allergy and clinical immunology has recently published a position paper with recommendations on allergy testing in children to improve the identification of allergy and quality of care . An earlier published study has shown that 76 out of 147 children could not be classified as having an ige - mediated disease or not, based on case history and physical examination alone . Similar results were found in a recently published study, where measurements of ige - antibodies, added to case history and physical examinations, highly improved the discrimination between ige- and non - ige - mediated diseases in young children . The results from our study confirm these findings and suggest that phadiatop infant could be a useful tool for discrimination between atopy / non - atopy . A positive phadiatop infant test should however be followed by allergen - specific antibody testing to a selected panel to identify the offending allergen(s) [19, 20]. The test seems to be at least as useful among the youngest children, below two years, as among children at 24 years of age . The youngest child in the study was 6 months, which confirms findings from other publications that allergen - specific ige - antibodies can be detected early in life [17, 18, 21]. These findings support the value of testing children with allergy - like symptoms at an early age . Family history has been considered an important risk factor for atopy and has been widely used for prediction of allergy and allergic diseases . The results from our study are in good agreement with the notion that allergy / atopy is extremely commonly found . On the other hand, the fact that 30% of the atopic children did not have a family history of allergy agrees with investigators claiming that a great portion of newly diagnosed allergics / atopics do not have a family history of allergy / atopy . Thus it could be stated that family history is no longer practical in predicting allergic disease . This study population represents a selected cohort as voksentoppen is a hospital having referred patients with allergy - like symptoms and allergic diseases from general paediatricians in the whole country, often of a more severe character . However, other studies have shown that phadiatop infant could be applied in populations with a lower prevalence of atopy, still demonstrating good performance characteristics [17, 18, 21]. The clinical appearance of allergy in our study is in agreement with the concept of the allergy march and supports what has been reported earlier from other studies [7, 8]. Thus, eczema was not common in the nonatopic group of children and only one of the 31 children with eczema was classified as nonatopic . The progression from eczema to other allergic problems eighty - six percent of the children with eczema and wheezing in combination with other symptoms were found in the older age group and as many as 82% were classified as atopic . The majority of children, all ages, presenting with wheezing were nonatopic (73%). Many infants and children who wheeze have transient conditions associated with diminished airway function and do not have an increased risk of asthma later in life . However, children with persistent wheezing, starting during the first years of life, and with an atopic heredity, should be considered being at risk for asthma later during childhood [11, 22, 23]. Therefore, an early diagnosis of ige sensitisation may be important for the choice of treatment to wheezing toddlers . Children with allergic symptoms usually present at a general paediatrician who needs to discriminate which patients have to be sent to an allergist for further evaluation . Rule - out tests with a high discriminating potential are suggested to have a gateway function to fulfil this differentiation and an important role to prevent the march of allergic children from the first to secondary level of care . The present study shows that phadiatop infant has a diagnostic performance with a high sensitivity and specificity . Thus, the test could be recommended as a complement to the clinical information in the differential diagnosis on ige - mediated disease in young children with allergy - like symptoms . Furthermore, the test could be useful in assisting primary care physicians in selecting atopic children at an early stage for further intervention or referral to an allergist . An early correct diagnosis will thus allow for better management and a possibility to delay or even prevent the onset of asthma in children with eczema and the avoidance of further deterioration of lung function in children with asthma [16, 25].
Mainly for safety reasons, many patients and their families are looking for a seizure detection system that is efficient, comfortable, and easy to use . This implies that such a system should have a high detection sensitivity (detect as many seizures as possible) and low false alarm rate; it should be unobtrusive, wireless, and in case of daytime use practically unnoticeable; and it should allow long - term use in a home situation without the presence of professionals . Alarming for safety reasons or reassurance is already used in fall detectors for the elderly, babyphones (reacting to sound or movement), and glucose monitors for persons with diabetes, . In addition to being used for alarming, a detection system could allow offline storage or online streaming of selected data, allowing follow - up on treatment efficacy or summoning emergency support, respectively . Our team is interested in the long - term observation of patients with epilepsy and detection of their seizures, . The focus lies on those convulsive seizures that can be dangerous because of their intensity (possible injuries), duration, or serious consequences such as brain damage, autonomic dysregulation, or suffocation . Clonic and clonic seizures with patients remaining in one place, their bed, mainly because of the need for supervision at night and because seizures often occur during sleep . As sensors need to be as unobtrusive as possible but still effective, small wireless accelerometers (measuring the acceleration of movement) are used and attached to the wrists and ankles of the patient . The goal of this study was to test the efficacy of the varia (video, accelerometry, and radar - induced activity recording) system and algorithms for tonic clonic and clonic seizures compared with that of semicontinuous monitoring by professional caregivers (keeping an eye on multiple patients), as well as the independence, robustness, comfort, and user - friendliness of the system . We therefore measure long term in a home environment without uncomfortable and stigmatizing electroencephalography (eeg) electrodes rather than in a video / eeg monitoring unit, and the system therefore needs to be able to store the data and to allow visual verification of detected events . Two patients were measured for one month in their rooms in an epilepsy center in flanders, belgium . As they returned home for the weekends and as the caregivers needed to get acquainted with the recording system, they were measured for a mean of 12 nights per patient . Patient 1 is an 8-year - old girl with epilepsy e.c.i . And different types of seizures that typically last no more than 3 min: tonic clonic seizures with or without faltering respiration, tonic seizures, and clonic seizures . She also exhibits myoclonic jerks manifesting as (head) nods or facial contractions with eye deviation . Seizures are noticed based on semicontinuous video monitoring and when a scream or breathing problem is heard . Patient 2 is a 9-year - old boy with lesional epilepsy due to herpes encephalitis, who is kept in bed with a restraining belt around the waist . Clonic seizures of various duration, tonic, clonic, and myoclonic seizures and (series of) spasms . The caregivers use a babyphone and semicontinuous video monitoring to keep an eye on him . Approval by the medical ethical commission of the antwerp university hospital, belgium and signed informed consent forms from all parents were obtained prior to inclusion in the study, which was performed in accordance with the 1964 declaration of helsinki . The system uses video, accelerometry (acm), and radar - induced activity recording and is therefore named varia . It consists of an axis m1011 camera (axis communications ab, sweden) and a hygrosens rad - mod motion sensor based on the radar principle (b + b thermo - technik gmbh, germany). The camera and radar are attached to a tripod that is placed close to the patient's bed . The radar is added for detection of movements in the direction of the sensor and for measurement through sheets . Radar technology is also used for detection of falling, wandering, sitting, or getting up, so we expect it to detect large and slow epileptic and nonepileptic movements, also those caused by other persons in the room . Four shimmer sensors with integrated three - axis acm (shimmer, ireland), streaming wireless (bluetooth) communication standards, are adjusted to allow recording of more than 10 h before batteries need recharging and are put in elastic bracelets worn around wrists and ankles . A laptop with a software application developed in labview (national instruments corporation, us) stores all movement data recorded by either camera, radar, or acm (fig . The technical aspects of the recording system will be published in more detail by bonroy et al . And are briefly described in the supplementary material . Clonic and clonic seizures based on a large database of video / electroencephalography (eeg) data were used . Results of a patient - specific (algorithm trained only on data of the patient itself), a nonpatient - specific (algorithm trained on data not including those of the patient itself), and a semipatient - specific (algorithm trained on data also including those of the patient itself) approach were compared with the notes of professional caregivers . For a full description on how to implement these methodologies in practice professional caregivers were asked to write down all seizures the patients had, with time and description in a form of a clinical report . They recognize epileptic seizures (one month training and yearly tuition), are awake during the whole night, know the patients, and are semicontinuously watching on average four patients with active epilepsy in front of a video screen, only leaving if one of them or one of the other (maximum) twelve patients need care . The events detected with the algorithms were visually inspected by a pediatric video / eeg specialist and a pediatric neurologist using the screening tool which is a graphical user interface developed in matlab (mathworks inc ., us) (fig . 2). The screening tool displays information on the number of movement events and on their duration . The acm signals, a distribution graph, and the video images are depicted for each event . Also, radar and video signals are shown, as well as the fraction (percentage) of movement within the event . The graph at the lower left corner sets out amplitude of all events based on acm signals and length of all events based on the three detection modalities . In other words, it shows the intensity and duration of the movement . After the application of each of the three developed seizure detection algorithms and during postprocessing, the following logical reasoning was used: rule 1: a margin of error of 5 min has been taken into account for the seizure reporting . As the caregiver might write down the seizure after having attended to the patient, we assume the seizure may have been noticed 5 min before or after the actual time in the notes.rule 2: only seizures longer than 10 s are considered as possibly dangerous and therefore candidates for the alarm system, hence shorter seizures are not counted when calculating detection performance.rule 3: an alarm is set after 7 s of continuous positive detection . Clonic seizures is not typically detected by acm; the tonic phase involves muscle contraction rather than limb movement.rule 4: an alarm within 1 min after a previous one is ignored . A future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm, and 1 min is considered too short for the caregiver to have checked on and left the patient after the first alarm.rule 5: alarms caused by the caregiver waking or handling the patient (medication administration) and installing (evening) or removing (morning) the system and acm are ignored . Again, at these times, the future real - time alarm system should be turned off by the caregiver . Rule 1: a margin of error of 5 min has been taken into account for the seizure reporting . As the caregiver might write down the seizure after having attended to the patient, we assume the seizure may have been noticed 5 min before or after the actual time in the notes . Rule 2: only seizures longer than 10 s are considered as possibly dangerous and therefore candidates for the alarm system, hence shorter seizures are not counted when calculating detection performance . Rule 3: an alarm is set after 7 s of continuous positive detection . Clonic seizures is not typically detected by acm; the tonic phase involves muscle contraction rather than limb movement . Rule 4: an alarm within 1 min after a previous one is ignored . A future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm, and 1 min is considered too short for the caregiver to have checked on and left the patient after the first alarm . Rule 5: alarms caused by the caregiver waking or handling the patient (medication administration) and installing (evening) or removing (morning) the system and acm are ignored . Again, at these times, the future real - time alarm system should be turned off by the caregiver . True positives (tp) are detected seizures with the extra seizures detected by the algorithm but not the caregiver mentioned in between brackets . False negatives (fn) are seizures reported by the caregiver but not detected by the algorithm . To calculate sensitivity, the formula tp / (tp + fn) was used . False positives (fp) are detections that were not seizures, which in some occasions were multiple alarms in one movement event . False detection rate (fdr) is calculated as the number of fp per night . Two patients were measured for one month in their rooms in an epilepsy center in flanders, belgium . As they returned home for the weekends and as the caregivers needed to get acquainted with the recording system, they were measured for a mean of 12 nights per patient . Patient 1 is an 8-year - old girl with epilepsy e.c.i . And different types of seizures that typically last no more than 3 min: tonic clonic seizures with or without faltering respiration, tonic seizures, and clonic seizures . She also exhibits myoclonic jerks manifesting as (head) nods or facial contractions with eye deviation . Seizures are noticed based on semicontinuous video monitoring and when a scream or breathing problem is heard . Patient 2 is a 9-year - old boy with lesional epilepsy due to herpes encephalitis, who is kept in bed with a restraining belt around the waist . Clonic seizures of various duration, tonic, clonic, and myoclonic seizures and (series of) spasms . The caregivers use a babyphone and semicontinuous video monitoring to keep an eye on him . Approval by the medical ethical commission of the antwerp university hospital, belgium and signed informed consent forms from all parents were obtained prior to inclusion in the study, which was performed in accordance with the 1964 declaration of helsinki . The system uses video, accelerometry (acm), and radar - induced activity recording and is therefore named varia . It consists of an axis m1011 camera (axis communications ab, sweden) and a hygrosens rad - mod motion sensor based on the radar principle (b + b thermo - technik gmbh, germany). The camera and radar are attached to a tripod that is placed close to the patient's bed . The radar is added for detection of movements in the direction of the sensor and for measurement through sheets . Radar technology is also used for detection of falling, wandering, sitting, or getting up, so we expect it to detect large and slow epileptic and nonepileptic movements, also those caused by other persons in the room . Four shimmer sensors with integrated three - axis acm (shimmer, ireland), streaming wireless (bluetooth) communication standards, are adjusted to allow recording of more than 10 h before batteries need recharging and are put in elastic bracelets worn around wrists and ankles . A laptop with a software application developed in labview (national instruments corporation, us) stores all movement data recorded by either camera, radar, or acm (fig . The technical aspects of the recording system will be published in more detail by bonroy et al . And are briefly described in the supplementary material . The algorithms developed by milosevic et al . For tonic clonic and clonic seizures based on a large database of video / electroencephalography (eeg) data were used . Results of a patient - specific (algorithm trained only on data of the patient itself), a nonpatient - specific (algorithm trained on data not including those of the patient itself), and a semipatient - specific (algorithm trained on data also including those of the patient itself) approach were compared with the notes of professional caregivers . For a full description on how to implement these methodologies in practice professional caregivers were asked to write down all seizures the patients had, with time and description in a form of a clinical report . They recognize epileptic seizures (one month training and yearly tuition), are awake during the whole night, know the patients, and are semicontinuously watching on average four patients with active epilepsy in front of a video screen, only leaving if one of them or one of the other (maximum) twelve patients need care . The events detected with the algorithms were visually inspected by a pediatric video / eeg specialist and a pediatric neurologist using the screening tool which is a graphical user interface developed in matlab (mathworks inc ., us) (fig . 2). The screening tool displays information on the number of movement events and on their duration . The acm signals, a distribution graph, and the video images are depicted for each event . Also, radar and video signals are shown, as well as the fraction (percentage) of movement within the event . The graph at the lower left corner sets out amplitude of all events based on acm signals and length of all events based on the three detection modalities . In other words, it shows the intensity and duration of the movement . After the application of each of the three developed seizure detection algorithms and during postprocessing, the following logical reasoning was used: rule 1: a margin of error of 5 min has been taken into account for the seizure reporting . As the caregiver might write down the seizure after having attended to the patient, we assume the seizure may have been noticed 5 min before or after the actual time in the notes.rule 2: only seizures longer than 10 s are considered as possibly dangerous and therefore candidates for the alarm system, hence shorter seizures are not counted when calculating detection performance.rule 3: an alarm is set after 7 s of continuous positive detection . Clonic seizures is not typically detected by acm; the tonic phase involves muscle contraction rather than limb movement.rule 4: an alarm within 1 min after a previous one is ignored . A future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm, and 1 min is considered too short for the caregiver to have checked on and left the patient after the first alarm.rule 5: alarms caused by the caregiver waking or handling the patient (medication administration) and installing (evening) or removing (morning) the system and acm are ignored . Again, at these times, the future real - time alarm system should be turned off by the caregiver . Rule 1: a margin of error of 5 min has been taken into account for the seizure reporting . As the caregiver might write down the seizure after having attended to the patient, we assume the seizure may have been noticed 5 min before or after the actual time in the notes . Rule 2: only seizures longer than 10 s are considered as possibly dangerous and therefore candidates for the alarm system, hence shorter seizures are not counted when calculating detection performance . Rule 3: an alarm is set after 7 s of continuous positive detection . Clonic seizures is not typically detected by acm; the tonic phase involves muscle contraction rather than limb movement . Rule 4: an alarm within 1 min after a previous one is ignored . A future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm, and 1 min is considered too short for the caregiver to have checked on and left the patient after the first alarm . Rule 5: alarms caused by the caregiver waking or handling the patient (medication administration) and installing (evening) or removing (morning) the system and acm are ignored . Again, at these times, the future real - time alarm system should be turned off by the caregiver . True positives (tp) are detected seizures with the extra seizures detected by the algorithm but not the caregiver mentioned in between brackets . False negatives (fn) are seizures reported by the caregiver but not detected by the algorithm . To calculate sensitivity, the formula tp / (tp + fn) was used . False positives (fp) are detections that were not seizures, which in some occasions were multiple alarms in one movement event . False detection rate (fdr) is calculated as the number of fp per night . Monitoring for 15 nights for patient 1 and nine nights for patient 2 resulted in 9586 movement events . In this first validation, 322 events (3.4% or 15 min) were discarded before algorithm application because the data of one or more accelerometers lasted less than 1 s or was completely missing . The results of the nonpatient - specific and semipatient - specific approaches can be found in table 1 . The nonpatient - specific approach resulted in a sensitivity of 90% and fdr of one per night for patient 1 and a sensitivity of 33% and fdr of almost two per night for patient 2 . The semipatient - specific approach gave somewhat better results with a sensitivity of 93% and fdr of a little over one per night for patient 1 and a sensitivity of 40% and fdr of one per night for patient 2 . Results of the patient - specific approach are not depicted as it was immediately clear that many seizures were missed, many false alarms occurred, and the approach was not stable in between trials (the results depended on which data were used for algorithm training). In this work, we validated algorithms for the detection of tonic clonic and clonic epileptic seizures with a new (wireless) system using video, wireless accelerometry (acm), and radar - induced activity recording, which is placed in a home environment for a prolonged period of time . Three methodologies were tested: patient - specific, nonpatient - specific, and semipatient - specific algorithms . It seems that combining a large amount of data of many (other) patients as in the last two methodologies, instead of using a limited amount of data of one specific patient as in the first approach, yields better classification results . The semipatient - specific algorithm gave the best results, including 13 extra seizures detected (31%) and eight seizures missed (19%) compared with professional caregivers' observations . Of these eight, four were not even recorded in any of the movement data . Possible explanations include a temporary defect in the system, mistakes in reporting by the caregiver (one seizure was reported at 5:20 a.m. but found at 6:20 a.m.), or short seizures involving only the head (not attached to an acm). The limitations of our setup are discomfort (four accelerometers), noncontinuously recorded data (only movement events), and lack of gold standard (no electroencephalography or eeg). They are not able to detect the tonic phase of tonic clonic seizures (which involves muscle contraction rather than limb movement), so if the clonic phase is too short, the seizure will be missed: the alarm is set after 7 s of positive detection, so if the clonic phase lasts 6 s and this whole part is detected, the alarm will not be set off . Finally, asymmetric tonic clonic or clonic seizures during which the movements of the limbs are not synchronized and seizures with a low frequency of clonic jerks are often missed, since the spatial and frequency content of these seizures differ from the majority of seizures in the training set (a large database of wired acm data synchronized with video / eeg). Fig . We compared the results of the semipatient - specific algorithm with those of the screening tool of bonroy et al . (submitted) where the most intense and longest movement events were visually inspected for seizures using the same datasets and the graphical user interface of fig . 2 . The screening tool can be used before a diagnosis is made to quickly and retrospectively look at a night and report abnormal nocturnal behavior, including seizures . Once the patient is diagnosed (with epilepsy) this implies that we have to reduce the false detection rate (fdr) and increase the sensitivity . The comparison results, presented in table 2, show an increase of sensitivity from 58% to 66% (five seizures were missed, but 14 more were detected) and a decrease of fdr per night from 15 to 0.7 . Because the method of bonroy et al . Selected a percentage of most abnormal events irrespective of the number of movements or seizures that night, this automatically resulted in more false positives, so comparison of fdr is not relevant . Moreover, 11 of the 16 false positives in our approach were due to a system failure (broken channel or high - frequency noise), hence clustered alarms can be used to indicate this kind of events and reduce the number of false alarms . Other publications reported on body - attached accelerometry for tonic clonic and clonic seizure detection . Lockman et al . Reached a sensitivity of 87% testing 6 patients with (only) 8 seizures; however, patterson et al . Only obtained a sensitivity of 31% it is difficult to compare with this study and obtained results, however, mainly because the gold standard video / eeg was not used to annotate the data . Furthermore, we did not review all video data (only where the algorithm or caregiver indicated a seizure), and some of the seizures reported by the caregivers were not even found in the (movement) data . Although setup without eeg is less optimal for seizure detection performance, it is optimal for testing the independence and robustness of the system and the patient - friendliness of the system in a long - term home environment . Also, the second part of the gold standard, namely video, is present in our system . Furthermore, a major advantage of our system is that it allows video storage for retrospective analysis . Of the commercially available seizure detection systems reported by van de vel et al ., some keep a log of the detected seizures: epiwatcher stores the time and duration of up to 20 detections, smartwatch,, keeps the time, duration, and movement patterns, epicare, saves the time and duration of an unspecified number of detections, and the mobile phone application epdetect logs the raw accelerometer data . Some studies report on video - based seizure detection (so without acm), but a disadvantage is the difficulty to measure through bed linen,, . In order to improve the accuracy of the device, we are convinced that a multimodal approach is needed, and literature research as well as user questioning has persuaded us that the most important signals to be monitored are movement (often an early manifestation of seizures) and heart rate changes (indicating clinically relevant seizures and part of the pathophysiological mechanism leading to sudden unexpected death in epilepsy or sudep). It is clear that the number of sensors should be reduced to one or two at the most . Attachment of a seizure detection device to the upper arm is less comfortable than to the lower arm and can easily move when for example changing clothes or not tightly attached; so for the moment, we will continue our research with wrist - attached devices . Detection of respiration changes is certainly useful, but these changes occur probably almost simultaneously with heart rhythm changes, respiration is a very slow signal, and heart rhythm changes are very easy to detect, which is why more studies focus on cardiac- than respiration - based detection, . Oxygen saturation measurement and electrodermal activity (eda) seem promising, but changes are detected more slowly than heart rhythm changes,, and eda is measured using electrodes (as are ecg and emg), which cause potential skin irritation, and there is a risk of losing the signal due to electrodes falling off or a bad contact with the skin, . Furthermore, emg and respiration cannot be measured at the wrist, and oxygen saturation cannot be measured yet without adding a finger cuff, even though reflectance oximetry might be an option in the future . All of these arguments lead us to focus our future research on movement detection by acm in combination with heart rate detection by ppg (photoplethysmography). The algorithm should be robust and independent of the number and functionality of used sensors, so that there is no rejection of the data, and to be able to use our future device patient - independently, we will be concentrating on adaptive (learning) algorithms . Not only efficacy of the device will be important but also comfort (including the reduction of sensors), user - friendliness (less setup steps), and therapeutic (e.g., medication changes) and social (e.g., quality of life including the impact of false alarms) impact . Therapeutic impact can be examined by using the logging function of a seizure detection device, but in order to look into social impact, the device obviously needs to allow online use (with real - time alarming). The latter brings along additional challenges such as the need for fast computation time, low energy consumption, large storage capacity, and miniaturization of the device, which we need to address . Finally, it would be interesting to compare the performance with that of commercially available seizure detection bracelets using accelerometry, such as smartwatch or epicare free, also tested in a home environment . This study demonstrates the first clinical results of an algorithm for the detection of tonic clonic and clonic seizures tested on two patients with epilepsy monitored in a long - term home environment with the varia system (video, accelerometry, and radar - induced activity recording) developed by our team . Compared with the professional caregivers' observations, a mean sensitivity of 61.97% and false detection rate of 1.44 per night were obtained with the nonpatient - specific approach and a mean sensitivity of 66.87% and false detection rate of 1.16 per night with the semipatient - specific approach . We believe that, while privacy protection needs to be taken into account, the addition of video to accelerometry allows a seizure detection system not only to alarm the family or (informal) caregiver but also allows retrospective (offline) or emergency (online) verification of detected events . Much work remains to develop a multimodal, learning, comfortable, and user - friendly device.
Angiomyolipoma (aml) is a benign mesenchymal tumour consisting of varying amounts of mature adipose tissue, smooth muscle, and thick walled blood vessels . It is relatively rare benign tumour appearing in about 0.3% of general population and accounts for 3% of solid renal masses . Extra renal amls (eramls) are rare tumours that present as incidentalomas upon imaging for other conditions . Case reports of extra renal aml are rare with <60 reported cases since they were first described by friis and hjortrup in 1982 . Our case described one of the largest ever reported myolipomas, which despite its size, can be rather asymptomatic . Magnetic resonance imaging (mri) revealed 9.5 cm 8 cm 2 cm right adrenal mass [figure 1]. The tumour was predominantly hyperintense with signal intensity similar to fat on t1/t2-weighted images and lost signal on the fat - suppressed sequences . A well - encapsulated, firm globular mass separate from the right kidney and no definable adrenal was found [figures 2 and 3]. Gross post - operative evaluation of the adrenal mass showed approximately 10 cm 8 cm 2 cm greyish white solid, smooth, and firm mass [figure 4]. Histological examination revealed benign adrenal cortical elements, with a predominance of mature benign adipose tissue interspersed with pockets of haemopoietic (erythro - myeloid) intermediates, and megakaryocytes which was confirmatory for the diagnosis of adrenal aml . Angiomyolipoma is a rare clinical entity mostly involving kidney and is part of the group of tumour known as tumours of perivascular epithelioid cell origin . The most widely accepted theory of origin is adrenocortical cell metaplasia in response to stimuli, such as necrosis, inflammation, infection or stress (meyer et al . ). The presence of perivascular epithelioid cells is characteristic of aml since these cells show immunoreactivity for muscle markers (epithelial membrane antigen, keratin, vimentin, desmin, and actin) and hmb-45 . Positive immunoreactivity for hmb-45, a monoclonal antibody, is characteristic of amls and can be used to differentiate amls from other similar appearing lesions . One is isolated aml, and the other is that is associated with tuberous sclerosis . Most patients of isolated cases are in the age group of 27 - 72 years, mean age being 43 years . Although the majority of eramls are benign, two cases of metastatic and recurrent eramls have been reported . The demonstration of fat density (hypodense) within an adrenal mass by computed tomography is virtually diagnostic of aml . Mri is sometimes required to demonstrate origin of the tumour, to define the tissue planes when the tumour is large and heterogeneous, and to distinguish benign from malignant lesions by comparing signal intensity ratios of adrenal to liver . A small, <5 cm, asymptomatic myelolipoma could be followed - up over 1 - 2 year period with imaging controls . On the contrary, asymptomatic lesion or a large> 5 cm myelolipoma should be surgically excised, since there are reports of spontaneous rupture and haemorrhage of the mass presented with life - threatening cardiovascular shock . Unstable patients may benefit from emergent tumour embolisation and a subsequently staged surgical resection . Since it is a benign disease, its prognosis is good . Nevertheless, follow - up is recommended because of atypical morphology . Currently, there is no agreed protocol on follow - up but an ultrasound three to 6 months following the surgery with annual clinical examination for large tumours is recommended . The adrenal myolipoma is a rare urological entity, which seems to increase its frequency, probably due to causes affecting primarily the function and physiology of the adrenals . Ever since its introduction by gagner in 1992, laparoscopic adrenalectomy has become the standard of care for the treatment of functioning and non - functioning adrenal tumours . Many authors have found a decrease in perioperative morbidity and convalescence after this procedure when compared with open surgery.
A 67-year - old male presented to the emergency eye centre (eec) complaining of a painful right eye of 5-day duration . Clinical examination revealed a visual acuity (va) of 6/9 with minimal conjunctival injection, anterior chamber (ac) cells 1 + and posterior synechiae (ps). A diagnosis of anterior uveitis was made and the patient was commenced on topical prednisolone 1% and topical cyclopentolate 1% . The response to this treatment was good and the steroids were slowly tapered over 1 month; however, before cessation of treatment, the anterior segment inflammation worsened . The patient represented with va 6/36 and ac cells 3 +, extensive ps and a posterior vitritis . 2, the topical treatment was increased and an urgent referral was made to the uveitis service . At review, 4 weeks after presentation, the anterior and posterior segment inflammation remained and a white mass was noted in the peripheral nasal retina [fig . 1 upper plate], almost at the ora serrata . A diagnosis of presumed toxoplasma chorioretinitis was made and treatment with pyrimethamine and sulfadiazine followed by oral clindamycin was initiated . On further questioning, it was discovered that there was the possibility of a previous pulmonary tuberculosis (tb) infection . He had had no previous treatment for active tuberculosis . In view of concern over possible reactivation of tb with an increase in oral steroids, quantiferon and induced sputum sampling were also performed (all negative) and the dose of prednisolone was increased to 60 mg daily . Blood cultures were also negative and there was no serological evidence of toxoplasma infection (toxoplasma latex <16). (upper plate) 10 mhz b - scan ultrasonography of subretinal mass (nasal peripheral retina - white arrow) (middle plate) hematoxylin and eosin stained section of the retinal biopsy showing brown fungal hyphae (black arrows) coursing through the abscess. (lower plate) masson fontana stain showing a positive melanin brown reaction . Thin arrows showing fungal septae and thick arrows indicate dichotomous branching of hyphae the patient responded to oral steroids and clindamycin, with a reduction in intraocular inflammation and improved va of 6/18 after 2 weeks treatment . However, when the oral prednisolone was gradually reduced and the clindamycin stopped (after 5 weeks treatment), there was an increase in intraocular inflammation and va reduced to 3/60 . A diagnostic pars plana vitrectomy was therefore performed with vitreous sampling, which revealed a chronic vitritis without infectious agent or neoplasia . No herpes family viruses or toxoplasma organisms were identified on polymerase chain reaction testing . In order to clinch the diagnosis, combined cataract surgery, retinal biopsy, and intraocular oil tamponade was performed . The nasal sub - retinal mass was biopsied using a 23-gauge (23 g) vitrector hand piece and samples sent to histopathology and microbiology . Close inspection showed brown pigmented septate branching hyphae that stained strongly with masson fontana melanin stain and with a periodic acid schiff (pas) and silver stain [fig . The presence of a melanin containing, septate, branching, and mycelial fungus on histopathology was diagnostic of phaeohyphomycosis infection, on morphology alone . Prolonged fungal culture of the biopsy was negative and 18s ribosomal subunit polymerase chain reaction (pcr) was also negative (most likely due to the presence of pcr inhibitors in the sample). The absence of a culture and pcr result did not permit precise speciation of the phaeohyphomycosis fungus . The patient remained under joint care and was treated with antifungal therapy for 6 months; however, no primary source of fungal infection was identified . Following retinal biopsy the patient developed a retinal fold secondary to proliferative vitreoretinopathy (pvr), which extended from the biopsy site to the optic disc . Vitrectomy, removal of silicone oil, epiretinal membrane peel, subretinal pvr removal and retinectomy were performed with laser retinopexy and intraocular gas tamponade . Unfortunately, the patient required two further operations for rhegmatogenous retinal detachment . Currently, the retina is flat with heavy silicone oil tamponade and the va is 2/60 . Further retinal biopsy taken during vitrectomy for retinal detachment revealed no fungal elements on microscopy and intravitreal amphotericin b was also injected perioperatively . Phaeohyphomycosis is a collective term that describes infection caused by darkly pigmented mycelial fungi, a feature secondary to the presence of melanin within their cell walls . The most common phaeohyphomycosis genera pathogenic to humans include exophilia, phialophora, wangiella, bipolaris, exserohilum, cladophialophora . Morphology by histopathology is sufficient to make a diagnosis of phaehyphomycosis, characterized by branching, septate hyphae with confirmation of melanin by tinctorial staining (masson fontana or an equivalent). Whilst histopathology permits a clear diagnosis of phaeohyphomycosis, the precise species does require pcr and cultures . Infection is presumed to be as a result of implantation following superficial trauma and exposure to contaminated soils or organic matter . Systemic infections result either from direct invasion from a more superficial site, such as the paranasal sinuses or by means of haematogenous dissemination . The central nervous system is most frequently affected but other sites including lung, bone, and joint or peritoneum may also be involved ., primary cerebral phaeohyphomycosis seems to ignore this rationale with more than half of cases being fully immunocompetent . Melanin within the cell walls of dematiaceous fungi may provide the reasons for this disparity; it has been extensively investigated as a virulence factor . However, this is the first time a subretinal abscess caused by phaeohyphomycosis has been documented in the medical literature . The most plausible theory involves a haematogenous route (metastatic infection) transmission from a peripheral nidus to the subretinal space, which in turn initiated a posterior and anterior segment uveitis . Patients with underlying lung disease appear to be at a increased risk of pulmonary phaeohyphomycosis . It is possible that, in this patient, the lung parenchyma may have been a source of infection, in the setting of chronic, low level immune suppression / modulation by the methotrexate therapy with reactivation of a previous self - limiting respiratory phaeomycotic infection, with subsequent dissemination . Posterior segment pathology is usually diagnosed by characteristic clinical signs, extensive systemic investigations and non - invasive ocular investigations such as fluorescein angiography . However, it is not uncommon for vitreoretinal surgeons to aid in the diagnosis of a posterior uveitis of unknown aetiology by sampling vitreous, retina or choroidal tissue, leading to increased diagnostic accuracy and prompt treatment . Reported 13 cases of retinochoroidal biopsy in unclear uveitis seven of the cases tissue biopsy helped direct specific treatment . Intraocular lymphoma, recurrence of lymphoblastic leukaemia, chronic scleritis, toxoplasmosis, and metastasis were diagnoses confirmed as a result of the tissue biopsies performed . Malignancy was also excluded in several cases, which allowed for a trial of medications such as oral steroids and anticytomegalovirus agents . In this case, it is difficult to postulate whether the final va and outcome have been improved by surgical intervention . It is clear that the patients intraocular inflammation was increasing rapidly and only high dose steroids subdued the response . Without sub - retinal biopsy, the specific treatment of this patient's phaeohyphomycosis with antifungal medication would not have been possible and years of treatment for idiopathic uveitis may have ensued.
In 1978, primary health care was seen as the solution to the inadequate illness management systems that had developed throughout the world . It was hoped that phc would address some of the major inequalities in health that were observed by a balanced system of treatment and disease prevention . The world health organization (who) envisaged that phc would take place as close as possible to where people live and work and be the first element of a continuing health care process . Additionally, health service collaboration and multi - professional partnerships were expected to replace professional boundaries and competition . To sum up, phc used family physician as the most important ways to increase equitable access and utilization of public health services . In any case, family physician, with the key elements of first contact, comprehensiveness, continuity, and coordination of patient care, is central to the health of any health care system . Recent government initiatives are supporting general practice to deliver health care through a variety of means and personnel . These initiatives have included expanding the roles of nurses in general practice and enabling better access to allied health services through general practitioners (gps). Evidence clearly indicates the effectiveness of teams in improving the outcomes, especially in chronic disease . Now, people in most of the countries use professional and educated nurses [called nurse practitioner (np)] besides family physician or are under their supervision for diagnosis, treatment, and specifically health education of the family . In accordance with the international council of nurses definition, a nurse practitioner is a registered nurse who has acquired the expert knowledge base, complex decision - making skills, and clinical competencies for expanded practice, the characteristics of which are shaped by the context and/or country in which s / he is credentialed to practice . The royal college of nursing (rcn) defines an np as a registered nurse who has undertaken a specific course of study of at least first - degree (honors) level and who: makes professionally autonomous decision, for which he or she is accountable;works collaboratively with other health care professionals;provides counseling and health education;screens patient for disease risk factors and early signs of illness; andhas the authority to admit or discharge patients from their caseload, and refers patients to other health care providers as appropriate . Makes professionally autonomous decision, for which he or she is accountable; works collaboratively with other health care professionals; provides counseling and health education; screens patient for disease risk factors and early signs of illness; and has the authority to admit or discharge patients from their caseload, and refers patients to other health care providers as appropriate . In new reform toward family physician in iran, there is not enough attention toward nurses, their services, and specifically their role in education and counseling . According to the health - for - all goals, the central concern of nurses in primary care should be the prevention of the disease by educating individuals and families on healthy lifestyles, and teaching community health workers and traditional birth practitioners to carry out many of the functions that nurses themselves have normally performed . Whereas a big part of the family physician program has not been realized in iran yet (especially in big cities with a population of over 20,000), most graduate nurses are now working in the school of nursing and midwifery, hospitals, and ambulatory clinics with limited responsibilities . In addition, most graduate nurses in our community can play an important role in preventing medicine and family physician team . Consequently, in spite of their ability and capacity, they have a brief role in our family physician team & their job description . The main goal of this study was to review the role and situation of nps in the new reform for better use of their capabilities in health care services . This is a qualitative, comparative (role of nurses in family physicians team), and cross - sectional study that was carried out with triangulation method in three stages in 2011 . In the first stage, we conducted a literature review to design a conceptual framework . The second stage was a comparative study on four countries that had great experiences in these two fields (family physician reform and nps) and included usa, uk, austria, and canada, based on purposive sampling . In the third stage, all experts were completely familiar with the health system and had academic and research experiences in nursing education system in iran . The survey on health systems in different countries indicates that many of the health sectors have begun their reforms with two main goals, including quality improvement and cost limiting . Family physician and referral system are among the few options that can maintain and even upgrade the quality of services, reducing the costs simultaneously . As each health system in each country has its specific referral system, iran is now on the verge of implementing a best fit referral system reform . So, it is crucial to well define the roles of each component, such as nurses, in this system . In the united states, nps are licensed by the state in which they practice, and have a national board certification (usually through the american nurses credentialing center or american academy of nurse practitioner). Most nps specialize in a particular field of medical care, and there are as many types of nps as there are medical specialties . Nps provide high - quality, cost - effective individualized care that is comparable to the health care provided by the physicians, and np services are often covered by insurance providers . Role of nurses in some organizations in canada, the nurses involved in clinics should participate in specific training course of diagnosis and management of health care after registration . Then, they are allowed to prescribe drugs, and lab and other diagnostic tests . Having access to patient medical record, nps can reduce unnecessary visits . According to the standards of practice for nps, the college of nurses of ontario states that they offer the full scope of primary health care practice, including consultation with physicians or other health care professionals when the client requires care beyond the nps scope of practice . Further, they are accountable for establishing a consultative relationship with a physician and consultation occurs with a family physician; however, nps may consult with a specialist physician, if appropriate to the situation and practice setting . No legislative or regulatory deterrent related to the scope of practice exists for the nps to refer to specialist physicians . In austria, nurses in nursing home and maternity do some of the medical procedures under the supervision of physicians . These nurses are educated, and can treat, inject drugs, and request for some diagnostic tests for their patients . In the united kingdom, the gps monopoly on primary care has been broken by nurse - led services, including nhs direct, which provides 24-h health advice by telephone, and nhs walk - in centers, which treat minor illness and injury . Practice nurses and nps increasingly substitute for gps in the care of minor illness and routine management of chronic diseases such as asthma, diabetes, and coronary heart disease . In england and wales, the role of the nps had evolved from that of undertaking traditional, medically prescribed nursing tasks in a treatment room to undertaking screening activities and health assessments of new patients, with over 50% of the nps managing chronic disease clinics . Six experts in two panels were asked to confirm the role of nurses in family physician reform . They suggested that special courses are needed to empower our graduated nurses for their new rules . Meanwhile, using nps as substitution of gps does not seem to be logically correct due to the gps workload and the social and cultural background in iran . The survey on health systems in different countries indicates that many of the health sectors have begun their reforms with two main goals, including quality improvement and cost limiting . Family physician and referral system are among the few options that can maintain and even upgrade the quality of services, reducing the costs simultaneously . As each health system in each country has its specific referral system, iran is now on the verge of implementing a best fit referral system reform . So, it is crucial to well define the roles of each component, such as nurses, in this system . In the united states, nps are licensed by the state in which they practice, and have a national board certification (usually through the american nurses credentialing center or american academy of nurse practitioner). Most nps specialize in a particular field of medical care, and there are as many types of nps as there are medical specialties . Nps provide high - quality, cost - effective individualized care that is comparable to the health care provided by the physicians, and np services are often covered by insurance providers . Role of nurses in some organizations in canada, the nurses involved in clinics should participate in specific training course of diagnosis and management of health care after registration . Then, they are allowed to prescribe drugs, and lab and other diagnostic tests . Having access to patient medical record, nps can reduce unnecessary visits . According to the standards of practice for nps, the college of nurses of ontario states that they offer the full scope of primary health care practice, including consultation with physicians or other health care professionals when the client requires care beyond the nps scope of practice . Further, they are accountable for establishing a consultative relationship with a physician and consultation occurs with a family physician; however, nps may consult with a specialist physician, if appropriate to the situation and practice setting . No legislative or regulatory deterrent related to the scope of practice exists for the nps to refer to specialist physicians . In austria, nurses in nursing home and maternity do some of the medical procedures under the supervision of physicians . These nurses are educated, and can treat, inject drugs, and request for some diagnostic tests for their patients . In the united kingdom, the gps monopoly on primary care has been broken by nurse - led services, including nhs direct, which provides 24-h health advice by telephone, and nhs walk - in centers, which treat minor illness and injury . Practice nurses and nps increasingly substitute for gps in the care of minor illness and routine management of chronic diseases such as asthma, diabetes, and coronary heart disease . In england and wales, the role of the nps had evolved from that of undertaking traditional, medically prescribed nursing tasks in a treatment room to undertaking screening activities and health assessments of new patients, with over 50% of the nps managing chronic disease clinics . Six experts in two panels were asked to confirm the role of nurses in family physician reform . They suggested that special courses are needed to empower our graduated nurses for their new rules . Meanwhile, using nps as substitution of gps does not seem to be logically correct due to the gps workload and the social and cultural background in iran . According to the who, in most countries, nurses are the most important group of health workers in terms of numbers, closeness to people and their health problems, and understanding of community needs . There is still debate in nursing and medical circles about what the focus of their role should be . For example, lauder, sharkey, and reel suggest that nps and gps in rural and remote areas should be interchangeable and that the focus should be on the competency of the person delivering care rather than the right of one discipline (medicine) to perform a particular role . In contrast, campbell believes that public acceptance of nurses as gp substitutes would be poor, and therefore suggests that a better role for nurses in primary care would be health education and illness prevention . On the other hand, in our country (iran) until now, nurses have not got a competent role in health sector reform . Given that consumers have limited understanding of phc, it is not surprising they do not articulate confidence in nps acting autonomously but rather as complementary to gps, perhaps undertaking initial assessment for triage purposes and providing ongoing management, education, and support under the gps delegation . They would also like them to be family oriented and holistic in their practice, supporting their emotional and social needs in the context of their family lives . It means that any effort to change nurses roles in health care services should accompany with some social and cultural changes toward their competencies . In iran, whereas a noticeable reform toward family physician is going on, redefining nurses role is essential . They can perform more active roles in associating with gps in the clinics of family physicians, both in urban and rural areas, even with higher degrees of autonomy . Their competencies for case management, education, treatment, and follow - up may repair many of the pitfalls we are confronting in health care services . Of course, if it has to be done, special courses are needed to empower graduated nurses for their new rules . Meanwhile, substitution of gps with nps does not seem to be a good suggestion due to gp workload and social and cultural background in iran . Thus, it can be concluded that in order to achieve the goals of family physicians reform and also utilize the maximum capacity of trained nurses, a fixed and definite place must be considered for them in the gp team to provide their services under the supervision of family physicians . Their active role not only enriches the family physician team, but also might be crucial in patients training, which is currently one of our weaknesses.
Despite advancements in renal replacement therapy, acute kidney injury (aki) is a frequent complication with severe implications for the critically ill patient . Published mortality rates for intensive care unit patients with aki range between 30 and 70%, and aki alone remains an independent risk factor for mortality even after adjustment for demographics and severity of illness [1, 2]. Kidney ischemia - reperfusion injury (iri) occurs in various clinical settings including shock, sepsis, organ transplantation, and vascular surgery . It has become apparent that clinically much of the high patient mortality can be attributed to the onset of systemic inflammatory response syndrome (sirs) and progression to multiple organ failure (mof). Since the 1950s pulmonary dysfunction was thought to result from increased permeability of congested pulmonary capillaries, and this was dubbed the uremic lung . Even in the acute setting, however, kidney dysfunction directly contributes to the onset of remote organ injury . For example, increased kidney ischemia time during complex aortic surgery is associated not only with acute and chronic renal failure, but also with an increased incidence of remote organ injury and death [46]. Clinical and translational laboratory studies have demonstrated the relevance of interactions between the injured kidney and distant organs, and complex mechanisms of crosstalk between injured kidneys and remote organs such as the lungs, liver, heart, gut, brain, and hematologic system have been identified . Recent data highlights the importance of both the innate and adaptive immune response, activation of proinflammatory cascades, and an alteration of transcriptional events in remote organs during ischemic aki . The purpose of this paper is to review emerging concepts of organ crosstalk and recent experimental data regarding the activation and systemic expression of proinflammatory pathways during ischemic aki . For a complete list of abbreviations used in this paper, please see table 1 kidney iri remains a major cause of aki in both native and transplanted organs and lacks a specific treatment aside from renal replacement therapy . The local effects of kidney iri begin in the most vulnerable regions of the organ, after which a cascade of microvascular inflammation propagates . Although 25% of cardiac output contributes to renal blood flow, only a fraction ultimately reaches the vasa recta of the renal medulla, with the majority reaching the renal cortex . Therefore, even slight alterations in total renal blood flow may lead to anoxic injury in the medulla, resulting in tubular dysfunction, salt wasting, and glomerular vasoconstriction through tubuloglomerular feedback [8, 9]. Kidney iri activates an inflammatory response which results in endothelial cell activation, leukocyte adhesion and entrapment, and compromised microvascular blood flow . Inflammation in the postischemic kidney triggers the upregulation of leukocyte adhesion molecules, toll - like receptors, and downstream transcription factors, which all contribute to disruption of the integrity of the renal vascular endothelium . Adhesion molecules such as integrins and selectins along with proinflammatory cytokines propagate cellular injury not only locally in the renal tubular epithelial cells but also travel to remote organs where genomic markers of injury are upregulated and phenotypic injury occurs . Unfortunately, selective inhibition of cytokines and adhesion molecules such as tumor necrosis factor- (tnf-) and intercellular adhesion molecule (icam-1) have failed to demonstrate global attenuation of both local and remote organ injury during experimental models of ischemic aki [1316]. However, -melanocyte - stimulating hormone (-msh), a cytokine with broad antiinflammatory, anticytotoxic, and antiapoptotic properties, has demonstrated success in treating the inflammatory phenotype in rodents . In this model, treatment with -msh has attenuated both renal and pulmonary injury during ischemic aki [13, 17]. The largely unsuccessful effort to ameliorate mof with specific anti - inflammatory therapeutics highlights the complexity of the systemic response to kidney iri . From these early experimental studies, it is possible that the multiple inflammatory pathways activated in each organ system represent a unique response to ischemic aki . Despite a paucity of data, several different pathophysiologic responses to ischemic aki in remote organs have been identified (figure 1). In the heart, increased expression of tnf and interleukin-1 (il-1) are associated with myocyte apoptosis . In the brain, increased expression of chemokines including keratinocyte chemoattractant (kc, a brain il-8 homologue) and granulocyte colony - stimulating factor (g - csf) are seen with increased vascular permeability . Additional responses in the lungs, gut, and liver have been discovered, and these individual organ responses will be detailed later in this paper . We will now focus on cellular mediators, specifically those involved in the innate and adaptive immune system, which may connect local ischemic aki with distant organ injury . The innate immune system plays an important role in mediating the inflammatory response during ischemic aki . Traditionally, innate immunity elicits an immediate, preprogrammed response to tissue injury that lacks immunologic memory . It is composed of plasma proteins (complement), cells (neutrophils, macrophages, and natural killer cells), and physical barriers . Proposed initiators of the innate immune response during iri include the activation of toll - like receptors (tlrs) and the release of reactive oxygen species (nitric oxide and superoxide anion) and mitochondrial products . The complement system, particularly the alternative pathway, is activated, stimulating release of cytokines and subsequent activation of neutrophils, endothelium, and macrophages . Tlrs are a family of transmembrane proteins which serve as major pattern recognition receptors, binding to a wide range of microbial products and endogenous ligands released as a consequence of injury . Tlr - dependent signaling serves as a rapid response mechanism to local tissue damage and has been implicated in early activation of the innate immune response during kidney iri [11, 20]. Renal tubular epithelial cells constitutively express tlr2 and tlr4, and iri results in selective upregulation of these tlrs . Experimental studies of kidney iri have demonstrated attenuation of renal structural and functional injury in both tlr2/ and tlr4/mice, implicating tlr2 and tlr4 signaling in renal damage [21, 22]. Tlr2 may also play a role in transplantation tolerance by decreasing the infiltration of t cells, dendritic cells, and macrophages . T cells may act during early iri despite lacking alloantigenic stimulation, which challenges the traditional function of t cells as exclusive mediators of the adaptive immune response . Significant evidence exists to support the role of t cells in gut, heart, and lung ischemia - reperfusion injury [2428]. Likewise, experimental data also points toward antigen - independent t - cell activation in ischemic aki through cytokines secreted by macrophages and dendritic cells, chemokines, oxygen free radicals and the complement system [7, 19, 29]. For example, mice deficient in cd4 + and cd8 + cells and athymic mice demonstrate protection from experimental ischemic aki; however, only adoptive transfer of t cells of the cd4 + type restores this injury [30, 31]. Not all t cells, however, perpetuate inflammatory damage during ischemic aki; some populations of t cells confer renal protection after iri, particularly the th2 phenotype of cd4 + t cells and t regulatory cells . Additional recent experimental data has characterized specific populations of t lymphocytes trafficking to remote organs that may facilitate organ crosstalk during ischemic aki . In a rodent model of kidney iri, a distinct influx of cd3 + (t) cells with a predominant cd8 + t cell subpopulation was identified 24 hours after experimental kidney iri . These pulmonary t cells acquired increased expression of activation markers suggesting that ischemic aki induced trafficking of activated t cells which contributed to pulmonary injury, specifically apoptosis . The specific role of subpopulations of t cells in the pathogenesis of ischemic aki remains a stimulating topic of current and future investigation, both in the innate and adaptive immune response to kidney iri . The adaptive immune system relies on the specificity of antigen receptors on both b and t cells that respond to millions of various antigenic molecular structures . Once stimulated by an antigen, b cells (humoral immunity) produce specific antibodies, perform opsonization to facilitate phagocytosis, and activate the complement system . The t - cell receptor (tcr) responds to presented antigen peptides by activating macrophages to kill phagocytosed microbes, directly destroying infected cells and by releasing cytokines to promote further response (cell - mediated immunity). Antigen - dependent t - cell activation has been demonstrated in experimental models of renal iri [34, 35]. For example, mice with a restricted tcr cell repertoire suffer less injury and promote a far diminished inflammatory response during renal iri . Additionally, athymic mice that subsequently underwent adoptive transfer with these tcr - restricted t cells failed to restore injury as severely as seen with transfer of wild - type t - cells . T cells also play a role in long term renal modification, as an increased number of both activated and effector - memory t cells have been observed in postischemic kidneys as long as 6 weeks after iri . These t cells may in fact recognize antigens released during kidney iri and subsequently target the kidney in an autoimmune response, leading to long - term progression of renal dysfunction . This hypothesis was demonstrated in a murine adoptive transfer model in which nave mice received t cells from mice who were 6 weeks after kidney iri and subsequently developed albuminuria . Little is known about the role of b cells in renal iri; however, b - cell - deficient mice demonstrated renal protection in the early phase of experimental iri . In summary, both the innate and adaptive immune responses play critical roles in the pathophysiology of ischemic aki . Either following antigen stimulation or in the presence of proinflammatory chemokines and oxygen free radicals, t cells undergo early activation and serve as a bridge between adaptive and innate immunity . This specific host immune response to kidney iri facilitates distant organ crosstalk along with soluble proinflammatory mediators and will be further discussed in the following paragraphs . The combination of aki with acute lung injury (ali) remains a formidable challenge to clinicians caring for critically ill patients . In the setting of mof, aki and ali occur more frequently together than any other combination of organ systems, and predicted mortality approaches 80% [40, 41]. This exceedingly high mortality cannot be attributed solely to volume overload; leukocyte trafficking, uremic toxins, and oxidative stress mechanisms all likely contribute to this devastating clinical syndrome . For the purposes of this paper, ali represents a pao2/fio2 ratio <300 combined with chest radiograph findings of acute bilateral infiltrates in the absence of elevated cardiac filling pressures, as defined by the america - european consensus conference on acute respiratory distress syndrome (ards). While the mechanisms for ischemic aki - induced ali remain incompletely understood, several studies point toward a self - propagating cycle with activation of proinflammatory and proapoptotic pathways (figure 2). Aki leads to lung injury and inflammation, and in turn, ali and its attendant hypoxemia and hypercapnia worsened by mechanical ventilation with high positive end - expiratory pressure leads to diminishing renal hemodynamics and function . Lung injury during ischemic aki features marked pulmonary vascular permeability, erythrocyte sludging in lung capillaries, interstitial edema, focal alveolar hemorrhage, and inflammatory cell infiltration [12, 43, 44]. Mechanisms for decreased alveolar fluid clearance include downregulation of pulmonary epithelial salt and water transporters including enac, na, k - atpase, and aquaporins during ischemic aki [4345]. This specific response likely contributes to the increased microvascular permeability and clinical pulmonary edema frequently encountered in the setting of ischemic aki and mof . Experimental studies have identified a distinct pulmonary functional response and genomic signature induced during ischemic aki which differs from that induced by uremia alone . A comprehensive genomic map and ontology analysis revealed many of these top differentially expressed genes participating in proinflammatory and proapoptotic pathways . Genes with early and sustained activation at 6 and 36 hours after ischemic aki included neutrophilic granule protein (ngp), serum amyloid a3 (saa3), and interleukin 1-receptor type ii (il-1r2). Further investigation by deng et al . Has also identified an early pulmonary inflammatory response with rapid activation of transcription factors nf-b and ap-1 during ischemic aki . By 4 hours, leukocytes play a fundamental role in the development of ali, and several studies have documented lung leukocyte activation and trafficking during experimental aki with early and sustained neutrophil sequestration [46, 47]. Additional experiments by klein et al . Have demonstrated early expression of the neutrophil chemokine keratinocyte - derived chemokine (kc / cxcl1) and macrophage inflammatory protein (mip-2/cxcl-2) along with increased pulmonary myeloperoxidase activity and pulmonary microvascular permeability during ischemic aki . The authors also examined interleukin-6, a neutrophil - recruiting chemokine previously identified as a candidate gene by preferential expression in whole lung tissue during ischemic aki, and determined it was critical for influencing lymphocyte trafficking and pulmonary permeability . While neutrophils are key mediators in several extrapulmonary models of ali such as sepsis and mesenteric iri, their role in ischemic aki - induced ali remains to be elucidated . Uremic neutrophils have even demonstrated a protective effect in the setting of ali . Clearly, leukocyte trafficking and the innate immune response play a complex and important role in mediating the pulmonary inflammatory response and dysfunction during renal iri . Though not traditionally associated with a proinflammatory response, pulmonary apoptosis plays a critical role in the genomic and phenotypic response to ischemic aki . Apoptosis of pulmonary epithelial and endothelial cells along with delayed leukocyte apoptosis occurs during ali [5052]. Pulmonary apoptosis disrupts tethering forces involved in cell - to - cell and cell - to - extracellular matrix interactions, leading to a loss of endothelial barrier function and increased vascular permeability . In a rodent model, our laboratory has characterized caspase - dependent pulmonary apoptosis which is abrogated by the administration of a broad - spectrum caspase inhibitor . Not only did caspase inhibition attenuate the pulmonary functional injury, but the proapoptotic response to ischemic aki appears to be mediated via a tnf receptor-1 (tnf - r-1) dependent pathway . Our research has also demonstrated t - cell - dependent pulmonary apoptosis during ischemic aki . Along with identification of activated t cells trafficking to the lungs during ischemic aki, we have found that pulmonary apoptosis was subsequently attenuated in t - cell - deficient animals . Further pursuit of the specific pathway for t cell and tnfr1-dependent pulmonary apoptosis remains an active focus of future investigation in our research laboratory . Cardiovascular collapse is one of the most common causes of death in the setting of aki, yet the mechanisms involved are not entirely understood . Kelly has demonstrated left ventricular dilation, increased left ventricular end diastolic and end systolic diameter, increased relaxation time, and decreased fractional shortening in an experimental model of ischemic aki . Mechanisms of cardiac injury during ischemic aki include cardiac myocyte apoptosis and neutrophil infiltration, which have been attributed to increased cardiac and systemic tnf, il-1, and icam-1 expression [56, 57]. When kidney ischemic time is decreased, cardiac il-1 and icam expression, along with myocyte necrosis, decreased correspondingly . Effects of ischemic aki on the central nervous system (cns) are evident clinically when mental status changes develop . Of note, renal replacement therapy fails to fully correct this cns manifestation of renal failure . Uremic toxins certainly contribute to many symptoms of encephalopathy; however, cellular and soluble inflammatory mediators have also been implicated . Preclinical data identified an increase of kc, g - csf, and glial fibrillary acidic protein (gfap) in the cerebral cortex and hippocampus of the brain, which may serve to recruit neutrophils to sites of neuronal damage . This may result from either increased local neuronal production or an alteration of the blood - brain barrier; further research is required to delineate the source . A cell - mediated proinflammatory response has also been identified with activation of microglial cells (brain macrophages) during ischemic aki . Ischemic aki is also implicated in oxidative stress, inflammation, apoptosis, and tissue damage in hepatocytes . Hepatic stellate cells (hscs) regulate leukocyte trafficking and the secretion of chemokines such as il-8, and crosstalk between hscs likely occurs via a c - jun n - terminal kinase pathway . Oxidative stress during ischemic aki causes hepatic malondialdehyde, an index of lipid peroxidation, to increase while total glutathione, an antioxidant, decreases . Proinflammatory cytokine tnf expression and hepatic cellular apoptosis is also evident during ischemic aki . Previous investigators and clinicians have labeled the gut as the motor of mof because of its ability to amplify the systemic sirs response in the setting of shock and gut hypoperfusion [6264]. These mechanisms include increased intestinal permeability, interactions between host and bacterial pathogens, and propagation of toxins to distant organs via the lymphatic system [63, 65] and could potentially play a role during ischemic aki . Clinical studies have long demonstrated the increased secretion of potassium by the colon and rectum in response to aki, and recent literature linked channel - inducing factor, a potassium channel regulator found in both the kidney and colon, to ischemic aki [67, 68]. The role of the gut in response to organ crosstalk during ischemic aki remains a fascinating topic of future investigation . Aki is a frequent complication amongst hospitalized patients, with grave implications in the setting of mof . Ischemic aki initiates a cascade of proinflammatory pathways, and through the release of soluble mediators and activation of the host innate and adaptive immune systems, it facilitates organ crosstalk and remote organ injury . As our understanding of the postischemic kidney's role in mediating organ crosstalk continues to evolve, further laboratory investigation into the remote organ response to this devastating injury
Plasma cell leukemia (pcl) is a rare disease that represents approximately 4% of plasma cell malignant disorders.1 diagnosis of pcl is established based on kyle s criteria, which includes an absolute plasma cell number accounting for greater than 2000/l or 20% of white blood cell differentiation.2 pcl consists of two variants: primary pcl presents in patients with no previous history of multiple myeloma (mm), while secondary pcl consists of a leukemic transformation in previously recognized mm.3 primary pcl is an extremely resistant, rapidly progressive, fatal disease, with a median overall survival of 6.8 months.4 there is no standard therapeutic strategy because none of the treatment options have been prospectively evaluated . We describe herein a successful case of newly diagnosed primary pcl, treated with a regimen that included bortezomib, followed by auto stem cell transplantation (asct) and nonmyeloablative allogeneic stem cell transplantation (allo - sct). A 37-year - old man with suspected primary pcl was sent from a nearby hospital to our institution in february 2010 . Table 1 shows the laboratory data of the initial medical examination, and table 2 shows the data on admission to our hospital . The white blood cell count and its differentiation met kyle s criteria . The patient had acute heart failure . His chest x - ray revealed cardiomegaly (cardiothoracic ratio was 59%) with slight pulmonary congestion . Amino - terminal pro - brain natriuretic peptide levels were elevated to 811.2 pg / ml . This acute heart failure was probably caused by cardiac amyloidosis, as cardiac ultrasound showed a granular sparkling pattern . These cells were negative for cyclin d1, cd3, cd5, cd10, cd20, cd56, cd79a, and chain, and positive for cd138 and chain . No immunoglobulin h / cyclin d1 (igh / bcl1) translocations were found in fluorescence in situ hybridization analysis . Treatment with a pad regimen (bortezomib 1.3 mg / m [days 1, 4, 8, and 11], adriamycin 1.3 mg / m [days 1, 4, 8, and 11], and dexamethasone 40 mg / body [days 14, 811, and 1518]) was started . As soon as the treatment was started, leukemic plasma cell levels rapidly decreased (figure 1). No organ dysfunction was detected, except that serum creatinine levels temporarily increased to 1.21 mg / dl . On day 27 of the first pad cycle after three pad cycles, we were unable to assess his bone marrow, because it remained dry tap . Serum /, a surrogate marker for the treatment effect of this case, became similar to normal levels . In addition, the granular sparkling pattern detected by cardiac ultrasound at the time of admission disappeared . The patient was then administered high - dose therapy (hdt) + asct . Before hdt + asct, his p eripheral blood stem cells containing 10.9 10/kg cd34-positive cells were harvested with high - dose cyclophosphamide . He received hdt + asct with a conditioning regimen of high - dose melphalan (100 mg / m [days 3 and 2]) and bortezomib (1 mg / m [days 7, 4, + 1, and + 4]). At the end of hdt + asct sixty - seven days after hdt + asct, the patient received allogeneic bone marrow stem cell transplantation (allo - bmt) from his human leukocyte antigen - matched brother, containing 2.1 10/pt kg nuclear cells and 1.44 10/pt kg cd34-positive cells . The conditioning regimen consisted of fludarabine (30 mg / m [day 6 to day 2]) and total body irradiation (2 gy [day 1]). Graft - versus - host disease (gvhd) prophylaxis included tacrolimus and short - term methotrexate . Twelve months after allo - bmt, the patient s serum / level was maintained at normal levels . He remained in a state of remission, with only the complication of mild skin gvhd . There are no established therapeutic regimens for pcl . Several regimens that include an alkylating agent are sufficient to improve the overall survival (os) of pcl patients . Jimnez - zepeda and domnguez reported a median os of 6.8 months for pcl patients treated with a common chemotherapy regimen consisting of vincristine, adriamycin, and dexamethasone, versus 2 months for those who received melphalan and prednisolone.5 it has also been reported that a combination of intermediate doses of melphalan and dexamethasone obtained the highest level of response in pcl patients . However, the median os was only 60 days for the cohort that achieved a partial or complete response.6 by comparison, bortezomib regimens show promise as induction regimens for pcl . Katodritou et al reported that a combination of bortezomib and dexamethasone prolonged os (median, 12 months).7 the efficacy of pad has been reported by chan et al.8 similar to the case presented by chan et al, our case, inducted with pad, showed a very impressive clinical course . As soon as pad was started, leukemic plasma cell levels rapidly decreased (figure 1). In addition, anemia and platelet depletion improved, and acute heart and kidney dysfunction rapidly recovered . After three pad cycles, serum / levels were normalized, without any adverse effects (figure 2). In the present case, the absolute values of the immunoglobulin / free light chains in the serum before and after therapy had to be very important although pad is effective for pcl, it remains to be determined whether long - term survival is improved, because of a lack of long - term follow - up . Immunomodulatory drugs (imids), such as thalidomide and lenalidomide, are as promising as bortezomib.9, the reasons we did not choose imids were that lenalidomide was not approved in japan at the onset of the patient s pcl, and we decided to leave an opportunity to use thalidomide at his relapse . Our case represented a potential curative strategy for pcl, with the safe introduction of allo - immunization with a n onmyeloablative regimen following pad and hdt + asct . For multiple mm, the most common malignant disease in plasma cells, asct is regarded as the standard therapy for newly diagnosed cases in patients younger than 65 years . However, few patients who undergo the procedure are free of disease for more than 10 years . On the other hand, allo - sct is a promising therapy . Because of the graft - versus - myeloma effect, a lower relapse rate and longer remissions have been reported in patients receiving allo - sct.11 presumably, pcl may be the same as mm . However, the high risk of transplant - related mortality (30%60%) is a limitation of the use of allo - sct for mm . To overcome this limitation, maloney et al reported that combining a cytoreductive autograft with a nonmyeloablative allograft lowered transplant - related mortality by approximately 15%.12 meanwhile, ramasamy et al reported that alemtuzumab - based reduced intensity conditioning allogeneic transplantation.13 although progression - free survival of their patient cohort was comparable to previously published data of reduced intensity conditioning allogeneic transplantation in myeloma, there is no plateau on the survival curves, with a significant transplant - related mortality of 21% . Although nonmyeloablative conditioning reduces transplant - related mortality, it is difficult for patients older than 65 years to use our strategy . For them we have a successful case of newly diagnosed primary pcl, treated with a regimen that included bortezomib and asct, followed by nonmyeloablative allo - sct . This strategy is promising for pcl, which, though an extremely resistant disease, may become curable.
Fitz hugh curtis syndrome is an extra - pelvic manifestation of pelvic inflammatory disease (pid) and is characterized by perihepatic adhesions between liver capsule and diaphragm or anterior peritoneal surface(13). Most fitz hugh curtis syndrome patients are women of child bearing age and rarely has the syndrome been reported in males . The predominant symptom is pain in the right upper quadrant, which may be confused with biliary disease an abdominal computed tomography (ct) scan may reveal subcapsular enhancement of the liver in arterial phase (4). We herein report a case of fitz hugh curtis syndrome in a male patient that was diagnosed via laparoscopy . A 29 year - old african american male with russel - silver dwarfism presented with one day history of diarrhea, nausea, vomiting, right side abdominal pain, and abdominal distention . The pain was constant, gradually increasing in severity, and not related to food intake . His medical history was significant for russel - silver dwarfism, calcium deficiency, cardiomegaly, and bilateral testicular implants for undescended testicles . The patient was sexually active only with his girlfriend and denied any history of sexually transmitted disease . On examination patient was afebrile and his vitals were stable . Laboratory workup revealed white blood cell (wbc) count of 14,000/l with normal liver function tests . Ct scan of the abdomen and pelvis showed a small amount of free fluid in pelvis; the proximal appendix appeared normal, however the distal appendix was not visualized . The liver capsule appeared normal and there was no subcapsular fluid collection (figure 1). Ct scan showing normal appearing liver capsule and no perihepatic fluid collection the patient was admitted to the surgical service . He was made nil per os (npo), and placed on intravenous fluids and pain medication . He was refusing any surgical intervention at this point . On hospital day 5, a repeat abdominal ct scan demonstrated a normal appearing liver, small bowel, large bowel, and appendix, with a mild increase in pelvic free fluid . As the patient's symptoms did not improve with conservative management, he ultimately agreed to undergo a diagnostic laparoscopy, and was taken to the operating room on hospital day 9 . The small bowel was run in a retrograde fashion starting at the caecum, and no stricture, mass, or perforation was noticed . Appendectomy was not performed as per patient'swishes . Following the procedure, the patient reported complete resolution of his symptoms . His diet was gradually advanced, which he tolerated well, and was discharged on post operative day 2 . Fitz -hugh curtis syndrome was first described in 1920 by carlos stajano . In the 1930's thomas fitz hugh and arthur curtis also described the syndrome and made a connection between right upper quadrant pain following a pelvic infection and violin string like perihepatic adhesions(5). The first case of gonococcal perihepatitis in a male the incidence ranges from 4% to 14% in women with pid, but is as high as 27% in adolescents with pid, whose less mature genitourinary tract anatomy makes them more susceptible to infection(1). The inflammation of the liver capsule has been attributed to the direct bacterial spread from an infected fallopian tube via the right paracolic gutter . In men, hematogenous and lymphatic spread to liver the predominant symptoms are right upper quadrant pain, tenderness, and pleuritic right sided chest pain(2). These symptoms can pose diagnostic challenges as they may be confused with biliary tract symptoms . In a clinical setting, the diagnosis is adequately established by excluding other possible causes of right upper quadrant pain . On laboratory examination, white blood cell count can be elevated in nearly half of the patients, while liver function tests are normal in most patients . Because urethral cultures frequently fail to demonstrate the presence of gonorrhea and chlamydia, the serologic microimmunofluorescence antibody test is helpful in diagnosis (2). Ct scan may show subcapsular fluid collection, thickening of hepatic capsule in the arterial phase, and wedging enhancement of the involved liver parenchyma in more than 50% of patients . Most cases of fitz hugh curtis syndrome are managed with antibiotics against gonorrhea and chlamydia . It occurs mostly in premenopausal women, however, cases in males have also been reported.
Chemical shifts are now routinely used as a source of local conformational restraints in the structure determination of proteins by nmr, due mostly to the widespread use of programs such as talos (cornilescu et al . 1999) and shiftor / preditor (neal et al . 2006; berjanskii et al . 2006). These programs share a common approach and output similar data; both search a database that correlates local patterns of chemical shifts with local conformation, and both provide backbone dihedral angle restraints for individual residues . This approach has been very successful, but has some limitations in the stringent criteria needed for selecting proteins or protein fragments to populate the database, i.e. Only those with both highly reliable chemical shift and structural data can be included . Although this may seem adequate for example, the talos database contains 186 proteins subdivided into over 24,000 tripeptide fragments (http://spin.niddk.nih.gov/nmrpipe/talos/)the sequence / conformation search space is very large, and the database coverage is unevenly distributed . As a result, rare combinations of amino - acid and conformation may be under - represented in the database, leading to significant under - prediction and even to errors outside of the heavily populated regions of the ramachandran map . We have adopted an alternative approach to extracting structural data from chemical shifts based on our simshiftdb algorithm . The original simshift was designed to test for structural similarities between proteins in a pair wise manner using chemical shifts to supplement sequence data . Simshift showed improved ability to detect distant structural relationships when compared to state - of - the - art methods based on the sequence alone . A natural further development of pairwise comparison was to adapt the simshift algorithm for database searching, resulting in simshiftdb (ginzinger et al ., simshiftdb provides a list of matching proteins in the database, scored by a measure of statistical significance . In effect it searches a synthetic chemical shift database of 13,000 proteins based on the astral library (chandonia et al . The matching sequence can be of any length, and structurally similar regions can be found ranging from small, locally similar fragments up to full domains . In principle, any structural alignment method can also be used to make predictions of local conformation by extracting torsion angles from matching regions of the target proteins, and it is this implementation of the simshiftdb algorithm we present here . We benchmark the program against talos as a standard for current methods and hhpred, a sequence search method based on hidden markov models (sding et al . We show that simshiftdb can significantly increase the amount of information that can be derived from chemical shifts . 2007a) routine for standardizing chemical shift referencing to produce a pipeline for analysis of chemical shift data . Full details of the simshiftdb algorithm have been published previously (see ginzinger et al . 2007b), but it is worthwhile giving a brief overview here (see fig . 1). Given a target protein, simshiftdb analyzes each possible pairing of the target protein with one of the template protein structures in the database . Here we use the proteins from the astral database (version 1.71, filtered for 95% sequence identity) to build the simshiftdb template database . As simshiftdb is based on the comparison of chemical shift data, the chemical shifts for all entries in the template database are back - calculated from the three - dimensional structure using shiftx (neal et al . 2003). For each combination of the target protein with a template from the database, top: an example for a set of local similarities for a target protein (s) and a template protein (t). Bottom: combination of a subset of local similarities to yield a self - consistent final alignmentstep 1: local similarities are found by looking for high scoring combinations of parts of the target protein sequence (s) with parts of the template protein sequence (t). For example, block b in the figure shows that the chemical shifts of the target protein sequence from index xmin to xmax are similar to the chemical shifts of the template protein sequence from index ymin to ymax . The similarity is calculated by summing the pairwise scores of the residues in the similar region in analogy to a pairwise sequence alignment . The pairwise similarity scores are given by the so - called chemical shift substitution matrices, which give a score for each combination of two residues with associated chemical shifts (for more details see ginzinger 2008).step 2: the set of local similarities from step 1 is taken as an input for step 2, where the most significant combination of blocks is identified, according to a statistical model of alignment scores (karlin and altschul 1993). Additionally, two blocks have to fulfill two constraints for their combination to be considered: an explanation of the simshift algorithm . Top: an example for a set of local similarities for a target protein (s) and a template protein (t). Bottom: combination of a subset of local similarities to yield a self - consistent final alignment step 1: local similarities are found by looking for high scoring combinations of parts of the target protein sequence (s) with parts of the template protein sequence (t). Fig . For example, block b in the figure shows that the chemical shifts of the target protein sequence from index xmin to xmax are similar to the chemical shifts of the template protein sequence from index ymin to ymax . The similarity is calculated by summing the pairwise scores of the residues in the similar region in analogy to a pairwise sequence alignment . The pairwise similarity scores are given by the so - called chemical shift substitution matrices, which give a score for each combination of two residues with associated chemical shifts (for more details see ginzinger 2008). Step 2: the set of local similarities from step 1 is taken as an input for step 2, where the most significant combination of blocks is identified, according to a statistical model of alignment scores (karlin and altschul 1993). Additionally, two blocks have to fulfill two constraints for their combination to be considered: blocks may not overlap in the target or in the template protein; this would otherwise result in an ambiguous alignment.as the three - dimensional structure of the template protein is known, we further require that the euclidean distance between the end of the first block and the beginning of the second block may be bridged (according to chemical restraints) by the relevant sequence of amino acids in the target protein . Blocks may not overlap in the target or in the template protein; this would otherwise result in an ambiguous alignment . As the three - dimensional structure of the template protein is known, we further require that the euclidean distance between the end of the first block and the beginning of the second block may be bridged (according to chemical restraints) by the relevant sequence of amino acids in the target protein . Finally, we calculate an e - value for the optimal combination of blocks . This e - value represents the number of alignments of equal or better quality, which are expected to occur by chance, given the distribution of the amino acids with associated chemical shifts in the target protein and the template database . Additionally, the e - value takes the size of the template database into account . According to the following evaluation, an e - value of <10 guarantees a high quality alignment . To test the performance of simshiftdb, a benchmark set has to be defined for which both chemical shifts and the three - dimensional structure of the protein are available . The bmrb (seavey et al . 1991) is the main public repository for chemical shift data . However, there is no consistent mapping to the structural databases, making it difficult to relate structural with chemical shift information reliably . Therefore a mapping between bmrb and astral is calculated based on amino acid sequence similarity . Every entry in the benchmark set has to fulfill the following constraints: a 100% sequence match to an astral entry.at least 100 residues with associated chemical shifts (to exclude very short protein fragments; e.g. Single helices). At least 100 residues with associated chemical shifts (to exclude very short protein fragments; e.g. Single helices). To identify protein structures corresponding to the respective bmrb entries, a blast - search (altschul et al . 1990) against the sequences from the astral database is conducted for each bmrb entry . If the full bmrb sequence can be matched without gaps against an astral sequence, the corresponding astral structure is assigned to the bmrb entry . As some entries in bmrb match more than one sequence in astral, one representative structure has to be chosen . 2004) provided for each astral entry, thereby selecting the structure with the best resolution . Through this procedure a benchmark set containing 144 entries was derived . When calculating the similarity score for two residues, simshiftdb is restricted to at most three chemical shifts from the following list: h, h, n, c, c, and c. thus it is important to select a combination of shifts to extract maximum information, and a priority for replacing missing shifts . To identify the most successful strategy, we tested all possible priorities for the six atom types, resulting in 6! The most successful priority was: c> c> h> c> h> n. this is the default priority, and is used in the following analysis . To evaluate the prediction accuracy, we applied the program to all entries in the benchmark set, using all proteins from the simshiftdb template database as potential templates . Subsequently, we used all alignments that achieved an e - value better than 10 to infer torsion angles for the target residue from the associated template residues . If residues of the target were mapped multiple times, we based the prediction on the highest scoring alignment . It is important to evaluate the performance of simshiftdb as a function of sequence similarity; therefore 9 evaluations were performed with the maximum allowed sequence similarity in the evaluated alignments set between 20 and 100% . About 70% of all torsion angles predicted using simshiftdb have a high accuracy (15 error). Another 10 to 20% of the predicted angles have an error of less than 30. therefore simshiftdb yields accurate results in 85 to 90% of the evaluated predictions . Through the use of the chemical shifts, this performance is nearly independent of the percentage of sequence identity in the respective alignments.fig . 2evaluation of torsion angle predictions calculated using simshiftdb, separated according to the maximum allowed sequence identity between query and target . The boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists evaluation of torsion angle predictions calculated using simshiftdb, separated according to the maximum allowed sequence identity between query and target . The boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists also of interest is the performance of simshiftdb on different types of secondary structure . Figures 3 and 4 show the difference between the secondary structure content in all predictions versus that in high quality and erroneous predictions, respectively . It can be seen that predictions for sheet (for both high quality and erroneous predictions) match well with the percentage observed in all predictions, and this match is largely independent of sequence similarity . In contrast, the percentage of high quality predictions in helix increases with decreasing sequence similarity, whereas the corresponding percentage in coil regions decreases . For erroneous predictions this seems logical, as the structures for coil regions are less reliable, and predictions are clearly harder to make than for secondary structure . This test shows empirically that simshiftdb has no significant bias when comparing performance in predicting helix versus sheet . The independence of sheet predictions from sequence similarity indicates that the chemical shift data is more diagnostic for sheet than for helix.fig . 3difference between secondary structure content in high quality predictions versus the content for all predictions . The bars show the difference in helix, sheet and coil content (red, yellow and grey, respectively). 4difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig . 3 difference between secondary structure content in high quality predictions versus the content for all predictions . The bars show the difference in helix, sheet and coil content (red, yellow and grey, respectively). The lines show the overall secondary structure content in all predictions difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig . 3 to show empirically that simshiftdb uses the information in the chemical shift data to yield more sensitive alignments, especially in the case of low sequence similarity, we compare simshiftdb to hhsearch (sding 2005). Hhsearch, a sensitive search tool based on hidden markov models, calculates alignments between proteins using the primary sequence complemented by sequence - based predictions of secondary structure . 2005), a protein structure prediction method based on hhsearch alignments, ranked second best in the casp7 (battey et al . Additionally, it is freely available for download and gives the user the possibility to define arbitrary template databases . Therefore it is perfectly suited to serve as a reference for purely sequence - based methods . To compare the performance of simshiftdb and hhsearch we used the benchmark set defined in the previous section . For both methods we ran each target protein against the simshiftdb template database and used alignments achieving an e - value better than 10 to predict torsion angles for the residues in the target protein . If residues were mapped multiple times, the prediction was based on the highest scoring alignment . The following notation is used for the presentation of the results:- a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5.two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5, or both predictions have an error> 30.missing predictions are treated as predictions with an error> 30. a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5. two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5, or both predictions have an error> 30. missing predictions are treated as predictions with an error> 30. figure 5 shows the results of this evaluation for alignments with maximal sequence identities ranging from 10 to 100% . There is a clear trend for simshiftdb to outperform hhsearch as the sequence identity decreases, demonstrating that simshiftdb uses the structural information in chemical shifts to improve alignments.fig . 5simshiftdb predictions compared to the respective hhsearch predictions, separated according to the maximum allowed sequence identity between query and target . The red line shows the percentage of predictions where simshiftdb performs better, the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality . The brown line corresponds to the number of torsion angles predicted (right axis) simshiftdb predictions compared to the respective hhsearch predictions, separated according to the maximum allowed sequence identity between query and target . The red line shows the percentage of predictions where simshiftdb performs better, the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality . The brown line corresponds to the number of torsion angles predicted (right axis) it is important to compare simshiftdb to the most prominent method for predicting torsion angles from chemical shifts, namely talos (cornilescu et al . Again we used the benchmark set defined earlier, and made torsion angle predictions based on simshiftdb alignments that achieved an e - value better than 10 . We then compared the quality of each torsion angle prediction to the corresponding talos prediction . Figure 6 shows that simshiftdb outperforms talos in at least 30% of all cases . It should be noted that there is likely to be a significant bias towards talos in these results, as many members of the benchmark set will have been calculated using talos restraints.fig . 6simshiftdb predictions compared to the respective talos predictions using the same notation as in fig . 5 simshiftdb predictions compared to the respective talos predictions using the same notation as in fig . 5 to test the performance of simshiftdb, a benchmark set has to be defined for which both chemical shifts and the three - dimensional structure of the protein are available . The bmrb (seavey et al . 1991) is the main public repository for chemical shift data . However, there is no consistent mapping to the structural databases, making it difficult to relate structural with chemical shift information reliably . Therefore a mapping between bmrb and astral is calculated based on amino acid sequence similarity . Every entry in the benchmark set has to fulfill the following constraints: a 100% sequence match to an astral entry.at least 100 residues with associated chemical shifts (to exclude very short protein fragments; e.g. Single helices). At least 100 residues with associated chemical shifts (to exclude very short protein fragments; e.g. Single helices). To identify protein structures corresponding to the respective bmrb entries, a blast - search (altschul et al . 1990) against the sequences from the astral database is conducted for each bmrb entry . If the full bmrb sequence can be matched without gaps against an astral sequence, the corresponding astral structure is assigned to the bmrb entry . As some entries in bmrb match more than one sequence in astral, one representative structure has to be chosen . 2004) provided for each astral entry, thereby selecting the structure with the best resolution . Through this procedure when calculating the similarity score for two residues, simshiftdb is restricted to at most three chemical shifts from the following list: h, h, n, c, c, and c. thus it is important to select a combination of shifts to extract maximum information, and a priority for replacing missing shifts . To identify the most successful strategy, we tested all possible priorities for the six atom types, resulting in 6! = 720 evaluations . The most successful priority was: c> c> h> c> h> n. this is the default priority, and is used in the following analysis . To evaluate the prediction accuracy, we applied the program to all entries in the benchmark set, using all proteins from the simshiftdb template database as potential templates . Subsequently, we used all alignments that achieved an e - value better than 10 to infer torsion angles for the target residue from the associated template residues . If residues of the target were mapped multiple times, we based the prediction on the highest scoring alignment . It is important to evaluate the performance of simshiftdb as a function of sequence similarity; therefore 9 evaluations were performed with the maximum allowed sequence similarity in the evaluated alignments set between 20 and 100% . About 70% of all torsion angles predicted using simshiftdb have a high accuracy (15 error). Another 10 to 20% of the predicted angles have an error of less than 30. therefore simshiftdb yields accurate results in 85 to 90% of the evaluated predictions . Through the use of the chemical shifts, this performance is nearly independent of the percentage of sequence identity in the respective alignments.fig . 2evaluation of torsion angle predictions calculated using simshiftdb, separated according to the maximum allowed sequence identity between query and target . The boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists evaluation of torsion angle predictions calculated using simshiftdb, separated according to the maximum allowed sequence identity between query and target . The boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists also of interest is the performance of simshiftdb on different types of secondary structure . Figures 3 and 4 show the difference between the secondary structure content in all predictions versus that in high quality and erroneous predictions, respectively . It can be seen that predictions for sheet (for both high quality and erroneous predictions) match well with the percentage observed in all predictions, and this match is largely independent of sequence similarity . In contrast, the percentage of high quality predictions in helix increases with decreasing sequence similarity, whereas the corresponding percentage in coil regions decreases . For erroneous predictions this seems logical, as the structures for coil regions are less reliable, and predictions are clearly harder to make than for secondary structure . This test shows empirically that simshiftdb has no significant bias when comparing performance in predicting helix versus sheet . The independence of sheet predictions from sequence similarity indicates that the chemical shift data is more diagnostic for sheet than for helix.fig . 3difference between secondary structure content in high quality predictions versus the content for all predictions . The bars show the difference in helix, sheet and coil content (red, yellow and grey, respectively). 4difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig . 3 difference between secondary structure content in high quality predictions versus the content for all predictions . The bars show the difference in helix, sheet and coil content (red, yellow and grey, respectively). The lines show the overall secondary structure content in all predictions difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig . 3 to show empirically that simshiftdb uses the information in the chemical shift data to yield more sensitive alignments, especially in the case of low sequence similarity, we compare simshiftdb to hhsearch (sding 2005). Hhsearch, a sensitive search tool based on hidden markov models, calculates alignments between proteins using the primary sequence complemented by sequence - based predictions of secondary structure . 2005), a protein structure prediction method based on hhsearch alignments, ranked second best in the casp7 (battey et al . 2007) experiment . Additionally, it is freely available for download and gives the user the possibility to define arbitrary template databases . Therefore it is perfectly suited to serve as a reference for purely sequence - based methods . To compare the performance of simshiftdb and hhsearch we used the benchmark set defined in the previous section . For both methods we ran each target protein against the simshiftdb template database and used alignments achieving an e - value better than 10 to predict torsion angles for the residues in the target protein . If residues were mapped multiple times, the prediction was based on the highest scoring alignment . The following notation is used for the presentation of the results:- a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5.two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5, or both predictions have an error> 30.missing predictions are treated as predictions with an error> 30. a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5. two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5, or both predictions have an error> 30. missing predictions are treated as predictions with an error> 30. figure 5 shows the results of this evaluation for alignments with maximal sequence identities ranging from 10 to 100% . There is a clear trend for simshiftdb to outperform hhsearch as the sequence identity decreases, demonstrating that simshiftdb uses the structural information in chemical shifts to improve alignments.fig . 5simshiftdb predictions compared to the respective hhsearch predictions, separated according to the maximum allowed sequence identity between query and target . The red line shows the percentage of predictions where simshiftdb performs better, the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality . The brown line corresponds to the number of torsion angles predicted (right axis) simshiftdb predictions compared to the respective hhsearch predictions, separated according to the maximum allowed sequence identity between query and target . The red line shows the percentage of predictions where simshiftdb performs better, the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality . It is important to compare simshiftdb to the most prominent method for predicting torsion angles from chemical shifts, namely talos (cornilescu et al . 1999). Again we used the benchmark set defined earlier, and made torsion angle predictions based on simshiftdb alignments that achieved an e - value better than 10 . We then compared the quality of each torsion angle prediction to the corresponding talos prediction . Figure 6 shows that simshiftdb outperforms talos in at least 30% of all cases . It should be noted that there is likely to be a significant bias towards talos in these results, as many members of the benchmark set will have been calculated using talos restraints.fig . 6simshiftdb predictions compared to the respective talos predictions using the same notation as in fig . 5 simshiftdb predictions compared to the respective talos predictions using the same notation as in fig . 5 we have presented simshiftdb and shown that the program is able to sensitively extract structural information from chemical shift data . This information is to a certain extent complementary to that from currently available tools . On one hand simshiftdb shows its strength especially in cases of low sequence similarity, which underlines the advantage of including chemical shift information in the alignment algorithm . On the other hand, we were able to show that one - third of the predictions by simshiftdb clearly have a higher quality than the corresponding talos predictions, and this is largely independent of sequence similarity . The main advantage of simshiftdb is derived from its superior coverage of the search space, due to the large and quickly adaptable template database . Simshiftdb outperforms talos especially in those cases where talos finds no predictions classified as good according to its selection criteria . Simshiftdb and talos are therefore complementary, and can be used in parallel to increase the number of available predictions . Ph1500c is a 78-residue homo - hexameric domain currently under investigation in our laboratories, and was chosen because it shows no significant sequence similarity to proteins of known structure . We used simshiftdb to search for templates matching ph1500c, using several different chemical shift priorities . The search identifies several templates at e - values around 10, which correspond to the region g17-f40 of ph1500c and consist of three - stranded -meander linked by two tight turns (fig . The consensus of simshiftdb predictions shows the first turn to be a standard type i -turn, while the second is a less commonly observed 5-residue -turn . An example template agrees very well with all structural data available in this region (fig . 7), and suggests local conformational details such as sidechain positions and local hydrogen bonding networks.fig . The left panel shows a representative template structure found in a simshiftdb search for ph1500c (1p22, residues a392-w415). Residues shown in green are predicted by talos to be in the -region of ramachandran space and the residue in yellow in the + -region, while for residues in white there is no prediction . Only one talos prediction is made for the six residues outside of canonical secondary structure, i.e. The two turns and the -bulge in the first strand . The right panel shows the structure calculated with all available nmr data, showing very good agreement to the template . The coloring is as above, with the addition of blue residues for residues in the -region of ramachandran space . A conserved sidechain hydrogen - bond acceptor (t101 in ph1500c) is shown in grey, highlighting the ability of simshiftdb searches to reveal fine structural detailsfig . 8the simshiftdb web - interface at the center for applied molecular engineering an example simshiftdb search result . The left panel shows a representative template structure found in a simshiftdb search for ph1500c (1p22, residues a392-w415). Residues shown in green are predicted by talos to be in the -region of ramachandran space and the residue in yellow in the + -region, while for residues in white there is no prediction . Only one talos prediction is made for the six residues outside of canonical secondary structure, i.e. The two turns and the -bulge in the first strand . The right panel shows the structure calculated with all available nmr data, showing very good agreement to the template . The coloring is as above, with the addition of blue residues for residues in the -region of ramachandran space . A conserved sidechain hydrogen - bond acceptor (t101 in ph1500c) is shown in grey, highlighting the ability of simshiftdb searches to reveal fine structural details the simshiftdb web - interface at the center for applied molecular engineering this example highlights the major difference in the simshiftdb and talos approaches, i.e. The length of the template structures found by simshiftdb when compared to the tripeptides used to make talos predictions . The second difference is the use of the e - value as a continuous measure of quality, rather than a discrete selection criterion based on a consensus of the ten best hits . In some cases there may be only one or two templates found for any region of the protein, but low e - value scores can nevertheless allow predictions with high confidence . We have established an accuracy of above 85% for simshiftdb predictions, based on our benchmark set of proteins . This may at first glance compare poorly to talos, where an accuracy of 9798% is reported . However, it must be considered that this value is based on single simshiftdb predictions, rather than the consensus of 10 predictions . Also, it is worth noting that talos is very accurate within secondary structure, and therefore the 23% of errors must be concentrated in the smaller fraction of other predictions . In our experience, these errors often result from predictions made out of structural context; e.g. For a residue in a -turn based on tripeptides from a helix . The wider context provided by simshiftdb results should therefore add both to the confidence of its predictions and those from talos . The optimum chemical shift priority found for simshiftdb searches is somewhat surprising in that it contains h, which is not generally regarded as containing much structural information . Perhaps this is due to some complementarities of the information from h and that from other shifts . It is worth noting, though, that the difference between the best priorities is small, and it may be worth testing a range of priorities . This is easily possible; although the program searches a database of 13,000 protein structures, an average simshiftdb run takes only 30 seconds on a standard laptop (intel t2500, 2.0 ghz, 1 gb ram). The different results are comparable using the calculated e - values, thereby enabling the user to select the most promising result.
Sutureless clear corneal incisions are now arguably the most common incisions made to perform cataract surgery with phacoemulsification, replacing scleral tunnel and limbal incisions . There are significant differences in the effects of clear corneal and scleral or limbal incisions, related to anatomical and physiological differences in the respective structures where the incisions are made . The cornea is comprised of a regular lamellar structure of collagen fibrils that stretch from limbus to limbus, arranged in a lattice formation; this is what provides the primary structural support of the cornea and accounts for its transparency . Vascular arcades are present, providing a potential source of fibroblasts . The differences in the healing effects of incisions at the limbus and the cornea have been previously discussed in the literature [24]. Limbal incisions appear to heal more quickly and are more resistant to deformation pressure than those in the cornea . Clear corneal incisions also appear to increase the likelihood of endophthalmitis, potentially for the reasons above . In short, there are no disadvantages to a limbal incision in terms of surgical safety . From a structural point of view it is known that incisions for cataract surgery will induce a flattening effect when made on (or near) the steep axis of the cornea . This effect is positively correlated with incision size (larger incisions generating more astigmatism, all other things being equal) and location (scleral or limbal incision inducing less astigmatism than clear corneal), though for small incisions the effect of location appears less critical [6, 7]. Wound construction also appears to have an effect, with square incisions reported to affect astigmatism the least . Data related to surgically induced astigmatism in the recent literature is primarily related to clear corneal incisions . The data reported here summarize the astigmatic changes produced with a small (2.2 mm) square posterior limbal incision . A retrospective patient chart review was conducted at one site (pe) for a study of toric and spherical iols . Those eyes with preoperative and postoperative keratometry results eyes were excluded if they had irregular (nonorthogonal) corneal astigmatism or if they had previous corneal surgery . This obviated the need for a specific informed consent or irb approval for the data collection undertaken here . In addition, all patients sign an acknowledgement that their deidentified phi data may be used for research purposes when they are seen in the practice . Comparative surgically induced astigmatism data were obtained from one of the authors (w. hill) from a website specifically designed to collect preoperative and postoperative keratometry data for the purposes of calculating surgically induced astigmatism . This site provides the necessary spreadsheet and instructions to surgeons for the calculation of sia . An option allows any surgeon who uses the spreadsheet to upload their data to the website for the purposes of aggregate analysis . Again, no phi was collected so informed consent was not required for this data set . Results from ph and the aggregate data from wh were tabulated in microsoft excel and analyzed using statistica statistical software . Differences between groups were calculated with t - tests and analysis of variance (anova) tests, with significance at p <0.05 . For the patients at one site (p. ernest) the data were corroborated with an automated keratometry reading and the automated keratometry feature of the iolmaster (carl zeiss meditec, jena, germany). The automated keratometry readings were repeated on all patients postoperatively, and the comparison to the preoperative automated keratometry was used to calculate sia . The surgical technique employed in the time period in which the retrospective data were collected was constant for all patients . All incisions were temporal, 2.2 mm square posterior limbal incisions, using a technique previously described in the literature . Surgical technique was not described in the aggregate sia data collected off the web, but incision type (e.g., clear corneal, limbal) was generally indicated, and the size of the incision was noted . These variables were collected so that surgeons could submit different preoperative and postoperative data sets for different incision sizes and locations . The review of available clinical records from pe yielded a total of 38 eyes that both met the criteria for inclusion and had available postoperative keratometry data for analysis . Surgically induced astigmatism was calculated as the vector difference between preoperative and postoperative anterior corneal astigmatism as measured with automated keratometry . The sia in this cohort averaged 0.25 d with a standard deviation of 0.14 d. a total of 1,712 eyes were available in the aggregate sia data from w. hill, from 51 different surgeons . The data were filtered to remove duplicates, include only incision sizes of 2.2 mm, and exclude those records where the incision type was not indicated . Where there were not at least 20 surgeries in any incision category (size and location) for a surgeon, those data were also deleted . After this data - screening step, 246 surgeries from 5 surgeons remained for comparative analysis . Average preoperative keratometry was not statistically significantly different between surgeons (p = 0.41); means ranged from 43.8 d to 44.5 d. the magnitude of preoperative corneal cylinder was also not statistically significantly different between surgeons (p = 0.13); means ranged from 0.79 d to 1.04 d. table 1 contains a summary of the surgically induced astigmatism by surgeon . Looking at the aggregate clear corneal incision data versus the ernest posterior limbal data, there was a statistically significant difference in the surgically induced astigmatism by incision type (p <0.001). Inside the clear corneal group as a post hoc test, the ernest data were compared to the surgeon with the lowest average sia from the clear corneal incision group (surgeon 5); there was a statistically significantly lower mean sia in the ernest cohort (p = 0.001). Note, too, that the standard deviation of the ernest cohort is less than half that of the cohorts of 4 of the 5 other surgeons, and 40% lower than surgeon 5 despite having less than half the sample size . Surgeons 1 to 5 used 2.2 mm clear corneal incisions, while pe used a 2.2 mm square posterior limbal incision . There was a statistically significant difference in sia by surgeon (p <0.001). Post hoc testing showed that the ernest data yielded a statistically significantly lower sia than the other surgeons' data . The results suggest that the magnitude and the variability of surgically induced astigmatism with small incision surgery (2.2 mm) is significantly lower if a posterior limbal incision is used instead of a clear corneal incision . Results for the clear corneal incisions used here as comparative data are consistent with those recently reported in the literature . Wilczynski et al . Reported sia values of 0.50 0.25 d with 1.7 mm and 1.8 mm surgical incisions . Holland reported sia values of 0.6 0.4 d for 2.4 mm clear corneal incisions . Masket reported that sia results were somewhat lower with his 2.2 mm clear corneal incisions (0.35 0.21 d) but it is worth noting that his incision was started with a groove at the temporal limbus . Some of the variability of sia data from surgeons other than ernest is likely related to differences in technique between surgeons . In addition, intrasurgeon variability may be slightly higher if surgeons did not measure their postoperative keratometry 1 month or more after surgery, as there is some change in keratometry expected in that first month . The minimization of the magnitude (and variability) of surgically induced astigmatism is important for modern day cataract surgery, particularly with the use of toric and multifocal intraocular lenses residual astigmatism after surgery will reduce the likelihood of spectacle independence for distance vision for these patients . Minimizing astigmatism for multifocal iols is equally important, as even small amounts of residual astigmatism can compromise the outcome with regard to uncorrected visual acuity at distance . Surgeons interested in reducing the magnitude and variability of induced astigmatism at the time of cataract surgery may want to consider the use of a 2.2 mm square posterior limbal incision.
The embryonic development of the cerebellum derives from the hindbrain from the fifth week, originating from the thickening of the dorsal alar plates . Anatomically it is formed by a median portion, the vermis, connected to two large lateral masses, the cerebellar hemispheres . The cisterna magna is located in the posterior fossa and occupies the space between the underside of the cerebellum, the posterior side of the bulb and the roof of the fourth ventricle . It continues caudally with the spinal subarachnoid space and binds to the fourth ventricle through the opening median . At ultrasonography the cerebellar hemispheres are usually low to moderately echogenic, connected above by an echogenic cerebellar tentorium . The vermis is a highly echogenic structure, which can be recognized as an oval density in the midline of the axial and sagittal plans . The cerebellum and cisterna magna can be observed on ultrasound around 11 weeks, but the formation of the cerebellar vermis ends around 18 weeks . For this reason, we must be careful in early diagnosis of the posterior fossa anomalies before this period, due to the possibility of a pathological image simulation [3, 4]. The measurement of the transverse cerebellar diameter by two - dimensional ultrasonography (2dus) is a very reliable parameter in the evaluation and early detection of intrauterine growth restriction . On the other hand, an adequate evaluation of the posterior fossa abnormalities allows the diagnosis of the cisterna magna as the dandy - walker complex, megacisterna magna, and blake's cyst . The three - dimensional ultrasonography (3dus) appeared as an important tool in the evaluation of fetal central nervous system in the late 1990s, because of the development of high resolution endocavitary volumetric transducers . Currently, with the development of new software like volume contrast imaging allows adequate evaluation of posterior fossa anomalies as well as the biometry of the cerebellar vermis [7, 8]. The three - dimensional extended imaging software (3d xi - medison, seoul, korea) is composed of three programs: multislice view, volume ct view, and oblique view . The multislice consists of multiple sequential and adjacent planes arranged in the set screen (sagittal, axial, and coronal). The reference plane, number of images arranged in the screen (1 1, 2 1, 3 2, 4 3, or 6 2), orientation and rotation of the image, the magnitude of the magnification, and depth and range between the planes (0.5 to 5.0 mm) can be adjusted according to the region of interest . A preliminary study evaluated the application of the multislice view in fetal central nervous system anomalies, showing potential benefits . Up to date there are no studies evaluating the measures of the fetal cerebellum and cisterna magna by 3dus using the multislice view . The objective of this study is to compare the measurements of transverse cerebellar and anterior - posterior cisterna magna diameter obtained by 2d and 3dus using the software 3d xi in normal pregnant women between 18 and 24 weeks . A prospective cross - sectional study between march 2010 and february 2011, involving 69 normal pregnant women between 18 and 24 weeks was performed . This study was approved by the research ethics committee of the irmandade da santa casa de misericrdia de so paulo, brazil, and the patients who agreed to participate signed a term of consent . This study was carried out at the department of obstetrics and gynecology, faculty of medical sciences of santa casa of so paulo (fcmscsp). The patients were randomly selected, and all evaluation made by a single examiner (fsbb), with five years experience in obstetric ultrasound . The examinations were performed on a sonoace x8 (samsung - medison, seoul, korea) device equipped with multifrequency volumetric convex transducer (37 mhz). The criteria for inclusion were (1) unique pregnancy with live fetus and (2) gestational age evaluated by last menstrual period and confirmed by ultrasound performed until the 14th week (crown - rump length - crl: 484 mm). Exclusion criteria were (1) pregnant women carrying fetuses with structural anomalies detected at the time of the examination and (2) pregnant women carrying chronic diseases that would interfere with fetal growth . Initially, a realtime 2d evaluation was performed in order to evaluate the biometry, morphology, and quantification of amniotic fluid volume . For the 2d measurement of transverse cerebellar and anterior - posterior cisterna magna diameter, it was performed a modified transversal slice of the fetal head slightly angled, through the thalamus, cerebellar hemispheres, cisterna magna, cave of septum pellucidum, the occipital bone, and nuchal fold . An insonation angle of the occipital bone was chosen, taking care that it was focused on an angle of 30. it was performed a single measurement of transverse cerebellar and antero - posterior of the fetal cisterna magna diameter in each mother, and this image is saved in the memory of the device . The three - dimensional volume acquisition was performed on the same 2d plane in which was performed the measurements of the transverse cerebellar diameter and anterior - posterior cisterna magna, to encompass the entire fetal skull (roi - region of interest) (figure 1(a)). In order to standardize all 3d measurements, the following preset was used on the device: scanning3d static; display mode three - dimensional extended imaging (multislice view); scanning speed slow; angle scanning70; overall gain of the device50% . After the three - dimensional scanning, the image was displayed in the multiplanar mode (axial, sagittal, and coronal) (figure 1(b)). The volumes were saved in the device memory and then stored on compact discs (cds) and transferred to a personal computer . The analyses were performed offline in the same apparatus in a time period of 30 to 120 days after the volumetric capture . For measurements of the transverse cerebellar and anterior - posterior cisterna magna diameters the program multislice view on 3 2 (two rows and three columns) was chosen, with slice thickness of 0.5 mm (figure 1(c)). The buttons 3 and 4 of the device were maneuvered, for navigation between tomographic slices, until in one of them the image of the cerebellum disappeared in higher plan than that of the posterior fossa (figure 1(d)). From this point, the 1 1 (one row and one column) option was selected and the number of the image was recorded . With the presence of only one tomographic slice on the screen, the button 4 of the apparatus was rotated clockwise or counterclockwise, depending on the fetus position, leading the exposition of a lower plan, successively until the outline of the cerebellum began to appear . Following, each plan of the cerebellum and the cisterna magna was measured, with a difference of 0.5 mm between them to the normal cerebellum outline disappeared and the bone of the skull base could be observed . On average, measurements of the transverse cerebellar diameter and the antero - posterior diameter of the cisterna magna were performed in 25 consecutive plans in each volume, being considered as the value of the measurement of the cerebellum and the cisterna magna by 3d xi method the highest value obtained . The average time for manipulation of the 3d volume measurement of the cerebellum and the cisterna magna was 180 seconds . The data were transferred to an excel 2007 (microsoft corp, redmond, wa, usa) spreadsheet and analyzed by the statistical package for social sciences (spss inc ., chicago, il, usa) version 19.0 for windows . For the 2d and 3d measurements of the transverse cerebellar diameter and antero - posterior cisterna magna, mean, median, maximum, and minimum values were calculated . And for the 3d measurements percentiles 5, 10, 90, and 95 were also calculated in each gestational age evaluated . To evaluate the difference between the two techniques the wilcoxon test was used . To evaluate the correlation of 2d and 3d, measurements of the transverse cerebellar diameter and antero - posterior cisterna magna as well as measurements of biparietal diameter (bpd) and head circumference (hc) according to the gestational age, the spearman's correlation coefficient (r) was used . In cases of high correlation, polynomial regression models, with adjustments by the coefficient of determination (r) the first examiner (fsbb) held a second 2d and 3d measures in 8 cases randomly selected 7 days after the first . For interobserver reproducibility, a second examiner (lsvf), with the same experience in obstetric ultrasound performed a third measure of these same 8 cases, and the results were armored one of the other . For this purpose, to calculate the reproducibility intraclass correlation coefficient (icc) and it is considered poor correlation icc <0.40; satisfactory iccs between 0.40 and 0.75 and excellent icc 0.75 . The bland - altman plots evaluate the average of measurements performed by one or two examiners plotted against the difference of their mean values with standard deviation 1.96 (limits of agreement). In all analysis we used a significance level (p) <0.05 . The 69 patients initially selected met the criteria for inclusion and exclusion, being allocated in the final statistical analysis . The age of the pregnant women ranged from 17 to 41 years, with an average of 26.93 years (standard deviation 6.10 years). The number of pregnancies ranged from 1 to 6, with an average of 2.19 pregnancies (standard deviation of 1.32 pregnancies). The average transverse and anterior - posterior cerebellum and cisterna magna diameter by 3dus ranged from 9.23 3.16 mm (18.0629.34) and 6.62 1.41 mm (4.1910.45), respectively . The average transverse diameter and antero - posterior cerebellum and cisterna magna by 2dus ranged from 22.33 3.16 mm (18.1429.10) and 5.60 1.33 mm (3.189.32), respectively . It was observed that on average the measurements obtained by 3dus were significantly higher, 0.76 and 1.02 mm for the length of the cerebellum and cisterna magna, respectively (p <0.001) (tables 1 and 2). There was a high correlation between the measurement of transverse cerebellar diameter by 3dus with gestational age (r = 0.940, p <0.001) as well as measures of dbp (r = 0.927, p <0.001) and hc (r = 0.938, p <0.001). The equation that best represented the correlation between the extent of the transverse cerebellar diameter and gestational age was of a second degree: dtc = 7.231 + 1.851 ga 0.027 ga2 (r = 0879). For the measurement of antero - posterior diameter of the cisterna magna, a low correlation between gestational age, bpd, and hc (r = 0.462, p <0.001; r = 0.430, p <0,001; r = 0.517, p <0.001, resp .) (figure 2) was observed . The low correlation between the antero - posterior diameter of the cisterna magna and gestational age did not allow the construction of polynomial regression models . Tables 3 and 4 show the percentiles 5, 10, 90, and 95 for measurements of the transverse and anterior - posterior cerebellum and cisterna magna diameter at each gestational age evaluated . An icc> 0.66 for all intra- and interobserver measurements of the cerebellum and cisterna magna length by 3dus was observed, with the exception of the intraobserver measurement of the length of cisterna magna (icc = 0.792, 0.668, 0.691, and 0.287, resp . ). The mean differences as well the limits of agreement for intraobserver and interobserver reproducibility of the cerebellum and cisterna magna length by 3dus were 0.16 mm (limits of agreement, 0.79; 1.11), 0.13 mm (limits of agreement, 1.22; 0.95), 0.08 mm (limits of agreement, 0.67; 0.82), and 0.12 mm (limits of agreement, 0.69; 0.93), respectively (figures 3 and 4). The posterior fossa cystic malformations include abnormalities of the meninges (arachnoid cyst, megacisterna magna) and cerebellum (dandy - walker malformation and variants). The prenatal diagnosis of these malformations is of great importance for an adequate followup of pregnancy as well as the relatives counseling . The postnatal results are in general very bad in the dandy - walker malformation and variants, mainly as result of associated anomalies . The postnatal result of megacisterna magna is better, especially if isolated . In this study, we compared the measurements of the transverse and antero - posterior cerebellum and cisterna magna diameters by 2d and 3dus between 18 and 24 weeks . This interval was defined by the fact that the transverse cerebellar diameter showed the highest correlation with gestational age, its measurement in millimeters is similar to the gestational age in weeks . Furthermore, the diagnosis of agenesis of the vermis, especially the partial cannot be performed before 18 weeks . In relation to the antero - posterior diameter of the cisterna magna, in general, in this period the ultrasound of the second trimester for malformations evaluation will be performed, its normal measure being less than 10 mm . In this study we observed that the transverse cerebellar and anterior - posterior cisterna magna diameters by 2d and 3dus increased from 18 to 24 weeks, however, the cisterna magna measurements by 3dus showed low correlation with the gestational age . In a cross - sectional study carried out by vinkesteijn et al . With 360 normal pregnant women between 17 and 34 weeks, the average of fetal transverse cerebellar diameter by 2dus was 22.1 mm from 18 to 24 weeks, very similar to our results obtained using 2dus (22.33 mm). In another cross - sectional study carried out by koktener et al . With 194 normal pregnant women between 16 and 24 weeks, the mean antero - posterior diameter of the cisterna magna by 2dus was 4.83 mm, lower than that obtained in our study (5.60 mm). The test to prove that the antero - posterior diameter of the cisterna magna increased from 18 to 24 weeks is important because the fixed value of 10 mm for the indication of normality cannot be real in more advanced gestational ages . We believe that this low correlation measurement of the cisterna magna by 3dus and gestational age is due to a greater difficulty in identifying the edges of the structure as it progresses toward the occipital bone . In general, the mean diameters of the transverse and anterior - posterior cerebellum and cisterna magna by 2dus were lower than those obtained by 3dus . In studies evaluating the volume of fetal cerebellum by 3dus, there was also an increase in this parameter with the gestational age, both by multiplanar and virtual organ computer - aided analysis (vocal) methods [1820]. There are no studies evaluating the volume of the fetal cisterna magna by 3dus throughout gestation . Other studies using other softwares such as three - dimensional volume contrast imaging c - plane have shown a positive correlation between the biometrics of the vermis and gestational age [821]. We observed in this study a statistically significant difference in the measurement of transverse and anterior - posterior cerebellum and cisterna magna by 2d and 3dus using the 3d xi (multislice view) software . The program multislice view (samsung - medison, seoul, korea) allows the evaluation of multiple plans of sequential and adjacent images arranged on the screen, being the thickness of the slices determined by the operator . In this study, we evaluated an average of 25 plans with 0.5 mm thickness between them, from the plan of the transverse cerebellar diameter measure to a plan in which the usual outline was lost and the bone of the skull base could be observed . We used as the final value the greatest measure found, unlike the 2dus in which we used a single measure . We believe that the measures taken by 3dus should be more reliable because it corrects any small displacement of the sound beam transducer, which can compromise the 2d measure . However, the biggest inconvenience of this technique, which makes its application in clinical practice difficult, is the long time required to perform the measurements, an average 180 seconds . We noticed in this study an icc value> 0.66 for all length measurements of the fetal cerebellum and cisterna magna through 3dus, except for the intraobserver measure of the cisterna magna (icc = 0.287). Such a result could assume a low reproducibility of the new method; however, by bland - altman plot it was observed that the intraobserver mean difference was similar to the cerebellum and cisterna magna (0.16 and 0.13 mm, resp . ). We believe that the low value of icc is due to the small number of cases we evaluated, and as this is a laborious technique with average rating of 25 plans per volume, with a mean time of 180 seconds, it may have contributed to this low value of icc . However, the real applicability of the method can only be testified in further studies evaluating pathological cases of fetal central nervous system . We believe that the great importance of this study is the evaluation of borderline cases, such as an anterior - posterior diameter of the fetal cisterna magna of 9.0 mm and a transverse cerebellar diameter of 16 mm at 18 weeks of gestational age . In these cases the 3dus with the program multislice view can help in decision making between normal and pathological cases, modifying the prenatal, and counseling of the parents . In summary, this was the first study that compared the measurements of the transverse and antero - posterior fetal cerebellum and cisterna magna diameters by 2d- and 3dus using the 3d xi (multislice view) software . The measurements the length of the fetal cerebellum and cisterna magna by 3dus were significantly higher than those obtained by 2dus . The length measurement of the cisterna magna by 3dus showed low correlation with gestational age . Measures the length of the cerebellum and cisterna magna by 3dus proved to be reproducible.
The protocol for this cross - sectional study was reviewed and approved by the institutional ethical committee for clinical research at the geneva university hospitals . All patients who had received a whole pancreas or an islet of langerhans transplant at the geneva university hospitals and who had regular follow - ups at our institution were asked to participate in the study . Patients were required to have at least partial graft function (as determined by positive c - peptide levels). Fifteen islet transplant recipients were enrolled in the islet group, which was further subdivided into an insulin - independent group (ii - isl) and an insulin - requiring group (ir - isl). Ten healthy nondiabetic volunteers were recruited to serve as control subjects . Finally, five patients with type 1 diabetes awaiting pancreas or islet transplant, all of whom demonstrated no c - peptide production at the time of study inclusion, served as negative control subjects . Written informed consent was obtained from all patients and control subjects prior to their participation in the study . Demographics and population characteristics all samples were obtained from patients during either their annual posttransplantation follow - up or one of their regularly scheduled outpatient consultations . The serum was labeled using the same anonymous codes assigned to the tubes at the time of the test . An arginine stimulation test was performed on all subjects to determine basal and stimulated pancreatic hormone levels . Blood was drawn at 10, 0, 2, 3, 4, 5, 7, and 10 min from intravenous injection of a 5 g arginine bolus . Patients who were back on insulin were required to discontinue slow - acting insulin at least 24 h prior to the test and rapid - acting insulin at least 6 h before the test . Serum levels of insulin, c - peptide, total proinsulin, and intact proinsulin (ipi) were then measured using commercially available elisas as described below . Acute insulin response (air) was calculated as the mean of the three highest values between 2 and 5 min minus the mean of the basal values at 10 and 0 min . The area under the curve (auc) between 0 and 10 min postinjection for insulin and c - peptide was calculated by the trapezoidal rule with the mean of the baseline values subtracted . Proinsulin fragments, including split-32,33 proinsulin, des-31,32 proinsulin, split-65,66 proinsulin, and des-64,65 proinsulin, were obtained by subtracting ipi from total proinsulin . Ratios of insulin to total proinsulin and insulin [insulin/(total proinsulin+insulin)] as well as proinsulin fragments to total proinsulin (proinsulin fragments / total proinsulin) were calculated to quantify the rate of proinsulin processing . Ratios representing peak stimulation were calculated as the mean of ratios for the three highest time points between 2 and 5 min after arginine stimulation . Insulin/(total proinsulin+insulin) ratio represents the quantity of fully processed insulin with respect to all circulating forms of the hormone . Commercially available elisas were used to measure hormone levels for insulin, c - peptide, total proinsulin, and ipi . The following kits were used: insulin (mercodia, uppsala, sweden), c - peptide (mercodia), total proinsulin (millipore, zug, switzerland), and ipi (millipore). Concentrations were determined using a standard curve with high- and low - level control subjects . Cross - reactivities of the assays were provided by the manufacturer and are as follows: the insulin assay cross - reacts 98% with des-64,65 proinsulin, <0.01% with c - peptide and proinsulin, and <0.05% with des-31,32 proinsulin and split-32,33 proinsulin; the c - peptide elisa cross - reacts <0.0006% with insulin, <1.8% with proinsulin, 10% with split-65,66 proinsulin, 74% with des-64,65 proinsulin, 3% with des-31,32 proinsulin, and 2% with split-32,33 proinsulin; the total proinsulin elisa cross - reacts 100% with intact and des-31,32 proinsulin, and 81% with des-64,65 proinsulin; the ipi elisa cross - reacts 36% with des-64,54 proinsulin, and it shows no cross - reactivity with des-31,32 proinsulin . The lower limits of detection of the assays were provided by the manufacturer and are as follows: the insulin assay has a detection limit of 6 pmol / l, the total proinsulin assay has a limit of 0.5 pmol / l, and the ipi assay has a lower limit of 0.1 pmol / l . All statistical analyses were performed using prism 6 software (graphpad software, la jolla, ca). For all analyses, values obtained from healthy subjects were used as control subjects . For data that were normally distributed, student t test was used to compare two groups, and one - way anova was used to compare between more than two groups . For data that had non - gaussian distributions, we used the mann - whitney test to compare between two groups and the kruskal - wallis test to compare between more than two groups . All samples were obtained from patients during either their annual posttransplantation follow - up or one of their regularly scheduled outpatient consultations . The serum was labeled using the same anonymous codes assigned to the tubes at the time of the test . An arginine stimulation test was performed on all subjects to determine basal and stimulated pancreatic hormone levels . Blood was drawn at 10, 0, 2, 3, 4, 5, 7, and 10 min from intravenous injection of a 5 g arginine bolus . Patients who were back on insulin were required to discontinue slow - acting insulin at least 24 h prior to the test and rapid - acting insulin at least 6 h before the test . Serum levels of insulin, c - peptide, total proinsulin, and intact proinsulin (ipi) were then measured using commercially available elisas as described below . Acute insulin response (air) was calculated as the mean of the three highest values between 2 and 5 min minus the mean of the basal values at 10 and 0 min . The area under the curve (auc) between 0 and 10 min postinjection for insulin and c - peptide was calculated by the trapezoidal rule with the mean of the baseline values subtracted . Proinsulin fragments, including split-32,33 proinsulin, des-31,32 proinsulin, split-65,66 proinsulin, and des-64,65 proinsulin, were obtained by subtracting ipi from total proinsulin . Ratios of insulin to total proinsulin and insulin [insulin/(total proinsulin+insulin)] as well as proinsulin fragments to total proinsulin (proinsulin fragments / total proinsulin) were calculated to quantify the rate of proinsulin processing . Ratios representing peak stimulation were calculated as the mean of ratios for the three highest time points between 2 and 5 min after arginine stimulation . Insulin/(total proinsulin+insulin) ratio represents the quantity of fully processed insulin with respect to all circulating forms of the hormone . Commercially available elisas were used to measure hormone levels for insulin, c - peptide, total proinsulin, and ipi . The following kits were used: insulin (mercodia, uppsala, sweden), c - peptide (mercodia), total proinsulin (millipore, zug, switzerland), and ipi (millipore). Concentrations were determined using a standard curve with high- and low - level control subjects . Cross - reactivities of the assays were provided by the manufacturer and are as follows: the insulin assay cross - reacts 98% with des-64,65 proinsulin, <0.01% with c - peptide and proinsulin, and <0.05% with des-31,32 proinsulin and split-32,33 proinsulin; the c - peptide elisa cross - reacts <0.0006% with insulin, <1.8% with proinsulin, 10% with split-65,66 proinsulin, 74% with des-64,65 proinsulin, 3% with des-31,32 proinsulin, and 2% with split-32,33 proinsulin; the total proinsulin elisa cross - reacts 100% with intact and des-31,32 proinsulin, and 81% with des-64,65 proinsulin; the ipi elisa cross - reacts 36% with des-64,54 proinsulin, and it shows no cross - reactivity with des-31,32 proinsulin . The lower limits of detection of the assays were provided by the manufacturer and are as follows: the insulin assay has a detection limit of 6 pmol / l, the c - peptide elisa has a limit of 15 pmol / l, the total proinsulin assay has a limit of 0.5 pmol / l, and the ipi assay has a lower limit of 0.1 pmol / l . All statistical analyses were performed using prism 6 software (graphpad software, la jolla, ca). For all analyses, values obtained from healthy subjects were used as control subjects . For data that were normally distributed, student t test was used to compare two groups, and one - way anova was used to compare between more than two groups . For data that had non - gaussian distributions, we used the mann - whitney test to compare between two groups and the kruskal - wallis test to compare between more than two groups . Subjects in the healthy control group were slightly younger than the rest of the study population (p <0.01). Islet recipients exhibited higher fasting glycemia and hba1c levels than other groups (p <0.05). Nonetheless, hba1c levels remained in the normal range for the ii - isl group . Creatinine levels were elevated in the pancreas and ir - isl groups with respect to the ii - isl and healthy control groups (p <0.005 vs. control, p <0.05 vs. ii - isl). Figure 1a and b shows the insulin and c - peptide response curves for all five groups . The insulin and c - peptide response curves maintain the same overall shape in all groups, with the exception of the diabetic control subjects who demonstrate no response to arginine stimulation . Insulin levels were higher in pancreas recipients but lower in islet recipients compared with healthy control subjects . Insulin levels in the pancreas group were significantly higher than those of the healthy control group . Airs for healthy control subjects, ir - isl, ii - isl, and pancreas groups were 217 163, 23 13, 125 90, and 266 the c - peptide acute responses in the same groups were 717 362, 91 53, 278 124, and 550 244 pmol / l, respectively (supplementary table 1). Mean acute c - peptide responses were similar in the healthy control group and pancreas group but significantly lower in the islet groups . The ir - isl transplantees showed a significantly decreased acute insulin and c - peptide response with respect to all other groups . There was no significant difference in air between the ii - isl and control groups . As expected, the diabetic control group showed negligible insulin and c - peptide levels . A: mean insulin levels during arginine stimulation of healthy control group, ir - isl group, ii - isl group, pancreas group, and diabetic control group . B: mean c - peptide levels during arginine stimulation of healthy control group, ir - isl group, ii - isl group, pancreas group, and diabetic control group . Auc values demonstrated similar distributions to acute responses in the various study groups (supplementary table 1). Auc values for insulin response in healthy control subjects, ir - isl, ii - isl, and pancreas groups were 1,332 959, 152 91, 774 528, and 1,540 707 pmol these values were higher in pancreas recipients and lower in islet recipients compared with healthy control subjects . Auc values for c - peptide response in healthy control subjects, ir - isl, ii - isl, and pancreas groups were 4,955 3,158, 630 428, 1,843 875, and 3,859 2,061 pmol the auc of c - peptide for the ir - isl group was significantly lower than in all other groups . Mean values for c - peptide and total proinsulin before and after arginine stimulation are shown in fig . These figures show that total proinsulin levels are reduced in the ir - isl group with respect to all other groups and follow a distribution similar to that of c - peptide . A: box plots of basal c - peptide and total proinsulin levels in healthy control group, ir - isl group, ii - isr group, and pancreas group before arginine injection (basal values). B: box plots of stimulated c - peptide and total proinsulin levels in healthy control group, ir - isl group, ii - isl group, and pancreas group during peak stimulation after arginine injection (stimulated values). Data are expressed as median (solid line), interquartile range (box), and range (whiskers). The healthy control group demonstrated lower basal proinsulin - processing rates than the pancreas (p <0.01) and ii - isl (p <0.05) groups [74 14% vs. 83 7% vs. 86 12%, respectively, for insulin/(total proinsulin+insulin) and 54 25% vs. 67 18% vs. 76 16%, respectively, for proinsulin fragments / total proinsulin]. The ir - isl group displayed a basal insulin/(total proinsulin+insulin) rate of 75 14% and a basal proinsulin fragments / total proinsulin rate of 54 25% . A: box plots of the proinsulin - processing ratios [(insulin / total proinsulin+insulin) and (proinsulin fragments / total proinsulin)] in healthy control group, ir - isl group, ii - isl group, and pancreas group before arginine injection (basal values). B: box plots of the proinsulin - processing ratios [(insulin / total proinsulin+insulin) and (proinsulin fragments / total proinsulin)] in healthy control group, ir - isl group, ii - isl group, and pancreas group during peak stimulation after arginine injection (stimulated values). Data are expressed as median (solid line), interquartile range (box), and range (whiskers). I / tp+i, insulin / total proinsulin+insulin; pf / tp, proinsulin fragments / total proinsulin . 3b . Interestingly, healthy control subjects, pancreas recipients, and ii - isl recipients all attain similarly elevated processing ratios after stimulation (93 2% vs. 93 3% vs. 93 5%, respectively, for insulin/(total proinsulin+insulin) and 80 7% vs. 77 9% vs. 76 12%, respectively, for proinsulin fragments / total proinsulin), while ir - isl transplant recipients ratios do not demonstrate a comparable increase . Although ir - isl proinsulin - processing ratios do increase slightly after stimulation to 87 4% and 58 18% for insulin/(total proinsulin + insulin) and proinsulin fragments / total proinsulin, respectively, these values remain significantly lower than all other groups (p <0.01 vs. control and pancreas groups, p <0.05 vs. ii - isl). Subjects in the healthy control group were slightly younger than the rest of the study population (p <0.01). Islet recipients exhibited higher fasting glycemia and hba1c levels than other groups (p <0.05). Nonetheless, hba1c levels remained in the normal range for the ii - isl group . Creatinine levels were elevated in the pancreas and ir - isl groups with respect to the ii - isl and healthy control groups (p <0.005 vs. control, p <0.05 vs. ii - isl). Figure 1a and b shows the insulin and c - peptide response curves for all five groups . The insulin and c - peptide response curves maintain the same overall shape in all groups, with the exception of the diabetic control subjects who demonstrate no response to arginine stimulation . Insulin levels were higher in pancreas recipients but lower in islet recipients compared with healthy control subjects . Insulin levels in the pancreas group were significantly higher than those of the healthy control group . Airs for healthy control subjects, ir - isl, ii - isl, and pancreas groups were 217 163, 23 13, 125 90, and 266 129 pmol / l, respectively . The c - peptide acute responses in the same groups were 717 362, 91 53, 278 124, and 550 244 pmol / l, respectively (supplementary table 1). Mean acute c - peptide responses were similar in the healthy control group and pancreas group but significantly lower in the islet groups . The ir - isl transplantees showed a significantly decreased acute insulin and c - peptide response with respect to all other groups . There was no significant difference in air between the ii - isl and control groups . As expected, the diabetic control group showed negligible insulin and c - peptide levels . A: mean insulin levels during arginine stimulation of healthy control group, ir - isl group, ii - isl group, pancreas group, and diabetic control group . B: mean c - peptide levels during arginine stimulation of healthy control group, ir - isl group, ii - isl group, pancreas group, and diabetic control group auc values demonstrated similar distributions to acute responses in the various study groups (supplementary table 1). Auc values for insulin response in healthy control subjects, ir - isl, ii - isl, and pancreas groups were 1,332 959, 152 91, 774 528, and 1,540 707 pmol these values were higher in pancreas recipients and lower in islet recipients compared with healthy control subjects . Auc values for c - peptide response in healthy control subjects, ir - isl, ii - isl, and pancreas groups were 4,955 3,158, 630 428, 1,843 875, and 3,859 2,061 pmol the auc of c - peptide for the ir - isl group was significantly lower than in all other groups . Mean values for c - peptide and total proinsulin before and after arginine stimulation are shown in fig . These figures show that total proinsulin levels are reduced in the ir - isl group with respect to all other groups and follow a distribution similar to that of c - peptide . A: box plots of basal c - peptide and total proinsulin levels in healthy control group, ir - isl group, ii - isr group, and pancreas group before arginine injection (basal values). B: box plots of stimulated c - peptide and total proinsulin levels in healthy control group, ir - isl group, ii - isl group, and pancreas group during peak stimulation after arginine injection (stimulated values). Data are expressed as median (solid line), interquartile range (box), and range (whiskers). The healthy control group demonstrated lower basal proinsulin - processing rates than the pancreas (p <0.01) and ii - isl (p <0.05) groups [74 14% vs. 83 7% vs. 86 12%, respectively, for insulin/(total proinsulin+insulin) and 54 25% vs. 67 18% vs. 76 16%, respectively, for proinsulin fragments / total proinsulin]. The ir - isl group displayed a basal insulin/(total proinsulin+insulin) rate of 75 14% and a basal proinsulin fragments / total proinsulin rate of 54 25% . A: box plots of the proinsulin - processing ratios [(insulin / total proinsulin+insulin) and (proinsulin fragments / total proinsulin)] in healthy control group, ir - isl group, ii - isl group, and pancreas group before arginine injection (basal values). B: box plots of the proinsulin - processing ratios [(insulin / total proinsulin+insulin) and (proinsulin fragments / total proinsulin)] in healthy control group, ir - isl group, ii - isl group, and pancreas group during peak stimulation after arginine injection (stimulated values). Data are expressed as median (solid line), interquartile range (box), and range (whiskers). I / tp+i, insulin / total proinsulin+insulin; pf / tp, proinsulin fragments / total proinsulin . 3b . Interestingly, healthy control subjects, pancreas recipients, and ii - isl recipients all attain similarly elevated processing ratios after stimulation (93 2% vs. 93 3% vs. 93 5%, respectively, for insulin/(total proinsulin+insulin) and 80 7% vs. 77 9% vs. 76 12%, respectively, for proinsulin fragments / total proinsulin), while ir - isl transplant recipients ratios do not demonstrate a comparable increase . Although ir - isl proinsulin - processing ratios do increase slightly after stimulation to 87 4% and 58 18% for insulin/(total proinsulin + insulin) and proinsulin fragments / total proinsulin, respectively, these values remain significantly lower than all other groups (p <0.01 vs. control and pancreas groups, p <0.05 vs. ii - isl). This study explores for the first time proinsulin processing in islet transplant recipients, not only in comparison with healthy control subjects, but also with respect to pancreas transplant recipients . We have shown, first, that the exposure to arginine stimulation is in fact similar in ii - isl transplant recipients and healthy control subjects . As expected, recipients of pancreas transplants with systemic venous drainage demonstrate higher insulin response to arginine than healthy control subjects . Second, there is a noticeable difference in the rate of basal proinsulin processing comparing ii - isl and ir - isl transplant recipients . Ii - isl patients have proinsulin - processing ratios similar to those of pancreas transplant recipients, which are significantly higher than those of healthy control and ir - isl patients . Third, after stimulation, ii - isl, pancreas, and healthy control subjects all increase their proinsulin - processing ratios to a similar level . In contrast, ir - isl patients are unable to achieve the same increase in proinsulin - processing rate . These data suggest that fully functional islet grafts are able to attain optimal proinsulin processing when stimulated but are required to sustain high levels of processing even during a metabolic fasting state in order to maintain glycemic control . Islet grafts requiring supplementation with insulin therapy, on the other hand, demonstrate low basal proinsulin - processing rates, which are unable to reach optimal levels even when stimulated . Interestingly, total proinsulin levels in this group (ir - isl) are low compared with other groups both before and after stimulation . However, the ratio of proinsulin to insulin in the ir - isl group after stimulation is disproportionately high . This suggests that partially functional islet grafts have a defect in proinsulin processing, which manifests itself as relative and not absolute hyperproinsulinemia . It should be noted that the calculation of proinsulin fragments may be slightly biased owing to elisa cross - reactivities . In theory, when we calculate proinsulin fragments as proinsulin fragments = total proinsulin ipi, we are assuming that all of the des-64,65 proinsulin has been measured by the total proinsulin assay and that none of it has been measured by the intact assay . In reality, we measure only 81% of des-64,65 with the total proinsulin assay and 36% with the ipi assay . . However, since the pc2 pathway responsible for the formation of des-64,65 proinsulin is not the primary pathway for the production of insulin, unmeasured levels of des-64,65 proinsulin are relatively insignificant with respect to des-31,32 proinsulin levels . It should also be noted that clearance of proinsulin and proinsulin fragments could be altered by diminished renal function . As seen in table 1, our ir - isl group demonstrated creatinine levels that were elevated compared with healthy control subjects . However, this difference was not significant comparing the ir - isl group to ii - isl and pancreas groups . In contrast, the reduced processing ratios observed in the ir - isl group are significantly lower than all other groups . Both absolute and relative hyperproinsulinemia have already been observed in settings of impaired glucose metabolism (1214). First, it has been suggested that dysfunctional -cells present a fundamental defect in the pathway responsible for processing of proinsulin to insulin (18). It is possible in the case of islet transplantation that manipulation of islets, introduction of -cells into a foreign microenvironment, or immunosuppressive drugs may render these cells inherently dysfunctional . A second theory suggests that hyperproinsulinemia occurs because of increased demand on -cells, which leads to insufficient time to complete proinsulin processing intracellularly before granule secretion occurs (15,19,20). It seems reasonable to suppose that a combination of insulin resistance and decreased -cell mass could lead to unattainable insulin needs and eventual -cell deterioration via exhaustion . Our data show that islet grafts initially present increased basal proinsulin processing with respect to healthy control subjects and that this processing deteriorates as graft function declines, a phenomenon that more readily supports the latter theory . We cannot, however, exclude the effect of prolonged glucotoxicity on -cell function as an additional possible explanation for the observed processing defects . Although our two islet groups had similar fasting glucose levels, hba1c was slightly increased in the ir - isl group, indicating that chronic glucotoxicity may have played a role in the functional deterioration of -cells within this subgroup . Ultimately, a longitudinal study on the same or similar populations might help to verify this hypothesis . Two studies have previously investigated proinsulin processing in islet transplant recipients (16,22). However, neither study used arginine stimulation to explore hormonal processing when under simulated metabolic stress and neither study compared islet and pancreas transplant recipients . It is interesting to note that these studies produced conflicting data and presented contrasting conclusions . We found that functional islet grafts display increased processing and relatively lower proinsulinemia at rest, with an inability to increase their processing rates further when stimulated . These grafts are essentially behaving the same during the metabolic fasting state as fully stimulated healthy control subjects . As islet grafts lose function, however, processing even during the fasting state becomes less effective and proinsulinemia increases . These findings directly correlate with the findings of mcdonald et al . And are supported by the data of klimek at al ., which include mostly ir - isl transplant recipients . Two other groups, fiorina et al . It would seem that there is a threshold level of demand beyond which -cells can no longer process proinsulin effectively . As stated earlier, we presume this to be due to an increased demand on decreased -cell mass . Further investigation is warranted to explain the exact mechanism responsible for the increased basal proinsulin processing seen in ii - isl grafts . Again, a longitudinal study of these patients might help shed more light on the issue . No study previously analyzed the stimulated hormonal response of pancreas grafts with respect to islet grafts . Our data show that pancreas grafts behave quite similarly to healthy control subjects but with elevated overall hormone levels . This is undoubtedly a reflection of the systemic venous pancreatic drainage of these patients, in whom there is no hepatic first - pass metabolism of pancreatic hormonal secretions (23). Pancreas grafts tend to show similar patterns of proinsulin processing to fully functional islet grafts . Whether this is peculiar to systemically drained pancreata cannot be elucidated from this data . This study used arginine stimulation rather than oral or intravenous glucose tolerance tests to test secretory reserve in our patient populations . Arginine stimulation provides the advantage of not inducing hyperglycemia, thus reducing the confounding influence of glucotoxicity to a minimum . Other investigators have found the intravenous glucose tolerance tests to be more robust and a better indicator of -cell status (24), but this method does not account for host insulin resistance . In summary, ii - isl transplant recipients can maintain basal metabolic parameters similar to healthy control subjects at the cost of a higher rate of basal proinsulin processing . Ir - isl transplant recipients demonstrate both lower insulin response and lower basal rates of proinsulin processing, which remain suboptimal even after arginine stimulation (i.e., loss of graft function is associated with less effective processing and relative hyperproinsulinemia). Finally, the higher airs of pancreas transplant recipients are a reflection of systemic venous drainage of endocrine secretions.
We have demonstrated that, melatonin is a bioprecursor of hypnotic acetyl metabolites produced by enzymatic acetylation of melatonin and 2-oxomelatonin under the control of acetyltransferases, most probably the nat enzymes . In 1994, in our laboratory, we developed a specific and highly sensitive gas chromatography - mass spectrometry (gc - ms) method to assay, simultaneously and distinct! ), plasma concentrations of endogenous melatonin (d0melatonin) and exogenous melatonin (d7-melatonin), in which 7 atoms of h have been substituted by 7 atoms of deuterium . Using the same human volunteers (12 young subjects in june 1994 and 12 elderly subjects in october 1994), we determined the pharmacokinetics of exogenous d7-melatonin, when given orally and intravenously, and the kinetics of the pineal secretion of endogenous d0-melatonin ., the results shown in figure 2 led to the following conclusions: secretion of melatonin by the pineal gland occurs only during the night . Pharmacokinetic analysis shows that the rate of melatonin secretion by the pineal gland is constant throughout the whole nocturnal pineal melatonin production, for the same subject . The beginning and end of melatonin secretion from the pineal gland are the same for each subject, whatever the season and night, length . Duration of melatonin pineal secretion is between 7.5 and 8 h. therefore, melatonin secretion and sleep are contemporaneous . There is a large interindividual variability in the amount, of melatonin released in plasma by the pineal gland during the night in young and old subjects alike . Results of previous related studies show that melatonin secretion, and therefore the presence of melatonin in the central nervous system (cns), is necessary for the induction and maintenance of nocturnal sleep . However, the presence of melatonin in the cns is insufficient for the induction and maintenance of sleep . Indeed, figure 3 and table i show results of observations in chicks in an alternate light (l)-dark (d) program (l / d, 12 h:12 h), in which the light phase lasted from 8.00 am until 8.00 pm . When melatonin was administered intramuscularly (pectoralis major muscle) during the light phase from 2.00 pm to 8.00 pm, the chicks did not . The absence of a hypnotic effect during the light phase correlated with the very low level of nat activity in the pineal glands of chicks measured at the same times . In contrast, when chicks were observed in a 7-day permanent light, program (l / l, 12 h:12 h), during which nat activity level was constantly higher, the administration of melatonin induced a significant, hypnotic effect . The duration of sleep (between 4 and 5 h) was much greater than that, observed with diazepam (between 1 and 2 h) when it . Was administered intramuscularly at the same dose (1 m per 100 g body weight, at 2.00 pm). These results lead to the following conclusions: the simultaneous presence of melatonin and nat in the cns (pineal gland) is a necessary and sufficient condition for the induction and maintenance of sleep . In contrast to the classic so - called hypnotic drugs (eg, benzodiazepines, barbiturates, zopiclone, and zolpidem), melatonin does not have direct, hypnotic properties related to its chemical structure . During the development of the gc - ms method for the assay of melatonin in plasma, our attention was focussed on the chemical reactivity of melatonin at position 3, which allows cyciization of the side chain after acylation . This proceeds by nucleophilic attack and leads to a fluoroacyl--carboline (figure 4). Considering our previous observations, we assumed that melatonin undergoes enzymatic acetylation during the night, under the control of nat, and that this leads to an n - acetyl--carboline, which we call carbo2 . We conclude that melatonin is a bioprecursor of hypnotic acetyl metabolites, such as carbo2 . Chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . In the middle of dark phase, they were treated with phjacetyl coenzyme a and melatonin (or 2-oxomelatonin) for 30 min . Figure 5 and figure 6 show that melatonin (or 2-oxomelatonin) undergoes an aeetylation that is significantly higher (p<0.002, in the middle of dark phase; p<0.0005, 1 h before end of dark phase [or p<0.00005 for 2oxomelatonin over the whole dark phase]) than that observed in controls (nonsignificant when melatonin was replaced by phosphate buffer). Gc - ms indicated the biosynthesis of [h]carbo2 for five chick pineal glands collected in the middle of dark phase (table ii). We have synthesized several acetyl derivatives, such as carbo2 (figure 7), which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program . The hypnotic activity of carbo2 has been observed and measured in chicks and beagles: in chicks, the tests were performed at 2.00 pm, in the middle of light phase, a time at which nat activity in the pineal gland is very low . The results are presented in table iii, together with some reference compounds . The essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan (as well as harmaline), which is the product of jv - deacetylation of compound carbo2, does not exhibit any hypnotic effect . In contrast, it induces excitatory effects in chicks by increasing locomotor activity in beagles, polysomnographic studies showed that when carbo2 was administered intravenously, it induced sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam (table iv). The most interesting feature, which provides more support for our assumption, is the eeg architecture of the sleep produced, which is similar to that of physiological sleep (see results with placebo in table iv), characterized by the significant proportion of slow - wave deep sleep and rapid eye movement (rem) sleep, in sharp contrast to the eeg sleep architecture observed with gabaergic (gaba, -aminobutyric acid) compounds, such as zolpidem or diazepam, which induce mainly drowsiness (light sleep) and little rem sleep . Chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . In the middle of dark phase, they were treated with phjacetyl coenzyme a and melatonin (or 2-oxomelatonin) for 30 min . Figure 5 and figure 6 show that melatonin (or 2-oxomelatonin) undergoes an aeetylation that is significantly higher (p<0.002, in the middle of dark phase; p<0.0005, 1 h before end of dark phase [or p<0.00005 for 2oxomelatonin over the whole dark phase]) than that observed in controls (nonsignificant when melatonin was replaced by phosphate buffer). Gc - ms indicated the biosynthesis of [h]carbo2 for five chick pineal glands collected in the middle of dark phase (table ii). We have synthesized several acetyl derivatives, such as carbo2 (figure 7), which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program . The hypnotic activity of carbo2 has been observed and measured in chicks and beagles: in chicks, the tests were performed at 2.00 pm, in the middle of light phase, a time at which nat activity in the pineal gland is very low . The essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan (as well as harmaline), which is the product of jv - deacetylation of compound carbo2, does not exhibit any hypnotic effect . In contrast, it induces excitatory effects in chicks by increasing locomotor activity in beagles, polysomnographic studies showed that when carbo2 was administered intravenously, it induced sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam (table iv). The most interesting feature, which provides more support for our assumption, is the eeg architecture of the sleep produced, which is similar to that of physiological sleep (see results with placebo in table iv), characterized by the significant proportion of slow - wave deep sleep and rapid eye movement (rem) sleep, in sharp contrast to the eeg sleep architecture observed with gabaergic (gaba, -aminobutyric acid) compounds, such as zolpidem or diazepam, which induce mainly drowsiness (light sleep) and little rem sleep . We have evidenced the role played by melatonin in both inducing and maintaining nocturnal sleep . Melatonin is the bioprecursor of hypnotic acetyl metabolites, such as carbo2, which result from the enzymatic acetylation of melatonin (and 2-oxomelatonin) by nat . Since insomnia and sleep disorders may be due to a lack of nat enzymes in the pineal gland, a therapeutic approach to sleep disorders could be suggested . Patients with insomnia may be treated by administering hypnotic acetyl metabolites of melatonin or their synthetic analogs.
Over the next several decades, the number of americans living to advanced ages will increase substantially . Although many individuals will age in relatively good health, a growing number will encounter challenges associated with the burdens of chronic conditions and associated disabilities [13]. This is especially so for the large numbers of women who will continue to outlive their male counterparts and likely live those additional years with chronic illnesses requiring day - to - day management [4, 5]. Further, with a dramatic increase of female baby boomers with obesity - related chronic conditions, accompanied by reduced fertility rates among this rapidly aging cohort, the additive or multiplicative effects of living with one or more chronic conditions are likely to result in a diminution of (1) individuals' capacity to adequately care for themselves, (2) caregivers to serve as efficient resources, and (3) healthcare providers to give adequate attention and guidance to complex patients with multiple chronic conditions (mccs). In line with the millions of older women struggling to manage the symptoms associated with chronic disease, there is growing recognition about the importance of self - care behavior, which is supported by strong epidemiological documentation regarding the positive association of self - care and health outcomes [710]. However, this issue transcends women's exposure to and understanding of pertinent information and their development of self - care skills . Older women with chronic conditions also need to assess their surrounding resources to develop the confidence and efficacy necessary to initiate and maintain self - care behavior [1113]. Self - care behavior are intended to draw upon one's physical, social, and healthcare environments to compensate for, or delay, physical limitations and chronic conditions from progressing into more severe disabilities . Further, self - care skills include (1) identifying strategies that enable older women to adopt and institute appropriate self - care behavior such as getting adequate exercise, eating healthy, or managing medications [15, 16]; (2) interacting with healthcare providers to obtain resources and referrals necessary to manage the progression of chronic conditions [10, 17, 18]; and (3) locating social and community resources to become more educated about their conditions and alleviate the stressors and frustration acting as barriers to self - care behavior [6, 19]. While a growing literature has identified general disparities related to self - care among women with chronic conditions based on their race / ethnicity [2022], socioeconomic status [2224], and residential rurality [22, 25], the extent to which self - care disparities exist based on these and other sociodemographics requires further investigation . Similarly, the role of education an indicator of socioeconomic status, healthcare access, and health behavior engagement emphasizes its importance when assessing disparities issues [26, 27]. Additional efforts are needed to examine the influence of sociodemographics on self - care skills and behavior among aging women, especially in the presence of perceptions about healthcare - related factors . Healthcare provider - patient interactions can foster self - care behavior although such interactions can also have a less - recognized negative effect on disease self - management . Women can feel frustrated and helpless when their physicians do not fully explain or clarify the causes of their disease or ways how to best manage their illness . Some patients report not having enough time to address their concerns, or that their physicians simply would not listen to them . Conversely, among patients with diabetes, those who have good communication with their physicians report feeling more involved in decision - making efforts to manage their condition more effectively . Also, patients who report their physicians provided adequate information about their conditions were more likely to better self - manage their illnesses . Healthcare professionals often encounter challenges to address their female patients' mcc alongside existing barriers to self - care behavior in their home or community environments . Regardless of the source or cause of these barriers, competing demands on the side of either the patient or provider have potential to create a recursive relationship resulting in disconnect, miscommunication, frustration, and fewer self - care practices . These occurrences may inevitably contribute to decreased health outcomes and rapid chronic disease progression, which highlights the importance of support mechanisms available to the patient . Effective self - care support mechanisms and resources identified to promote self - care behavior include traditional in - person, familial, and community support systems (e.g., support groups and faith - based organizations) [6, 3135]; however, an emergence in technology - based support mechanisms has been shown to enable individuals with chronic conditions to access and utilize self - care information, despite traditional barriers [18, 3639]. Further, the active seeking of self - care support and resources has been shown to enhance self - care behavior among individuals with chronic conditions [12, 38, 40]. The preferred and/or utilized support mechanisms differ by population subgroup and type of chronic condition . Evidence shows that racial / ethnic minorities with chronic conditions report increased in - person support mechanisms compared to their non - hispanic white counterparts [41, 42]. Conversely, those residing in rural areas have shown improvement in their self - care behavior through the use of internet - based support mechanisms, which may be critical to overcome traditional challenges associated with geographic isolation, less healthcare resources, and longer travel distances to healthcare services [25, 44]. The advancing study of self - care behavior has identified factors influencing the adoption and maintenance of self - care behavior for different populations and people of all ages, with more recent attention paid to lifestyle relative to disease self - care behavior [17, 45, 46]. To advance the translation of research to practice, this secondary data analysis assesses issues surrounding self - care barriers, healthcare - related frustrations, and perceived supports among middle - aged and older adults with one or more chronic conditions . In an effort to further understand the multilevel influences on perceived barriers to self - care, this study will examine the roles of healthcare frustrations and doctor - patient interactions alongside other simultaneously occurring contextual factors (see figure 1). More specifically, the purposes of this study were to (1) describe sociodemographic variables, health indicators, healthcare - related frustrations, and perceptions of physician support among middle - aged and older adult women with one or more chronic conditions and (2) identify these factors' association with reporting the need to help learning how to take better care of their health among this female population . The national council on aging (ncoa), with support from atlantic philanthropies and the california healthcare foundation (chcf), commissioned the ncoa chronic care survey, which offers unique insight into the lives of americans with chronic health conditions . The ncoa chronic care survey is a nationally representative probability survey of 960 community - dwelling men and women aged 44 years and older with at least one chronic condition . Lake research partners utilized telephone - based interviewing to collect data using random digit dialing (rdd) sampling techniques, which oversampled those aged 65 and older and hispanics / latinos . The dataset was weighted by age, race, and region to reflect the overall population of americans 44 + with chronic condition(s). Margin of error is greater when analyzing smaller subgroups within the sample . To be eligible for inclusion in the ncoa chronic care survey, participants had to report having at least one chronic condition at the time of the study . Participants were screened for chronic condition(s) with the following question(s): have you ever been told by a doctor, nurse, or other health professional that you have (name of chronic condition)? Chronic conditions included in the screening process included heart disease, cancer, stroke, diabetes, arthritis, asthma, hypertension or high blood pressure, emphysema, chronic bronchitis, depression, anxiety, and others . Only participants who reported yes to at least one of these items of the 960 adults age 44 years and older in this sample, only women were included in study analyses (n = 427; 44.5%). Of these women, an additional 140 cases (32.8%) more specifically, cases were omitted for incomplete data on rurality (n = 48), frustration with the healthcare system (n = 25), perceived physician support (n = 18), self - reported chronic conditions (n = 17), marital status (n = 8), using the internet for general support (n = 7), race / ethnicity (n = 6), education (n = 4), perceived barriers to self - care (n = 4), and activity limitations (n = 3). The analytic sample for this study contained 287 community - dwelling women, aged 44 years and older who self - reported having at least one chronic condition . When comparing characteristics of women omitted from the study (n = 140) with those in the analytic sample (n = 287), a significantly larger proportion of the analytic sample was younger (= 4.98, p = 0.026), non - hispanic white (= 4.60, p = 0.032), and married (= 11.78, p = 0.001). Participants were asked to self - report their perceived barriers to self - care using an item intended to measure their need for help to learn how to take better care of their health . More specifically, participants were asked to rate their level of agreement to the following statement: i need help learning how to take better care of my health in a way that works for me and my life . Responses were scored on a 5-point likert - type scale ranging from strongly disagree to strongly agree . Based on the frequency distribution, participant responses were then dichotomized into two categories: disagree (scored 0) and agree (scored 1). Participants were asked to report their frustrations with healthcare interactions using items intended to measure their feelings about repeatedly having to describe their conditions at each doctor visit, the self - care instructions they received from the healthcare provider, the time spent interacting with the healthcare provider, and having a friend or family member attend physician's visits with them . For example, participants were asked to rate their level of agreement to statements like how often do you feel tired of describing your same conditions and problems every time you go to a hospital or doctor's office? And how often do you wish you had a friend or family member who could go to the doctor with you? Responses were scored using a 3-point likert - type scale with categories of never (scored 0), occasionally the healthcare - related frustration scale (ranging from 0 to 12) was created using these six items . All items loaded on one factor and the items were summed into a single - composite score (= 0.766). Higher scores for the healthcare - related frustration scale indicate a higher level of frustration with healthcare interactions . Based on the frequency distribution, the highest tertile (i.e., representing the highest frustration levels) served as the referent group . Participants were asked to self - report the degree to which their physician engages them in treatment - related problem - solving / decision - making, refers them to other healthcare services and professionals, and asks if they understand their medications and associated regimens . For example, participants were asked to rate their level of agreement to statements like how often does your physician ask for your ideas about how you can take care of your health problems? And how often does your physician talk to other doctors and nurses who are taking care of you? Responses were scored using a 5-point likert - type scale with categories of never (scored 1), rarely (scored 2), occasionally (scored 3), frequently (scored 4), and always (scored 5). The perceived physician support scale (ranging from 6 to 30) was created using these six items . All items loaded on one factor and the items were summed into a single - composite score (= 0.776). Higher scores for the perceived physician support scale indicate a higher level of perceived physician support . Based on the frequency distribution, this scale was converted into tertiles for the analytic purposes . The lowest tertile (i.e., representing the lowest perceived support levels) served as the referent group . Participants were asked to self - report aspects of their current health status using items intended to measure activity limitations, the number of prescription medications taken regularly each day, and the number of physician visits in the previous 12 months . Participants were asked are you limited in any way in any activities because of physical, mental, or emotional problems? Responses were scored as no (scored 0) or yes (scored 1). Participants were also asked in the past 12 months, how many times have you, yourself made a doctor visit? Participants were asked to self - report their perceptions about their use of the internet for general support related to managing their health problems . Participants were asked how much do you rely on the internet for ongoing help and support with your health problems? Responses were scored on a 4-point likert - type scale with categories of not at all (scored 0), a little (scored 1), some (scored 2), and a lot based on the frequency distribution, participant responses were then dichotomized into two categories: no (scored 0; indicating that they do not rely on the internet at all) and yes (scored 1; indicating that they rely on the internet at least a little). Participants were also asked how often do you feel you get the help and support you need to improve your health and manage your health problems? Responses were scored using a 5-point likert - type scale with categories of never (scored 0), rarely (scored 1), occasionally (scored 2), frequently (scored 3), and always sociodemographic variables in this study included age (i.e., 4464 years, 65 + years); race / ethnicity (i.e., non - hispanic white, non - white); education level (i.e., high school or less, some college or more); marital status (i.e., unmarried, married); and residential rurality (i.e., urban, suburban, and rural). Frequencies were calculated for all major study variables, which were initially examined in relationship to participants' age group (4464 years, 65 + years) and whether they reported needing help learning how to take better care of their health (yes or no). Pearson's tests were performed to assess the independence between the dependent variable and categorized independent variables . Logistic regression was performed to examine how sociodemographics, health indicators, perceived support, and frustrations were associated with reporting the need to help learning how to take better care of their health (i.e., not needing help served as the referent group). Sample characteristics of study participants are presented in table 1 . Of the 287 females participating in this study, over 65% of participants were between the ages of 44 and 64 years and 34.4% were aged 65 years and older . Respondents were disproportionately non - hispanic white (88.5%), married (69.0%), and had an education level of some college or more (61.7%). Fifty percent of the study population resided in suburban areas, 25.2% resided in urban areas, and 24.8% resided in rural areas . Approximately 33% of participants reported being limited from activities because of physical, mental, or emotional problems . On average, participants reported taking 3.67 (3.83) medications daily and visiting a physician 3.07 (1.94) times in the previous 12 months . Over 45% of participants reported relying on the internet for ongoing help and support to manage their health problems, and 67% of participants reported frequently or always getting the help and support they need to improve their health and manage their health problems . Compared to women aged 65 years and older, a significantly larger proportion of participants aged 4464 years had some college education or more (= 7.14, p = 0.008) and relied on the internet for ongoing support to manage their health problems (= 21.15, p <0.001). A significantly larger proportion of participants who reported needing help learning how to better care for their health problems were non - white (= 4.78, p = 0.029) and had a high school education or less (= 4.23, p = 0.040). Healthcare - related frustration scale characteristics are presented in table 2 . Of those who reported healthcare - related frustrations, 16.4% reported frequently wishing their doctor had more time to spend talking to them; 16.0% reported frequently feeling tired of describing their same conditions and problems every time they go to a hospital or doctor's office; 8.7% reported frequently wishing they had a friend or family member who could go to the doctor with them; and 8.3% reported frequently being tired of feeling on their own when it comes to taking care of their health problems . Fewer respondents (5.6%) reported frequently feeling their doctor does not realize what it is really like for them at home trying to take care of their health problems or (5.2%) frequently leaving the hospital or doctor's office feeling confused about what they should do . On average conversely, when comparing frustrations by whether the participant reported needing help learning how to better care for their health problems, those needing help reported significantly higher scores on the healthcare - related frustration scale (t = 4.79, p <0.001) and higher frustration levels for five of the six individual scale items (t = 25.94, p <0.001). Perceived physician support scale characteristics are presented in table 3 . Of those who reported receiving physician support, 50.9% reported their physician always helped them get an appointment they needed; 46.5% reported their physician always asked if they understood their medications when their doctor prescribed them; 15.7% reported their physician always talked to other doctors and nurses who were taking care of them; and another 15.7% reported their physician always made plans to contact them after a visit to see how they were doing . Fewer respondents (13.3%) reported their physician always told them about other people who could help them with their health problems or (13.2%) their physician always asked for their ideas about how they can take care of their health problems . On average, participants scored 18.70 (5.84) on the perceived physician support scale . Conversely, when comparing support by whether the participant reported needing help learning how to better care for their health problems, a significantly smaller proportion of those needing help reported asked for their ideas about how they can take care of their health problems (= 10.48, p = 0.033). Table 4 displays the results of the logistic regression analysis explaining factors associated with participants reporting they need help learning how to better care for their health problems (i.e., not needing help served as the referent group). Participants who were non - white were significantly more likely to report needing help learning how to better care for their health problems (compared to non - hispanic whites, or = 2.26, p = 0.049), whereas those with some college or more were significantly less likely to report needing help learning how to better care for their health problems (compared to those with high school or less education, or = 0.55, p = 0.044). Compared to participants with the highest level (tertile) of healthcare - related frustrations, those with middle (or = 0.17, p <0.001) and lowest (or = 0.44, p = 0.017) frustration levels were significantly less likely to report needing help learning how to better care for their health problems, respectively . Compared to participants with the lowest level (tertile) of perceived physician support, those with the highest level of perceived support were significantly less likely to report needing help learning how to better care for their health problems (or = 0.49, p = 0.033). Despite our concerns that the majority of older women would likely experience self - care problems [4850], our analyses revealed that only about one - third of the women in our sample reported needing help learning how to take better care of their chronic conditions . But consistent with health disparities in the epidemiology of chronic illnesses [22, 24], persistent health disparities related to self - care behavior were noted among minorities and those with less education . The rural healthcare disparity often reported in other studies [25, 5153] was not observed in this study, nor were unmarried women disadvantaged relative to their married counterparts . These findings suggest middle - aged and older women in this sample were comparable in terms of having a variety of social supports for learning how to take care of their health . Additionally, despite the previous assumption that older women might be disadvantaged relative to younger baby - boomers, no significant differences in help needed based on age group were reported . Similarly, our proxy measures for disease magnitude and severity (i.e., number of medications, physician visits, and limitations in activities) did not differentiate those needing help . However, the existence of an age - based digital divide was observed [5456], with older women less likely to use online / technology as a resource for chronic condition self - management . Further investigation is needed to examine differences in the types of methods / strategies in which these older women engage when caring for their health conditions outside of the healthcare setting . This study helps elucidate the complex relationships among contextual factors, healthcare frustrations, and patient - provider interactions and support and points to potential opportunities for intervention . The lack of a significant relationship between age and healthcare - related frustrations or perceptions about physician support may be attributed to the fact that the sample was selected based on the presence of chronic conditions . However, the lack of a significant relationship is consistent with the fact that participation in community - based disease self - management programs is not limited to those of only older ages; rather, program enrollment is based on the participant's chronic disease status . This study reveals two strong modifiable correlates of women needing help learning how to care for their chronic conditions, even after controlling for sociodemographic and health status indicators in multivariate analyses: healthcare - related frustrations and perceived physician support . The majority of identified frustrations were significantly related to middle - aged and older women's perceptions of needing help learning how to care for their health, which is supported in other studies [8, 16, 58, 59]. The recent emphasis on patient - centered care and medical homes is designed to help reduce such frustrations and hence can be expected to help boost women's self - efficacy to care for their own health conditions . Additionally, perceived physician support was another significant factor for knowing how to self - manage chronic conditions, especially in terms of patient activation as exemplified by wanting physicians to ask for your ideas about how you can take care of your health problems . This reinforces previous research about the importance of perceived physician support for motivating patients to engage in healthier lifestyles and recommended medical regimens [9, 31, 61, 62]. From the health professional point of view, fostering beliefs of patient support can be accomplished through continuing education units (ceus) or expanded emphasis during medical school training to develop strategies in which clinicians can engage and implement to be supportive, listen to their patients, and solicit their patients' thoughts so they have an active role in their healthcare team [29, 63]. From the patient point of view, widely available evidence - based self - management programs include elements within their curricula to teach older women how they can more effectively communicate with their healthcare providers . Considering perspectives from each side of the healthcare interaction is essential to improve self - management both within and outside of the healthcare setting, which has implications for reducing disease mismanagement, unnecessary healthcare utilization (e.g., emergency room use), and medical costs . This secondary dataset did not contain all variables necessary to fully contextualize barriers and challenges associated with chronic disease self - care behavior among this aging population . However, this national study contained many variables of interest to address the current research gaps in knowledge about associations of healthcare frustrations, physician support, and self - care needs to chronic disease management . Substantial numbers of participants were lost due to incomplete data on particular scale items, resulting in an analytical sample that was systematically different from the full sample (e.g., younger, married, and more white) and potentially limited the generalizability of study findings . This is especially true in that the reduction of cases in the analytic sample reduced the potential proportion of older adults in the study from 42.1% to 33.4%, which may especially influence generalizability of findings to populations aged 65 years and older . Study participants reported a variety of chronic conditions, but subanalyses based on disease type were not performed because the sample size was inadequate to make such comparisons . Recognizing that needed self - care behavior may differ based on women's particular condition diagnoses, disease stage, and the number of comorbidities in which they are diagnosed, future studies should strive to compare about barriers to self - care, perceived physician support, and frustrations with the healthcare system by their chronic condition profile . Further, because women's ability to cope with and adjust to their disease self - care may differ based on their available resources and socioeconomic status, future studies should examine such variables to determine their relationship with self - care disparities . Another study limitation reducing the ability to widely generalize findings to the greater female community was the self - report and cross - sectional nature of these data . However, the sample was derived from random digit dialing and included items that deeply explore the challenges and frustrations with chronic condition self - management, which are not typically seen in other studies that investigate correlations between self - care behavior and other healthcare or physical health indicators . Thus, this study contributes to a fuller understanding of the complex interrelationships that exist between self - care strategies, provider - patient interactions, and policies / programs in community contexts . The current study adds to the existing science base by examining barriers to self - care with a new lens, exploring healthcare - related frustrations and perceptions of physician support, and how these perceptions relate to various life domains, including diverse health status and sociodemographic contexts . Identifying these common and unique challenges and correlates of these challenges, with a representative national population, advance our current knowledge about self - care issues among middle - aged and older women . Learning more about healthcare - related frustrations of and self - management supports utilized by middle - aged and older women has the potential to help program deliverers, healthcare providers, and health agencies provide the services and resources that women with chronic conditions want and think are helpful . Findings from this investigation has potential to inform and guide modifications in the implementation and dissemination of evidence - based programs for older women to better match individuals with programs that meet their needs.
Patients with schizophrenia (scz) exhibit a wide variety of cognitive deficits, particularly with memory (barch and ceaser, 2012; ranganath et al ., 2008). These deficits are predictive of overall functional outcome and clinical remission (bodnar et al ., 2008; green, 2006; green et al ., 2004; kahn and keefe, 2013), and it has recently been suggested that schizophrenia should be viewed primarily as a cognitive disorder (kahn and keefe, 2013). Understanding the nature of the memory deficits is therefore a critical goal when moving towards improved clinical interventions in scz . When considering episodic memory, one area in which patients with scz have a substantial deficit is with source monitoring (identifying the context in which a stimulus was encountered; johnson et al ., 1993). For successful source monitoring it is necessary to bind elements of a memory together into a memory trace, along with their context (spatial context, temporal context, etc . ). Patients demonstrate source monitoring deficits even when stimulus recognition is preserved (danion et al ., 1999; this deficit in source monitoring is in many ways not surprising given that patients with scz also demonstrate difficulties with relational or associative memory (binding items together during memory encoding, and later recalling which stimuli were presented together), while object memory is not as severely impaired (achim et al . Most typically, source monitoring problems in scz are considered in the context of attributing events from internal (self) to external sources . Patients with predominant hallucinations and thought disorder have a greater tendency or bias to attribute internally generated events to an external source, while still being able to correctly identify externally generated stimuli (brunelin et al . This bias for scz to misattribute internal sources has been observed in the absence of recognition memory deficits or false positive responses (fisher et al ., 2008). Interestingly, in a repetitive magnetic stimulation trial of low frequency (1 hz, inhibitory) stimulation to the left temporal parietal junction over 5 consecutive days not only improved auditory hallucinations but also resulted in an improvement in source monitoring compared to sham stimulation (brunelin et al ., 2006). The improvement in auditory hallucination was marginally correlated with the improvement in source monitoring, further suggesting the relationship between source monitoring and hallucinations . Patients with schizophrenia have also been found to have a deficit in source memory (remembering the context in which experimental stimuli occurred; brebion et al ., 2002; the deficit in source memory has been related to deficits in binding contextual cues together into a holistic memory representation (diaz - asper et al ., 2008; waters et al ., 2004), which is an essential component of source memory . Source memory errors are present in scz even for short - term recognition (when source information is tested immediately, minimizing the need to recollect information stored in long - term memory), suggesting that source memory deficits in scz may be related to encoding errors rather than problems in recognition (achim et al ., 2011). (2009) performed a list learning task in scz and found that patients who hallucinate had more intrusions (indicating a word was part of the memory set when it was not) than non - hallucinating patients . This finding was related to source misattribution (patients attributing an internally generated stimulus to an external source, the original memory set). Overall, scz appears to have a noteworthy deficit in source memory which is likely intricately related to other memory and cognitive processes, such as contextual binding and episodic memory . Within healthy controls, source memory involves a range of cortical regions known to be involved in episodic memory, including the medial temporal lobes, prefrontal cortex, and parietal cortex . Increased hippocampal activity has been related to trials in which the source was correctly identified (davachi et al ., 2003; ranganath et al ., 2004), probably due to the role of the hippocampus in relational binding (davachi, 2006). One of the first fmri studies to examine source memory found greater left prefrontal activity for source memory and for old new recognition (nolde et al ., 1998), with subsequent studies finding activity in the left lateral prefrontal cortex associated with source memory for a variety of stimuli types (mitchell and johnson, 2009). Prefrontal lesions have been found to disrupt the self - initiation of processes which promote feature binding (stuss and benson, 1986), and left prefrontal damage is associated with deficits in source monitoring (duarte et al ., 2005). Prefrontal activity during source recognition may be more involved in the evaluation of source information (e.g. Does this stimuli fit with source x) rather than retrieving source information per se (mitchell et al ., activity in medial parietal areas (intraparietal sulcus and precuneus) has been suggested to be present regardless of the type of source information being assessed (uncapher et al ., 2006), while activity in lateral parietal areas may be more dependent on how well the information has been encoded (wheeler and buckner, 2004) and/or to attentional processes (cabeza, 2008). Examining declines in source memory with age has proven fruitful for examining structural and functional correlates of source memory, with evidence that age - related decline in source memory is related to decreased activity mainly in the prefrontal and medial temporal lobes (see mitchell and johnson, 2009, for review). (2006) examined source monitoring in scz using a level - of - processing framework . Patients were presented words with either deep (semantic) or shallow (orthographic) encoding instructions . During recognition, when participants successfully identified a previously encountered stimuli they were asked under which encoding condition the word was encountered (the source memory aspect being recalling the context in which the word was encountered, in this case, which encoding condition). When contrasting correct vs. incorrect source, both patients and controls activated areas of prefrontal and parietal cortices . Patients showed activity in the middle and superior temporal gyrus, thalamus, and parahippocampal gyrus, which was correlated with more severe positive and negative symptoms independent of memory performance . Other neuroimaging studies of source monitoring in scz have focused on deficits related to attributing stimuli as self - generated or externally - generated (reality monitoring). Deficits in reality monitoring appear to involve the medial prefrontal cortex (subramaniam et al ., 2012; vinogradov et al ., 2008; wang et al ., 2011). Following computerized training to improve source monitoring, activity was found to be increased in the medial prefrontal cortex (subramaniam et al ., 2012). The purpose of this study was to examine the neural correlates of source memory in schizophrenia . While most previous studies of source memory (in both controls and schizophrenia) have used less ecologically valid task (e.g. Identifying the color of the stimuli during encoding), we utilized a paradigm involving encounters (with a person and an object in a specific place) within a realistic 3d environment (burgess et al ., 2001; king et al ., 2005), which may better evaluate source memory networks used in everyday life . We examined participants with early schizophrenia (within the first 4.5 years of treatment, with 75% of patients within the first 2 years) thus minimizing potential confounds associated with prolonged illness such as cognitive decline, social isolation and long - term medication effects . During fmri scanning a source recognition memory task was employed, which was contrasted with an object memory task . By directly comparing source memory to object memory, we can identify regions in the cortex which are specific to source memory compared to object memory and determine if any deficits observed in schizophrenia are source - memory specific . We hypothesized that patients would show deficits in source memory relative to object memory and may show compensatory activation in source memory retrieval contrasts . Furthermore, as deficits in source memory have been associated with auditory hallucinations (woodward et al ., 2007) we expected to observe relationships between hallucinations and the neural activity of source memory retrieval . All participants with scz were treated at the douglas mental health university institute in montreal, canada, at the prevention and early intervention program for psychoses, a specialized service providing treatment to individuals aged 1435 years from a local catchment area . Individuals with an iq> 70 who had not taken antipsychotic medication for more than 1 month were consecutively admitted as in- or out - patients . Patients were assessed with the scale for assessment of positive symptoms (saps) (andreasen, 1984) and the scale for assessment of negative symptoms (sans) (andreasen, 1983) at numerous time - points following clinic admission (baseline; at 1, 2, 3, 6, 9, and 12 months; and every 6 months thereafter). (2003) or visit http://www.douglas.qc.ca/pages/view?section_id=165 for more details . For the neuroimaging study, only individuals aged 1830 years with no previous history of neurological disease, head trauma causing loss of consciousness, or lifetime diagnosis of substance dependence twenty - five people with schizophrenia spectrum disorders were recruited, diagnosed according to the structured clinical interview for dsm - iv (first et al ., 1997) and confirmed between two senior research psychiatrists (a.m. and r.j . ). Twenty - four healthy controls were recruited through advertisements in local newspapers and were included only if they had no current or previous history of any axis i disorders, neurological diseases, or head trauma causing loss of consciousness, and no first - degree family members with schizophrenia or related schizophrenia - spectrum disorders . All patients provided written informed consent, and the study was approved by the research ethics boards of the douglas hospital research centre and the montreal neurological institute . Participants performed an encoding task (outside the mri) using a modified version of the virtual city developed by burgess and colleagues (burgess et al ., 2001; king et al ., 2005), created using 3ds max (3ds max, 2011) and unity software (unity, 2011). Participants navigated through a 3d virtual city, following a path indicated by green arrows to the site of an encounter (an encoding trial, a character at a location). After approaching within five virtual meters of the character, the participant's view was frozen and the character stepped aside, and a life - sized object appeared on a small table displayed to the right . Participants were instructed to pay careful attention to the object, character and location, and try to remember for a later memory test . After a 5 s study delay the person and object disappeared, and participants then followed the arrows to the next encounter . There were a total of 20 encounters, each with a unique person, location and object . A total of 80 recognition trials were administered, in which participants were shown an image consisting of a typical viewpoint encountered within the virtual city (in one of 20 places where encounters occurred), with a person in the center of the screen, and two objects, one on each side of the person . Participants were then asked one of four possible recognition questions: (1) person (which object was paired with this person), (2) place (which object did you view in this location), (3) object (which object was viewed in the city; the other object was new), and (4) bright (which object is brighter in appearance). Participants responded on an mri - compatible button box to indicate which item (left or right) was selected . For the person condition, the place was not associated with either object, while in the place condition the person was not associated with either object . The person and place conditions were designed to access source memory (in what context was an object encountered) while the object condition assesses object memory (old vs. new). Including two source memory conditions allows us to better understand if the observed activity is modality specific . The bright condition was not considered in this analysis as we were specifically interested in source vs. object memory . Images (with encoding question) were presented on the screen for 8000 ms, with a 20008000 ms isi (in 100 ms increments), with an average trial length of 13 s. stimuli were presented and results were recorded through e - prime 1.0 software . 1 . A practice route was designed to allow participants to become familiar with the arrow key and mouse, and to practice following the arrows and encounter two characters with objects in independent locales . Participants also practiced answering two of each of the four forced - choice recognition questions regarding the objects collected . Echo - planar images were collected on a siemens 3 t tim trio mri (tr = 2000 ms, te = 30 ms, flip angle = 90, 36 slices of 4 mm thick, 64 64 voxel plane with an fov of 256 mm 4 mm slices). Each bold run was preceded by 4 volumes that were later discarded to allow a magnetic steady state . The anatomical scan was an mprage (tr = 2300 ms, te = 2.98 ms, fov 256, 1 1 1 mm voxels, flip angle = 9) and lasted 5.21 min . Data analysis was conducted using spm 8 (wellcome department of cognitive neurology, london, uk). Data was motion corrected by realigning to the 3rd tr, normalized to the icbm template (and resampled at 2 2 2 mm voxel size) and smoothed with an 8 mm isotropic gaussian kernel . The general linear model was implemented by convolving a standard hemodynamic response function and its first temporal derivative and dispersion . Events were defined based upon recognition question (person, place, object, or bright), with incorrect answers modeled as distinct event types and excluded from further analysis . Accuracy data was not available for three controls and four scz patients due to technical problems during initial data collection . For these participants, contrasts were person vs. object and place vs. object (both performed bidirectionally) to identify voxels which are differentially activated by source or object memory . A second level analysis was performed separately for each group (controls or scz) using a one sample t - test . Corrections for multiple comparisons were performed at the cluster level using an individual voxel threshold of p <0.001 (uncorrected). A monte - carlo simulation of 1000 iterations (slotnick et al ., 2003) resulted in a cluster extent threshold of 49 resampled voxels . To test for differences between groups, an independent - samples t - test was performed, using the contrast value derived for each participant from the above contrasts . Given that between - group differences often have smaller effect sizes, we used a slightly more liberal single - voxel threshold of p <0.005, but corrected to p <0.01 at the cluster level (resulting in an extent threshold of 97 voxels). A regression analysis in patients was performed to examine the relationship between source memory and clinical symptoms . As patients were stabilized and undergoing treatment at the time of scanning, few patients were actively experiencing positive symptoms as of the assessment closest to the date of scanning . As a result, the data did not possess sufficient variability for a regression analysis (the majority of cases had global scores of 0 or 1 on the saps at closest assessment). Furthermore, positive symptoms are often highly responsive to treatment (malla et al ., 2006; robinson et al ., 1999). As such any patient displaying continued positive symptoms may represent treatment resistant patients or those with a more severe illness, rather than relate to the symptoms themselves per se . Instead, we utilized scores from the assessment at the initial visit to the clinic, prior to treatment onset . The presence of specific symptoms at initial clinical assessment was considered as a proxy of how prone to those symptoms each patient may be . We propose that the pre - treatment ratings best represent the underlying neurobiology and clinical characteristics of individual patients, as they show their symptom characteristics in an untreated state of illness . While it is not possible to make several such assessments prior to treatment onset (which would best capture the potential symptom profile of each patient), such an approach may allow for a data exploration which separates patients who are prone to certain symptoms (such as hallucinations) from those who have experienced less or never experienced those symptoms while in an untreated state . As such, this can be conceptualized as a trait based approach to symptom evaluation . Saps scores for global hallucinations and global thought disorder were entered into a whole - brain regression model with the person> object and place> object contrasts . The global delusion score was not included as most patients were highly delusional at baseline . In order to account for differences in time since initial diagnosis, the interval between baseline assessment and mri scan (in days) was entered as a covariate in the second - level regression analysis . Cluster threshold for the regression was 49 voxels at p <0.001 uncorrected (voxel threshold). There were no significant differences in age or gender distribution between groups, though patients had a marginally significant lower parental ses and significantly fewer years of education . Accuracy in the person, place and object conditions was assessed using independent samples t - test . Schizophrenia patients had significantly lower accuracy in the person condition, t(40) = 2.247, p = 0.03, but not for place, t(40) = 0.44, p = 0.66, or object, t(40) = 0.07, p = 0.95 . Numerous cortical regions showed significant increases in neural activity for retrieval of source memory over retrieval of object memory, similar to the pattern observed in previous studies using a similar paradigm (burgess et al ., 2001; king et al ., 2005). Activated regions in the source memory contrasts (person> object and place> object) included bilateral activity around the parietal occipital sulcus (including the precuneus and retrosplenial cortex) extending into the superior parietal cortex, left inferior frontal gyrus (vlpfc), fusiform gyrus bilaterally, and the occipital cortex . Activity in the person> object contrast included the head of the caudate nucleus, the right inferior frontal (vlpfc), and the medial aspect of the superior frontal gyrus . Object> place showed widespread activity in the medial frontal cortex and parietal cortices (supramarginal gyrus bilaterally) and smaller clusters in the frontal and occipital cortices . For the object> person contrast, activity was observed in the left and right angular gyrus . 2 . When examining source vs. object retrieval contrasts in scz, a smaller number of significantly activated voxels were observed relative to the activity maps of controls . In the person> object contrast only smaller clusters in the head of the caudate and occipital cortex were significant . In place> object, mainly posterior activity was observed (e.g. Parietal occipital sulcus and fusiform cortex). In both the object> person and object> place, scz showed a right inferior frontal (vlpfc) activity which was not observed in controls . 2 . When comparing between groups, controls demonstrated regions of greater differences between conditions than scz for person> object condition (including bilaterally in the precuneus and superior parietal, and left and right inferior frontal in the vlpfc) and for place> object (bilaterally in the superior parietal, and the left precuneus). Schizophrenia patients had greater differences between conditions than controls when considering place> object across a wide range of areas . The group comparison was run using contrast values, which can be positive (e.g. If place> object) or negative (e.g. If object> place). Thus, while we observed greater activity in scz than control in the place> object contrast, it is not clear from the activation maps if such a difference is due to changes in activity in either object or place . In order to visualize the results for each condition, beta values were extracted for an roi of 11 voxels (9 in - plane voxels surrounding the selected voxel and one above and one below). Rois were selected to represent a range of representative patterns of activity across the brain (e.g. Regions in which controls showed differences from scz in both person> object and place> object, and regions in which scz showed greater activity in place> object). Which controls had greater differences between conditions than scz, controls are showing increased activity to the source memory conditions (person and/or place) relative to object, while values in scz do not show any such differentiation . Interestingly, in regions in which we observed greater differences between conditions (in this case, place> object) in scz than controls, we again observe little differences between source memory and object memory in scz . Thus, it is not that patients with scz are showing greater activity in the place condition, but that they are failing to modulate brain activity in the same way as control participants . Results of the regression analysis with positive symptoms are presented in table 6 and fig . We observed significant negative correlations with the global hallucination score from the saps (at first assessment) and place> object contrast, in the right midtemporal gyrus, left prefrontal cortex, and right cerebellum (in a region noted in at least one lesional case study to be associated with source memory deficits; tamagni et al ., 2010). In order to examine the relationship between recognition memory performance and neural activity, spearman's correlations were run on global hallucination score, memory performance in the place and object conditions, and values from the three clusters . While hallucinations did not significantly correlate with performance in either condition (place, rho = .228, p = 0.32; object, rho = 0.28, p = 0.22), there was a marginally significant correlation between performance in the object condition and value in the dlpfc cluster (rho = 0.38, p = 0.088). This study examined differences in the neural correlates of source memory in patients with schizophrenia and controls . We examined this issue using a virtual reality paradigm, which has improved ecological validity when assessing the source of a memory, as compared to other studies which have used less ecologically valid tasks . Furthermore, we examined a group of patients, who were within the first 4.5 years of treatment avoiding potential issues associated with illness chronicity such as cognitive decline, prolonged exposure to medications (although medication exposure remains an issue), social isolation, and sedentary lifestyle (pelletier et al ., 2005). While patients with scz demonstrated some activation in the source memory contrasts, the extent and magnitude of activity were substantially less than those in controls . Even in regions in which the difference appeared to be in the directions of scz> controls, it seems to be the case that controls differentiate between source and object memory while patients do not . As discussed below, this finding may be true of other forms of memory, and may therefore be representative of the underlying deficit across a range of memory subtypes . More specifically, patients with scz may fail to activate cortical regions which facilitate elaborative memory processes . This is consistent with previous findings of relatively intact object memory in scz (achim et al . 2009), but deficits in source memory (johnson et al ., 1993) and associative memory (achim and lepage, 2003). It may be that the vr environment minimizes these behavioral differences, in that the place condition may be easier than person as locations were more distinct from each other relative to the 3d rendered characters . Alternatively, our recent - onset sample may have better preserved function than the more enduring schizophrenia samples often examined . However, this lack of behavioral difference has a positive aspect, in that it removes performance as a potentially major confound in the fmri analysis . Had patients with schizophrenia demonstrated profound deficits in performance, it would beg the question if any observed neural activity differences were due to disease pathology or simply related to poor performance (and as such would be similar to poor performing healthy controls). We did observe a performance difference in the person condition, and interestingly very little significant activity in the schizophrenia group for person> object . However, some activations observed in the group analysis, particularly in the posterior regions, were present in both person> object and place> object, suggesting that these differences were not at all modality specific . Negative correlations were observed between the difference in neural activity in place> object and global hallucinations, measured at intake baseline using the saps . This suggests that the propensity of an individual to experience hallucination may modulate differences between source and object memory in these areas . We did not observe significant correlations with global thought disorder, which is not surprising as hallucinations but not thought disorder are generally associated with source memory (woodward et al ., 2007). While these regions did not directly overlap activity observed in the healthy control group, the left frontal cluster was proximal to significant activity in place> object in controls, while the middle - temporal cluster was proximal to significant voxels in the object> place clusters in controls, suggesting that these regions are at least similar to those observed in healthy controls . However, the lack of direct overlap and given the nature of the result (greater difference with more hallucinations), it is possible that these regions represent malfunctioning cortical regions which are more active in patients who have experienced hallucinations as part of their disorder . This over - activation within these regions when considering source information may play an important role in generating hallucinations, which are essentially the misattribution of internally generated stimuli to an external source . At least one study has found relationships between the left prefrontal cortex and the right temporal cortex while patients are actively experiencing hallucinations (hoffman et al ., 2011), and a meta - analysis suggests that these regions among others are frequently active during hallucination (jardri et al ., 2011). As such, the regions found to be significantly active in our regression are consistent with the existing literature on the neurobiology of hallucinations . While many studies examining hallucinations have utilized either general state based measurements (approximately how much the participant is hallucinating in general at the time) or direct analysis of overall activity during hallucinations, we have attempted to utilize a our sample of patients early in treatment makes such an approach possible with minimal confounding for chronicity (which we attempted to control for by including length of treatment as a covariate in the regression). Our results overall suggest a relationship between mal - adaptive neural activity related to source memory retrieval and how prone participants are to hallucinatory symptoms, further verifying the relationship between source memory and hallucinations (woodward et al ., 2007). However, some limitations must be considered as well . While we have attempted to use baseline scores as a trait measure of how prone an individual is to hallucinations, it is well known that clinical symptoms can vary across time . As such, the appropriate method for evaluating trait symptoms is to take repeated symptom measurements over time (mathalon and ford, 2012). It would be preferable to have several assessments of pre - treatment symptoms to have a complete picture of the symptom profile of a given patient, but this is not possible as it would require delaying treatment . As such we utilized the best available measure to assess pre - treatment symptom profile, which produced results which are consistent with the existing literature on those symptoms . However, it is important to remain considerate of the limitations of our measures when considering these results . Source memory can be considered a form of associative / relational memory, as participants are binding elements together during encoding and storing these elements together as part of a larger whole . A deficit in source memory can be viewed as a failure to associate a memory item with its context . In the case of this study in particular, our source memory paradigm is not far removed from studies examining associative memory (achim et al ., 2007; achim and lepage, 2005; murray and ranganath, 2007), which is known to be more impaired in scz than object memory (achim and lepage, 2003). Some studies have suggested that patients with schizophrenia do not properly differentiate between associative and item memory (achim et al . 2012), in keeping with suggestions that source memory impairments are part of associative memory impairments (achim et al ., 2011). Our present study focused on source retrieval, and as such we cannot definitively determine if the differences observed in our contrasts are driven by deficits in encoding (in that the information was not properly moved into memory) or retrieval (in that the memories may have been encoded but are not properly accessed), or a combination of both (which seems likely given the plurality of evidence for memory deficits in scz). (2011) found deficits in source memory even when using short term recall, minimizing the need for retrieval of information from long - term memory . This suggests that at least part of the source memory deficit in scz is related to problems with encoding . Our finding of an overall pattern of lack of differentiation between conditions may be a fairly consistent finding in the cognitive neuroscience of memory in scz, regardless if one considers activity during encoding or retrieval / recognition . Core regions required for task performance may be relativity intact in scz, while deficits in activity will be observed in that is to say, the deficits in memory (and possibly cognition in general) are related to a lack of engagement of extended cortical regions . These extended regions are not critically required for minimal task performance, but instead serve to enhance performance (such as regions involved in cognitive control processes). Healthy controls will tend to utilize such areas automatically, while patients with schizophrenia will fail to do so . For example, patients with scz have been shown to have impairments in initiating elaborative encoding processes which may be beneficial during associative encoding (brebion et al . However, when patients are specifically instructed to utilize effective encoding strategies they show an improvement in memory performance, demonstrating that patients with scz can utilize such encoding strategies when specifically instructed but fail to do so spontaneously (brebion et al ., 1997). This pattern of results is similar to that in patients with prefrontal cortical lesions (alexander et al ., 2003). Bonner - jackson et al . (2008) examined memory strategies in schizophrenia by contrasting intentional but unstructured encoding (simply instructing participant to memorize words) with an externally imposed deep encoding strategy (having participants perform an abstract / concrete judgement on words, deep semantic encoding which facilitates memory encoding; craik and lockhart, 1972). They observed group encoding interactions in several regions, including the left inferior frontal gyrus and precuneus, with the most common finding being a difference between controls and scz in the incidental, unstructured encoding condition . For example, in the left inferior frontal cortex, scz patients showed no activity for unstructured encoding, but substantial activity for deep encoding . That is to say, when scz patients were provided a structured encoding strategy they activated this region, but failed to do so spontaneously . Likewise, controls showed significantly better memory performance than scz patients for the deep encoding condition . Further support for the importance in strategic and/or cognitive control comes from findings of changes and/or normalization of activity in scz following cognitive training (hooker et al ., 2012; penades et al ., within both encoding and recognition studies in schizophrenia, the most prevalent finding is a decrease in the extent or magnitude of activity in scz patients relative to controls, although many studies also report findings in the direction of scz> controls (ragland et al ., 2009). While it is likely that scz often shows compensatory networks or inefficient over - activation during cognitive tasks, it can be difficult to judge from many of the published studies on cognition in scz . Many papers report differences in group activities without also reporting the parameter estimates which accompany those changes . If we had done so in this study (by only presenting the activation maps in fig . 3 and not the beta values) we may have concluded that scz showed greater activity in some regions for place vs. object and concluded that this was compensatory of maladaptive over - activation . However, when examining the beta values, we realize that this is not the case but instead these are regions where controls show greater activity for object over place (often in the form of a decrease in activity for place). This highlights the importance of carefully examining parameter estimates when performing between - group comparisons . It is possible that in at least some cases in the existing literature, the so - called compensatory activity may instead be a lack of activity relative to the control group, such as was the case in this study . Such ambiguity and misinterpretation can be avoided if studies fully report parameter estimates for contrasts which differ between groups . We have reported the results of a study looking at source memory retrieval in scz using a virtual reality environment on a group of patients relatively early into treatment . While controls activated a large, extensive network for source relative to object retrieval, scz showed a marked reduction in activity . This reduction was borne out in the group comparison, and appears to be related to the fact that patients with scz are failing to activate these regions or differentiate between object and source retrieval . Despite the large - scale group differences, patients were still able to perform the source retrieval tasks, suggesting that at least some of the core system involved in source retrieval is intact . Instead, we propose that the observed differences are related to supporting regions which are not critical to task performance but instead facilitate source retrieval . That is, patients may generally fail to engage extended cortical networks which facilitate task performance and facilitate overall cognitive functioning.
Ras is a multifunctional signaling molecule acting as an essential component of signal transduction pathways that regulate cellular physiology (campbell et al . Members of the ras family of proteins that are constitutively activated by point mutations play a major role in the onset of a large number of human cancers, including those originating from skeletal muscle tissue (yoo et al . These skeletal muscle tumors are incapable of differentiation and thus do not withdraw from the cell cycle . Up to 35% of these tumors contain activating ras point mutations, suggesting a major involvement of ras in rhabdomyosarcomas (stratton et al . Constitutively active ras mutants stimulate secretion of growth and angiogenic factors (rak et al . 1995), potentially allowing neoplastic cells to overcome growth restrictions in their normal tissue environment . In skeletal muscle cells, activated ras mutants have been shown to promote secretion of an unidentified factor that can repress myogenic differentiation and may participate in the development of rhabdomyosarcomas (weyman and wolfman 1997). Of particular interest is the observation that cultured human embryonal rhabdomyosarcoma cells express the fgf-2 gene and produce biologically active fgf-2 (schweigerer et al . Release of fgf-2 may stimulate the growth and neovascularization of human rhabdomyosarcomas and contribute to tumor development . Although ectopic expression of oncogenic ha - ras in myogenic cell lines represses terminal differentiation, it is not reported to elicit a proliferative response (olson et al . 1987; konieczny et al . 1989; weyman and wolfman 1997). From these studies, it has been concluded that ras inhibits muscle differentiation without affecting proliferative response pathways . Fgfs are likely candidates for such factors since they play critical roles in regulation of skeletal muscle differentiation in cultured cells (linkhart et al . 1981; allen et al . 1985; kardami et al . 1985; seed and hauschka 1988; rando and blau 1994; flanagan - steet et al . 2000), in skeletal muscle development in vivo (flanagan - steet et al . 2000), and in skeletal muscle regeneration (anderson et al . 1995; floss et al . Mm14 myoblasts express fgf-1, -2, -6, and -7 but are absolutely dependent on exogenously supplied fgfs to repress myogenesis and promote cell proliferation (clegg et al . Fgf-2 is one of four fgfs that do not possess signal peptides and do not use the classical er / golgi - dependent secretory pathways for export from the cell (florkiewicz et al . Since ectopically expressed ha - ras can repress differentiation of mm14 cells (fedorov et al . 1998), we asked if ha - ras was capable of stimulating proliferation in mm14 cells . Here we report that constitutively active ras stimulates mm14 myoblast proliferation via a novel mechanism that is dependent on export of endogenously produced fgf-2 and subsequent release or activation of the exported fgf-2 . Moreover, we also found that the signaling pathways used by oncogenic ras to stimulate proliferation and repress differentiation in myogenic cells are distinct and mediated independently . Mouse mm14 cells (lim and hauschka 1984) were cultured as described previously (kudla et al . Baf3/fr1 cells, a baf3 cell clone stably expressing fgf receptor (fgfr)-1, was cultured as described by ornitz et al . 1992 . Wehi3 cells were purchased from the american type culture collection and baf3/fr1 cells (ornitz et al . Human recombinant fgf-2 was purified from a yeast strain expressing this growth factor (rapraeger et al . Heparin, nacl, and naclo3 were purchased from sigma - aldrich . A monoclonal anti 1993) and anticysteine - rich fgfr control antibody (zuber et al . 1997) were used as described previously (hannon et al . Dna was transiently transfected into mm14 cells by a calcium phosphate dna precipitation method as described previously (kudla et al . Equivalent dna concentrations were maintained by the addition of a pbssk+ (stratagene) plasmid . The pdcr - h - ras (g12v) expression vector encoding a constitutively active mutant of human ha - ras, rasg12v (white et al . Mm14 cells were grown on 6-well plates to a density of 5 10 and transfected with the indicated expression vectors or control plasmids . Cells were trypsinized (0.05% trypsin, 0.53 mm edta) and replated at clonal density (1,000 cells per 10-cm plate) 1 h after transfection . The cells were maintained in the presence or absence of fgf-2 (0.3 nm unless otherwise indicated), cultured for 36 h, then processed for -galactosidase histochemistry as described elsewhere (sanes et al . A differentiation - sensitive muscle - specific reporter activity assay was used to determine the extent of mm14 differentiation after transient transfection . The reporter contained the firefly luciferase gene driven by a muscle - specific promoter (msp; human anti - cardiac actin promoter) (kudla et al . Transfected cells were plated at 10 cells per well in 6-well plates and incubated for 36 h in growth media without fgf . Cells were then washed once with pbs (ph 7.2) and incubated for 1 h at room temperature in 1 ml of baf3/fr1 growth medium supplemented with 50 g / ml of heparin . Baf3/fr1 cells (10 cells per well in 24-well plates) were incubated in the collected medium for 72 h. the number of living cells in each well was quantified by counting the number of cells that exclude trypan blue . Mouse mm14 cells (lim and hauschka 1984) were cultured as described previously (kudla et al . Baf3/fr1 cells, a baf3 cell clone stably expressing fgf receptor (fgfr)-1, was cultured as described by ornitz et al . 1992 . Wehi3 cells were purchased from the american type culture collection and baf3/fr1 cells (ornitz et al . Human recombinant fgf-2 was purified from a yeast strain expressing this growth factor (rapraeger et al . Heparin, nacl, and naclo3 were purchased from sigma - aldrich . A monoclonal anti 1993) and anticysteine - rich fgfr control antibody (zuber et al . 1997) were used as described previously (hannon et al . Dna was transiently transfected into mm14 cells by a calcium phosphate dna precipitation method as described previously (kudla et al . Equivalent dna concentrations were maintained by the addition of a pbssk+ (stratagene) plasmid . The pdcr - h - ras (g12v) expression vector encoding a constitutively active mutant of human ha - ras, rasg12v (white et al . Mm14 cells were grown on 6-well plates to a density of 5 10 and transfected with the indicated expression vectors or control plasmids . Cells were trypsinized (0.05% trypsin, 0.53 mm edta) and replated at clonal density (1,000 cells per 10-cm plate) 1 h after transfection . The cells were maintained in the presence or absence of fgf-2 (0.3 nm unless otherwise indicated), cultured for 36 h, then processed for -galactosidase histochemistry as described elsewhere (sanes et al . A differentiation - sensitive muscle - specific reporter activity assay was used to determine the extent of mm14 differentiation after transient transfection . The reporter contained the firefly luciferase gene driven by a muscle - specific promoter (msp; human anti - cardiac actin promoter) (kudla et al . To determine their fgf-2 export capabilities, transfected cells were plated at 10 cells per well in 6-well plates and incubated for 36 h in growth media without fgf . Cells were then washed once with pbs (ph 7.2) and incubated for 1 h at room temperature in 1 ml of baf3/fr1 growth medium supplemented with 50 g / ml of heparin . The medium was then collected and filtered through a 0.2-m filter . Baf3/fr1 cells (10 cells per well in 24-well plates) were incubated in the collected medium for 72 h. the number of living cells in each well was quantified by counting the number of cells that exclude trypan blue . Mm14 cells are absolutely dependent on exogenously supplied fgfs to repress myogenesis and promote proliferation, yet they express several fgfs (hannon et al . 1996), suggesting that the endogenously produced fgfs are inaccessible to fgfr-1 . In addition, we have established that distinct fgfr-1 signaling pathways mediate the proliferative and differentiation inhibitory responses in mm14 cells (kudla et al . Ectopic ha - ras expression stimulated proliferation and repressed differentiation of mm14 cells in the absence of exogenous fgf (fig . 1). Activated ras appears to replace only fgf - dependent signaling events since mm14 cells transfected with oncogenic ras were unable to proliferate in growth medium with reduced (2.5%) serum (data not shown). We hypothesized that ha - ras may induce fgf export or secretion of endogenously produced fgfs and therefore treated mm14 cells expressing rasg12v with agents that block fgf signaling . Addition of suramin, a negatively charged polysulfonated binaphthyl urea used as a general heparin - binding growth factor antagonist (lozano et al . 1998), to cells ectopically expressing oncogenic ras inhibited proliferation in a dose - dependent manner (fig . Fgf-2 antibody completely abolished the capacity of ha - ras to stimulate proliferation (fig . 2 a), whereas addition of a control monoclonal antibody had no effect (fig . Unexpectedly, we found that the ability of ha - ras to stimulate proliferation appears dependent on extracellularly supplied fgf-2 . Fgf signaling is dependent on heparan sulfate, which involves the interaction of heparan sulfate with both fgf and the fgfr tyrosine kinases (rapraeger et al . In addition, heparan sulfate proteoglycans (hspgs) participate in fgf storage, sequestration, and release (rifkin and moscatelli 1989). Treatment of mm14 cells with sodium chlorate, a reversible inhibitor of intracellular sulfation, prevents fgf binding and induces terminal differentiation (rapraeger et al . Incubation of oncogenic ras - transfected mm14 cells with heparitinase (not shown) or sodium chlorate significantly decreases cell proliferation (fig . 3 a). Both heparitinase (not shown) and chlorate - induced inhibition of mm14 cell proliferation was rescued by addition of heparin (50 g / ml), indicating that the effect is heparan sulfate specific (fig . 3 a). Surprisingly, addition of 600 pm fgf-2 to chlorate - treated ha - ras transfected cells promoted proliferation, ameliorating the inhibitory chlorate effect (fig . This was unexpected since addition of fgf-2 had no effect on chlorate - treated parental mm14 cells or mm14 cells transfected with a pcdna3 vector control (fig . 3 b). The requirement for hspgs and the ability to overcome this requirement with high concentrations of exogenously added fgf-2 suggests a more complicated role for hspgs in addition to their known requirement for signaling . Taken together, our data demonstrate that rasg12v induces proliferation of skeletal muscle cells and that induction of proliferation requires an autocrine fgf-2 response . Little is known regarding the mechanisms involved in fgf-2 export since fgf-2 has no signal peptide sequence and is not secreted through the established golgi - dependent secretory pathway (mignatti et al . 1992). Instead, an unusual atp - dependent pathway that includes the na / k - atpase appears to be involved (florkiewicz et al . Whether oncogenic ras is directly involved in regulating fgf-2 export, we asked if mm14 cells expressing ha - ras exhibited increased levels of extracellular fgf-2 . Although transfection of mm14 cells with fgf-2 results in export of biologically active fgf-2 (hannon et al . 1996), this extracellular fgf-2 cannot be detected in the tissue culture media, presumably due to its strong association with membrane - bound and extracellular matrix associated heparan sulfate . One assay utilizes heparin treatment of mm14 cells to release bound fgf-2, which is then assayed on baf3 cells expressing fgfr-1 (baf3/fr1). Baf3 cells are pre - b cells that undergo apoptosis after interleukin 3 withdrawal and do not express either fgfrs or hspgs . As such, these cells are unresponsive to fgfs, unless they ectopically express fgfrs and heparin is added as an hspg substitute (ornitz et al . We found that both ras - g12v and control (pcdna3)-transfected mm14 cells release similar levels of factor(s) that support baf3/fr1 survival and promote baf3/fr1 proliferation (fig . A second assay involves cotransfection of mm14 cells with a construct encoding an fgf-2luciferase fusion protein and either ras - g12v or a control vector . The results from this assay are indistinguishable from the baf3/fr1 cell assay, suggesting that similar levels of fgf-2 are exported by control and ha - ras transfected cells (data not shown). We conclude that oncogenic ras does not affect the level of fgf-2 export from mm14 cells . Although mm14 cells produce fgf-2 and export fgf-2 that can be recovered in an active form, this fgf-2 is not normally available to the cells (hannon et al . Thus, our data suggest that exported fgf-2 is normally retained in an inactive form on the cell surface . We propose that ha - ras activates this inactive extracellular pool of fgf-2 either by promoting its release from hspgs or by providing a mechanism for fgf-2 to gain access to cell surface fgfr-1 . Although the mechanisms involved are not understood, the ability of the ha - ras mutant to promote proliferation is dependent on exogenous fgf-2 and subsequent fgf-2mediated signaling events . The ability of oncogenic ras to inhibit skeletal muscle differentiation has been well documented, but the ras effector mediating repression of differentiation is not known (ramocki et al . We wanted to determine if the ability of ras to effectively inhibit mm14 differentiation was dependent on extracellular fgf-2, as is the proliferation response . Fgf-2 antibody did not affect the ability of ras - g12v to repress myogenesis (fig . These data are consistent with our results published previously, which demonstrate that independent fgf signaling events mediate repression of differentiation and proliferation (kudla et al . Thus, similar to fgfr, ha - ras appears to utilize independent signaling mechanisms to repress terminal differentiation and promote proliferation . Although the downstream signaling events that mediate the repression of myogenesis by ras are not understood, it is well documented that oncogenic ras stimulates secretion of growth factors and angiogenic factors (rak et al . Moreover, ectopic expression of activated ras releases a factor that represses myogenic differentiation (weyman and wolfman 1997). Data presented in this report argue that oncogenic ras may be involved in the activation or release of extracellularly localized fgf-2 that is normally sequestered in a biologically inactive state . It is noteworthy that all of the extracellular activity observed is neutralized by a specific blocking fgf-2 monoclonal antibody, since proliferating mm14 cells synthesize fgf-1, -2, -6, and -7 (hannon et al . 1996). Although detectable, fgf-2 mrna is present at extremely low concentrations in many adult tissues despite the presence of high levels of fgf-2 activity, suggesting a mechanism for retaining or storing fgf-2 in a biologically inert form (baird et al . Oncogenic ras appears to be involved in activating or releasing inactive, extracellularly localized fgf-2 . The fgf-2 produced by skeletal muscle cells cannot be detected in the tissue culture medium, forming the basis for previous conclusions that the ras - secreted myogenic inhibitory factor was not an fgf (weyman and wolfman 1997). The ras - dependent proliferation factor we identified is released by a heparin wash and its activity is abrogated by treatment with either chlorate, heparitinase, suramin, or a monoclonal anti fgf-2 antibody, implying that the proliferation factor is fgf-2 . Together with the prevalence of oncogenic ras mutants in rhabdomyosarcomas and the involvement of fgfs in regulation of myogenesis, our data suggest that fgf-2 may be a critical factor for supporting ras - dependent growth of rhabdomyosarcomas.
Lipomas represent uncommon neoplasms of the oral cavity; only 1% to 5% of cases occur at this site . Based on their histopathological features of conspicuous multiple components, lipomas can be divided into some subclasses [2, 3], and the most common is the fibrolipoma [1, 3, 4]. Cases reporting on lipoma with osteo / chondroid differentiaion were only 16 cases as a result of retrieving literature from 1960 to 2008 . Further, other names of this subclassification exist in literature; osteo / chondrolipoma, ossifying / osseous lipoma, lipoma with chondro / osseous metaplasia, and lipoma with cartilaginous / osseous change . The obscure etiology of osseous / chondroid differentiaion led to confusion of the name . To clarify the etiology of osseous / chondroid differentiaion in lipoma, the authors report additional 2 cases of oral lipoma / fibrolipoma with osseous and/or chondroid differentiation and describe the clinical, histopathological, and immunohistological features of these . Review of the literature and clinicopathological analysis from the files of the oral pathology department, nihon university school of dentistry at matsudo from 1995 to 2007 of these cases were performed and compared . Two cases that had been diagnosed as lipoma with osteo / chondroid differentiation were retrieved from the files mentioned earlier . In these cases, sections of 2 cases were deparaffinized in xylene and dehydrated in tris - buffered saline (ph 7.6). Primary antibodies against e29 (epithelial membrane antigen (ema)), qbend 10 (cd34 class ii), ki - s5 (ki-67 antigen), 5.8a (myo d1) were purchased from a commercial source (dako, denmark), and sc-12488 (runx-2), sc-20095 (sox-9), sc-71992 (s-100 chain), sc-71993 (s-100 chain), sc-55520 (fgf-1), and sc-57494 (vegf) were purchased from a commercial source (santa cruz biotechnology, inc, usa). For detection of the antigen, the dextran polymer method (chem mate envision kit, dako, denmark) was used . To improve detection, the deparaffinized sections were pretreated by microwave heating with citrate buffer (ph 6). The primary antibodies were generally used at a dilution of 1: 50, and the incubation time was 1 hour at room temperature . Positive controls consisted of specimens of schwannoma for s-100 and; squamous cell carcinoma for ema, cd34 and ki-67; ossifying fibroma for fgf-1 and runx-2, normal lung tissue for sox-9; and inflammatory granulation tissue for myod-1 . As a negative control, mouse igg1 (ki-67, vegf, cd34, s-100), igg2a (ema, s-100) and igg2b (fgf-1), goat igg (runx-2) and rabbit igg (sox-9) were used instead of the primary antibodies . At least 500 nuclei were counted in 5 high - power (x400) fields and ki-67 labeling index was calculated . Mean intratumor microvessel density was obtained by calculating the average counts of these 5 fields . The protocol was approved by the committee on studies involving human beings of nihon university school of dentistry at matsudo (ec 05 - 002). Clinical data were retrieved from patient records, and all cases were reviewed microscopically and subclassified . The literature from 1960 to 2008 of lipoma with osseous / chondroid differentiation was reviewed . Case 1a 28-year - old woman presented with a painless relatively hard mass on the dorsal surface in the midline of the tongue that had recently grew slightly . The patient's mother first noticed a tiny nodule when the patient was 6 months old . Examination showed a well - defined hard nodular mass, approximately 16 16 9 mm in size, which was sharply demarcated within the muscles of the tongue and freely mobile . A yellowish tinge was visible through the overlying mucous membrane and the lesion was firm on palpation . Clinical diagnosis of lipoma with calculus was made and the tumor was completely excised from the tongue under general anesthesia . Gross examination showed a yellowish soft to hard smooth mass measuring 16 15 12 mm in size . Microscopically, the tumor consisted of a well - circumscribed mass of fatty tissue with a cellular chondroid component . Chondromatous nodules within uniform adipose tissue were seen in large areas of mature fat cells supported by fibrous connective tissue (figure 1). Pathological diagnosis was made as lipoma with chondroid differentiation . A 28-year - old woman presented with a painless relatively hard mass on the dorsal surface in the midline of the tongue that had recently grew slightly . The patient's mother first noticed a tiny nodule when the patient was 6 months old . Examination showed a well - defined hard nodular mass, approximately 16 16 9 mm in size, which was sharply demarcated within the muscles of the tongue and freely mobile . A yellowish tinge was visible through the overlying mucous membrane and the lesion was firm on palpation . Clinical diagnosis of lipoma with calculus was made and the tumor was completely excised from the tongue under general anesthesia . Gross examination showed a yellowish soft to hard smooth mass measuring 16 15 12 mm in size . Microscopically, the tumor consisted of a well - circumscribed mass of fatty tissue with a cellular chondroid component . Chondromatous nodules within uniform adipose tissue were seen in large areas of mature fat cells supported by fibrous connective tissue (figure 1). Case 2a 59-year - old man presented with a relatively painless hard mass on the left side of the lower labial vestibule surface, which appeared the sense of incompatibility 2 months prior to consultation . Examination showed a well- defined hard nodular mass, approximately 5 mm in diameter, and freely mobile, and covered with mucosa of normal aspect and color . There was no tenderness, no sign of inflammation . To retrieve more - detailed relativity with the surrounding tissue, ct was taken and showed a lesion in the left side of the lower lip that appeared to be a small mass with areas of little calcification within . Gross examination showed a yellowish soft to hard smooth mass measuring 9 5 5 mm in size . Microscopically, the tumor consisted of a well - circumscribed mass of mature fat cells supported by fibrous connective tissue septa and myxoid tissue characterized by spindle cells . A focal island of consecutive chondroid and woven bone component was surrounded by spindle / fusiform - shaped mesenchymal cells throughout the lesion (figure 2). Pathological diagnosis was made as fibrolipoma with osseous / chondroid differentiation . A 59-year - old man presented with a relatively painless hard mass on the left side of the lower labial vestibule surface, which appeared the sense of incompatibility 2 months prior to consultation . Examination showed a well- defined hard nodular mass, approximately 5 mm in diameter, and freely mobile, and covered with mucosa of normal aspect and color . There was no tenderness, no sign of inflammation . To retrieve more - detailed relativity with the surrounding tissue, ct was taken and showed a lesion in the left side of the lower lip that appeared to be a small mass with areas of little calcification within . Gross examination showed a yellowish soft to hard smooth mass measuring 9 5 5 mm in size . Microscopically, the tumor consisted of a well - circumscribed mass of mature fat cells supported by fibrous connective tissue septa and myxoid tissue characterized by spindle cells . A focal island of consecutive chondroid and woven bone component was surrounded by spindle / fusiform - shaped mesenchymal cells throughout the lesion (figure 2). The patients made an uneventful recovery, and after 15 years (case 1) and 1-year (case 2) follow - up, there was no sign of recurrence . All chondrocytes were stained for s-100 &, some for runx-2 (figure 3(a)) and some of the outer layer for sox-9 . Spindle cells were divided into 2 groups; peripheral spindle cells around osteo / chondroid tissue and distant spindle cells inside the myxoid area . Peripheral spindle cells around chondroid tissue stained diffusely for s-100 & and sox-9 (figure 3(b)), though peripheral spindle cells around osteoid tissue only stained for runx-2 (figure 3(c)). Distant spindle cells inside the myxoid area stained diffusely for s-100 and focally for s-100, myod, sox-9, and cd34 . As for the number of vessels detected by cd34, there were 5.0 vessels in this tumor, and there was no differences among the inside areas . None of the cases stained for either ema or fgf-1 or with vegf . Between 1995 and 2007, the files for all cases of oral lipoma at this department showed 909 cases of nonepithelial tumors in the oral soft tissue . Of all the lipoma cases, 27 occurred in males and 19 in females; their mean age was 53.8 years (range: 2872 years). Most lesions were located in the buccal mucosa (n = 16), margin of tongue (n = 10), lower lip (n = 7), gingiva (n = 4), and others . Microscopically, 28 cases (61.0%) were classified as lipoma and 18 (39.0%) as fibrolipoma . The literature from 1960 to 2008 of lipoma with osseous / chondroid differentiation was reviewed . Fourteen reports (including 16 cases) were described, and our cases are number 17 and 18 . Of all cases, 10 occurred in males and 8 in females; their mean age was 52.4 years (range: 2181 years). Most lesions were situated in the tongue (n = 6), lower lip (n = 5) and other areas . Microscopically, 6 cases (33.3%) had an osseous component, 11 cases (61.1%) had a chondroid component, and 1 case (our case, 5.6%) had an osseous / chondroid component . According to our review of the english - language literature, which included our cases, 6 cases were described as lipoma with osseous component [57, 9, 14, 15], 11 cases as chondroid component [4, 813, 16, 17] and 1 case as fibrolipoma with osseous / chondroid components . Our case 2 is namely the first report of fibrolipoma with osseous / chondroid differentiation . Osteolipomas are less common than chondrolipomas in the whole body, and almost the same tendency was described in oral lesion . Lipoma with osseous / chondroid differentiaion is extremely rare and occurs mainly in large long - standing lipomas [15, 19]. Case 1 also showed 28-year duration, and this might cause the osseous / chondroid to change into lipoma . As with many tumors, further indistinct etiology of osseous / chondroid change in lipoma has been discussed and most researchers mentioned that their origin is from different types of undifferentiated mesenchymal cells . The hypothesis is that the neoplastic transformation occurs in multipotential undifferentiated mesenchymal cells that later differentiate into lipoblasts, chondroblasts, or osteoblasts and fibroblasts . Another hypothesis is that only the adipose cells have a neoplastic transformation, and that the cartilage and bone is produced by differentiation of undifferentiated mesenchymal cells of stroma in chondroblasts or osteoblasts . Immunohistochemical results in this study indicated that there was no proliferative characteristic in mesenchymal (spindle) cells with small positive rates by ki-67 . Chondrocytes and peripheral spindle cells around chondroid tissue with positive findings for s-100,, and sox-9 had chondromatous characteristics . Sox-9 belongs to the sox (sry - related high - mobility group box) family of transcription factors and is a key regulator of developmental processes including chondrogenesis . Runx-2 is essential for skeletal mineralization when it stimulates osteoblast differentiation of mesenchymal stem cell and mature chondrocyte differentiation . Positive findings for runx-2 in spindle cells around osteoid components and some of chondrocytes indicated osteoblast / chondrocyte characteristics of them . These characteristics are synthesized and thought to be differentiation on account of small positive rate for ki-67 and negative findings for vegf and fgf-1 in this study . Osseous / chondroid formation occurred in the central part of these tumors in which a small amount of vessels existed . Multipotential undifferentiated mesenchymal cells had undergone differentiation to osteoblast / chondroblast by topical modification, such as nutrition disorder or asphyxia . The bone formation adjacent to chondroid tissue should not be dystrophic calcification because of its positive findings for runx-2 in spindle cells surrounding chondrocytes . These immunohistochemical results supported the conclusion that endochondral ossification and/or perichondral ossification had occurred in these tumor . These results supported lipoma with osseous / chondroid differentiation . Lipomas are benign mesenchymal neoplasms composed of mature adipocytes and are rare soft tissue tumors with a 2.2% incidence rate of the whole body . A 20% incidence rate of lipoma has been reported in the head and neck region . A 5% incidence rate in oral lesion of this study showed lower than in the head and neck region, but this rate is almost the same tendency compared to previous reports [1, 20, 21]. The average age of the patient of lipoma was 53.8 years old, and male - to - female ratio was 1.4: 1.0 in this study . Our study supports the current reports that lipomas are generally more common in males than in females (soft tissue), and occurred in adult patients most often between the ages of 40 and 60 years [1, 3]. The predominant locations were the buccal mucosa and the margin of the tongue and the lower lip, where there is plenty of adipose tissue . The patients' characteristics and location distribution, cheek and tongue, were similar to previous reports [1, 20, 22]. Microscopically, 28 cases (61.0%) were classified as lipoma and 18 (39.0%) as fibrolipoma . Fujimara have reported, but our case 2 is the first report of fibrolipoma with osseous / chondroid differentiation . The two cases this reports contributed to pile up of rare lipoma / fibrolipoma with osseous / chondroid differentiation case, and, in addition, immunohistochemical result is contributed to the clarification of the etiology.
Leydig cell tumours are rare stromal tumours of the testis accounting for 13% of testicular neoplasms . They exhibit a peak incidence in the preadolescent as well as in the older (> 50 years) age groups [1, 2, 3, 4]. The diagnosis of a malignant leydig cell tumour is not always easy since no definite histological criteria for malignancy exist . A 35-year - old man presented to our hospital with a 3-month history of a painless left testicular mass . Blood concentrations of chorionic gonadotrophin, fetoprotein and human placental lactogen were within the reference range . A left radical orchidectomy was performed and the specimen submitted for histopathological examination . On microscopic examination, a malignant leydig cell tumour was found . The cells displayed acidophilic cytoplasm, intranuclear inclusions and increased mitotic activity (> 3/10 hpf). Many cells with large atypical pleomorphic nuclei could also be observed (fig . 1, fig . 2). There was no angiolymphatic invasion, foci of necrosis or extension beyond the capsule of the testis . The immunohistochemical study showed that the tumour cells were positive for vimentin, melan a and inhibin, and negative for ckae1, ckae3, s100p, p63, cea, afp and actin . 3). Retroperitoneal lymph node dissection was suggested, but the patient declined further surgery . Leydig cell tumours are rare and only about 3% of them are bilateral . About 1520% of the patients already present with metastatic disease, particularly in the lymph nodes, lung and liver . These tumours may be hormonally active, and gynaecomastia is seen in 30% of the cases [2, 3, 6]. Leydig cell tumours in an undescended testis may exhibit only manifestation of endocrinological disorders such as gynaecomastia, impotence and loss of libido . However, among the 480 cases of leydig cell tumours reported in the literature, only 20 cases were associated with cryptorchidism, and there is no evidence that undescended testes are more prone to develop leydig cell tumours [2, 5, 7]. Features associated with malignancy include a large tumour size (5.7 cm), nuclear atypia, a mitotic count of> 3/10 hpf, foci of necrosis, angiolymphatic invasion, infiltrative margins, dna aneuploidy and an increased expression of ki67/mib-1 and p53 [2, 3, 8, 9, 10]. Our patient was young and did not fit into either of the known age incidence peaks . Microscopic features such as the severe nuclear atypia and increased mitotic activity (> 3/10 hpf) favoured the diagnosis of malignancy . However, there was no angiolymphatic invasion or extension beyond the capsule of the testis . Also, the small size of the tumour was remarkable.
The prevalences of asthma, allergic rhinitis and allergic eye disease in antalya, on the south coast of turkey, have been reported as 8.2%, 10.8% and 7.5%, respectively . Allergic diseases are most likely due to complex interactions between largely unknown genetic and environmental factors [36]. The micro - array techniques for the detection of specific ige have improved the diagnostic procedures for allergic diseases . A detailed knowledge of the sensitization pattern may have relevant implications for the prescription of specific immunotherapy . A number of epidemiologic studies have also supported a relationship between allergic rhinoconjunctivitis and diet, hygiene, and life - style, suggesting that environmental factors also impact the development of allergic rhinoconjunctivitis . Apoptosis is an active physiological process that can cause an inflammatory reaction and tissue damage, and is fundamental to maturation and homeostasis in the immune system . It can be induced passively, through lack of essential survival signals, or actively, through ligand - induced trimerization of specific death receptors of the tumor necrosis factor (tnf) receptor family, such as fas, the tnf receptor, or the tnf - related apoptosis - inducing ligand (trail) receptor . Trail also is able to prevent apoptosis through the actions of its decoy receptors, dcr-1 and dcr-2 . Various regulators of trail include fadd, iaps, bcl-2s, p53, and flips . Trail is present in cells involved in asthma, including eosinophils, mast cells, fibroblasts, and airway epithelial cells . It is expressed in airway remodeling and may be linked with the pathways of transforming growth factor beta1, which is thought to cause damage to the epithelium . The repair process of the epithelium is hindered as a result of increased apoptosis induced by tgf - beta1, which overlaps with the pathways of trail . Trail is also seen as the basis for a miracle drug for cancer because of its ability to selectively kill cancer cells . It has previously been reported that negative selection of t cells in the thymus is controlled by trail . For example, mice deficient in trail had a severe defect in thymic deletion of t cells and were hypersensitive to collagen - induced arthritis . Mast cells activation through fc epsilon ri cross - linking has a pivotal role in the initiation of allergic reactions . Ige - dependent activation increases trail - induced caspase-8 and caspase-3 cleavage, and regulates human mast cell apoptosis by fine - tuning anti - apoptotic and pro - apoptotic factors . Our study aimed to identify the role of strail in the pathophysiology of allergic rhinoconjunctivitis, and to explore whether allergen - specific subcutaneous immunotherapy treatment of allergic rhinoconjunctivitis patients altered any observed effect of strail . The study was conducted in antalya, turkey between 9 january 2009 and 28 january 2010 . The study was approved by the local ethics committee, and written consent was obtained from all patients and healthy volunteers . All patients were followed in the immunology and allergy clinic of antalya education and research hospital . Subjects with kidney disease, heart disease, liver disease, diabetes mellitus, cancer status, obesity, (body mass index (bmi) 30 kgm), and autoimmune disease were excluded clinically and serologically . The first group of 25 patients included 11 male and 14 female subjects with allergic rhinoconjunctivitis, having a combined mean age of 38.5612.03 years . The first group included 2 measurements in the same patients; group - ia represents data recorded before subcutaneous allergen - specific immunotherapy, and group - ib shows data recorded 12 months after the subcutaneous allergen - specific immunotherapy . All patients received immunotherapy every 4 weeks . The symptoms and severity of allergic reactions were recorded before and after treatment . A second group of 25 healthy individuals (11 male and 14 female) (group ii) had a mean age of 38.2312.21 years (table 1). Subjects with kidney disease, heart disease, liver disease, diabetes mellitus, cancer status, obesity (body mass index (bmi) 30 kgm), and autoimmune disease were excluded clinically and serologically . Blood samples were collected into 5 ml plain vacutainer tubes and centrifuged at 3000 g for 10 min . Serum ige levels, hepatitis markers (hbs ag, anti hbs, anti hcv) were evaluated in all patients . Total and specific ige levels were enumerated by fluoroenzyme immunoassay (immunocap - feia) using an immunocap kit (phar - macia, uppsala, sweden). Values above 100 ku / l and 0.35 ku / l for total and specific ige levels were considered abnormal . Serum strail levels in all individuals (patients and healthy controls) were measured by a sandwich enzyme - linked immunosorbent assay (diaclone, france). Skin prick tests on the forearm were performed in all patients, using standardized latex extract containing high ammonia natural rubber latex, and a full set of 10 common allergens . In addition, venom spt was performed on 1 patient based on the subject s clinical history . Student s t test was used for comparison of controls and patient groups (group ia and group ib). The study was conducted in antalya, turkey between 9 january 2009 and 28 january 2010 . The study was approved by the local ethics committee, and written consent was obtained from all patients and healthy volunteers . All patients were followed in the immunology and allergy clinic of antalya education and research hospital . Subjects with kidney disease, heart disease, liver disease, diabetes mellitus, cancer status, obesity, (body mass index (bmi) 30 kgm), and autoimmune disease were excluded clinically and serologically . The first group of 25 patients included 11 male and 14 female subjects with allergic rhinoconjunctivitis, having a combined mean age of 38.5612.03 years . The first group included 2 measurements in the same patients; group - ia represents data recorded before subcutaneous allergen - specific immunotherapy, and group - ib shows data recorded 12 months after the subcutaneous allergen - specific immunotherapy . All patients received immunotherapy every 4 weeks . The symptoms and severity of allergic reactions were recorded before and after treatment . A second group of 25 healthy individuals (11 male and 14 female) (group ii) had a mean age of 38.2312.21 years (table 1). Subjects with kidney disease, heart disease, liver disease, diabetes mellitus, cancer status, obesity (body mass index (bmi) 30 kgm), and autoimmune disease were excluded clinically and serologically . Blood samples were collected into 5 ml plain vacutainer tubes and centrifuged at 3000 g for 10 min . Serum ige levels, hepatitis markers (hbs ag, anti hbs, anti hcv) were evaluated in all patients . Total and specific ige levels were enumerated by fluoroenzyme immunoassay (immunocap - feia) using an immunocap kit (phar - macia, uppsala, sweden). Values above 100 ku / l and 0.35 ku / l for total and specific ige levels were considered abnormal . Serum strail levels in all individuals (patients and healthy controls) were measured by a sandwich enzyme - linked immunosorbent assay (diaclone, france). Skin prick tests on the forearm were performed in all patients, using standardized latex extract containing high ammonia natural rubber latex, and a full set of 10 common allergens . In addition, venom spt was performed on 1 patient based on the subject s clinical history . Student s t test was used for comparison of controls and patient groups (group ia and group ib). In this clinical follow - up study, 25 patients were already receiving subcutaneous immunotherapy, and these subjects were included for further analysis at our clinic . The mean ige levels were: group ia 699.505 iu / ml; group ib 164.115 iu / ml; and group ii 41.08 iu / ml . There was a statistically significant difference between the values of the research group before and after immunotherapy (p=0/0005). Prick tests in all patients in group i were detected in mite, olive and grass allergy . These results correlated with specific ige, and hepatitis markers were negative in all patients . As shown in figure 1, significant difference was seen in the mean values of strail in allergic rhinitis patients before immunotherapy (n=25; 939.85352.52 pg / ml), afterc immunotherapy (n=25; 628.93170.5 pg / ml) and healthy controls (n=25; 612.64135.6 pg / ml). There was a statistically significant difference between the values of the research group before and after immunotherapy (p=0.001). While there was a statistically significant difference between the pre - treated group and control group (p<0.0001), there was no difference between the post - treated group and healthy individuals (p=0.801). As shown in table 3, there was a statistically significant difference between the values of the research group before and after immunotherapy (grasses mixture, barley mixture, oleaauropeae, d. pteronyssinus, d. farinae) in this clinical follow - up study, 25 patients were already receiving subcutaneous immunotherapy, and these subjects were included for further analysis at our clinic . The mean ige levels were: group ia 699.505 iu / ml; group ib 164.115 iu / ml; and group ii 41.08 iu / ml . There was a statistically significant difference between the values of the research group before and after immunotherapy (p=0/0005). Prick tests in all patients in group i were detected in mite, olive and grass allergy . These results correlated with specific ige, and hepatitis markers were negative in all patients . As shown in figure 1, significant difference was seen in the mean values of strail in allergic rhinitis patients before immunotherapy (n=25; 939.85352.52 pg / ml), afterc immunotherapy (n=25; 628.93170.5 pg / ml) and healthy controls (n=25; 612.64135.6 pg / ml). There was a statistically significant difference between the values of the research group before and after immunotherapy (p=0.001). While there was a statistically significant difference between the pre - treated group and control group (p<0.0001), there was no difference between the post - treated group and healthy individuals (p=0.801). As shown in table 3, there was a statistically significant difference between the values of the research group before and after immunotherapy (grasses mixture, barley mixture, oleaauropeae, d. pteronyssinus, d. farinae) allergen - specific immunotherapy has been used in the management of allergic diseases for nearly 100 years . The results of our previous study suggest that in our region 51.8% of the allergens determined by prick test are mite and 42.3% are pollens . Pollen positivity rate of the cases that had immune - therapy was 61.8%, and mite positivity rate was 60.4% . Allergens are known to exhibit regional variation in expression, which suggests that the allergen profiles and skin prick tests should be designed with reference to individual locales . Allergen - specific immunotherapy treatment significantly reduced the nasal symptom score across all group i patients studied . In this study, spt was a statistically significant difference between the values of the research group before and after allergen - specific subcutaneous immunotherapy (grasses mixture, barley mixture, oleaauropeae, d. pteronyssinus, d. farinae). Our earlier studies provided a novel perspective on severe persistent allergic asthma and the effect of anti - ige treatment, using as markers serum soluble tnf - related apoptosis - inducing ligand, total antioxidant capacity, hydrogen peroxide, malondialdehyde, ceruloplasmine oxidase activity, high sensitive c reactive protein and total nitric oxide concentrations measurements [1316]. In our previous study we found that the trail levels in variances of the patients who had the effective anti ige treatment were significantly lower than the healthy controls . Engagement of the fas / fasl system has not yet been shown to contribute to increase apoptosis . Recent interest has focused on the molecule trail, which is involved in the pathophysiology of different diseases, including cancer, diabetes mellitus, autoimmune diseases, and inflammation [1721]. Trail also present in cells that involved in asthma including eosinophils, mast cells, fibroblasts, and airway epithelial cells . It is expressed in airway remodeling and may be linked with the pathways of transforming growth factor - beta (tgf-), which is thought to cause damage to the epithelium . The repair process of the epithelium is hindered as a result of increased apoptosis induced by tgf - beta, which overlaps with the pathways of trail . These results reflect the different mechanism(s) in the pathogenesis of allergic diseases by the regulation of apoptosis . Desloratadine (dcl) is a non - sedating antihistamine approved for the treatment of allergic rhinitis . Patients in groups i - a and b had a dcl usage history during the exacerbation phase from may to november . Blood samples from the patients were obtained in january, when they would be expected to have fewer allergic symptoms . Mast cells (mc) play a key role in allergy and are involved in several chronic inflammatory diseases . Furthermore, they are involved in innate immunity and in tissue repair [2427]. The regulation of mc numbers, as of any other normal cells, depends on both their generation rate and survival time within tissues . The critical event in allergic reactions is allergen - induced crosslinking of specific ige molecules bound to fc_ri receptors on the mc surface, which triggers mc degranulation and release of inflammatory mediators . Non - ige - mediated activation may also contribute to continued degranulation of mc during the late phase of allergic reactions . Allergic mechanism are involved in the increased susceptibility of human mc to trail - induced apoptosis after ige - dependent activation . In this study, ige levels had a statistically significant difference between the values of the research group before and after allergen - specific subcutaneous immunotherapy . Taken together, our results and those of others, suggest that characterization of the specific receptor systems activated, and the pro - inflammatory factors regulated, by trail in vivo may lead to the development of novel therapeutic strategies for diseases as diverse as infection, autoimmunity, and allergy.
Calcimimetics provide an alternative approach to the traditional therapy with active vitamin d and phosphate binders for the management of secondary hyperparathyroidism (shpt). Type ii calcimimetics are positive allosteric modulators of the parathyroid gland calcium - sensing receptor (car) that increase its sensitivity to serum calcium, so that lower concentrations of calcium are sufficient to mediate signalling through the car . This suggests that, despite the reduced car expression observed in hyperplastic parathyroid tissues, calcimimetics have the potential to control even advanced disease by enhancing the function of the remaining car [47]. In addition, calcimimetics have the potential to influence both car expression and car functions that have been implicated in the progression of disease, particularly effects on parathyroid hormone (pth), cell proliferation and hyperplasia . These characteristics suggest that targeting the car presents a therapeutic option for the stabilization of early disease, as well as remaining a viable therapeutic target in the face of advancing disease . In addition to being effective in patients with mild to moderate disease, calcimimetics may be the most viable option for chronic kidney disease (ckd) patients with advanced hyperparathyroidism, who are often refractory to standard calcitriol therapy [810]. The data below summarize preclinical findings providing a rationale for the clinical use of calcimimetics . Administration of cinacalcet or the calcimimetic r-568 to 5/6 nephrectomized rats prevented hyperplasia or increased car expression . Interestingly, in an in vitro study, the calcimimetic cinacalcet suppressed pth secretion in human parathyroid cells with advanced hyperparathyroidism, even though car expression was significantly diminished . Expression of vitamin d receptor (vdr) mrna is reduced in the parathyroid gland of hypocalcaemic rats compared with normocalcaemic rats . Because the vdr is a transcriptional repressor of pth recent studies indicate that type ii calcimimetics, in the presence of low concentrations of calcium, can up - regulate vdr mrna . Rats injected with a calcimimetic (r-568, 1 mg / kg intravenously 3 and 6 h before euthanasia) exhibited an increase in vdr mrna expression compared with controls (p <0.001), with a marked increase observed when r-568 and calcitriol were coadministered (figure 1). Rats were treated with r-568 (1 mg / kg intravenously 3 and 6 h before euthanasia; n = 22), calcitriol (10 pmol / kg intraperitoneally every 30 min for 5.5 h before euthanasia; n = 22), or a combination of r-568 and calcitriol (n = 22) before euthanasia and measurement of vdr mrna by quantitative real - time polymerase chain reaction . A control group (n = 20) received no treatment . Mean serum concentrations of ionized calcium and pth are also included . Data are mean standard error . Adapted with permission from rodriguez et al . . These data indicate that, in addition to regulating pth levels and restoring car density, calcimimetics can up - regulate vdr expression in parathyroid cells . This effect of calcimimetics on vdr expression is of clinical importance because it may result in an additive interaction between calcimimetic and vitamin d sterol therapy . Although parathyroid cells are relatively quiescent under normal conditions, the mineral and hormonal imbalances characteristic of shpt, particularly low serum calcium and high serum phosphorus, drive cell proliferation leading to polyclonal and oligoclonal cell expansion . The importance of maintaining calcium homeostasis is evidenced in weanling rats fed a low - calcium diet for 10 days, in which the number of proliferating cell nuclear antigen (pcna)-positive parathyroid cells was increased sixfold compared with controls . Calcimimetics, in the presence of low serum calcium, can hold proliferation in check, as evidenced by decreased parathyroid cell proliferation in uraemic rats following treatment and their ability to cause regression of advanced hyperplasia in some experimental animal models . Long - term administration of the calcimimetic cinacalcet significantly reduced the number of proliferating (pcna - positive) cells and glandular hyperplasia, as measured by weight, in 5/6 nephrectomized rats . This effect was parathyroid tissue - specific and was due to a direct effect on cell growth, rather than to induction of apoptosis . Taken together, the mechanistic data presented here, and reviewed in greater detail in another article in this supplement (riccardi and martin), suggest that calcimimetics have the potential to retard shpt disease progression . Preclinical studies in uraemic rats indicate that calcimimetics can modify the defining parameters in shpt by significantly lowering pth and blood - ionized calcium in a dose - dependent manner without inducing significant changes in serum phosphate . Administration of cinacalcet or the calcimimetic r-568 to 5/6 nephrectomized rats prevented hyperplasia or increased car expression . Interestingly, in an in vitro study, the calcimimetic cinacalcet suppressed pth secretion in human parathyroid cells with advanced hyperparathyroidism, even though car expression was significantly diminished . Expression of vitamin d receptor (vdr) mrna is reduced in the parathyroid gland of hypocalcaemic rats compared with normocalcaemic rats . Because the vdr is a transcriptional repressor of pth recent studies indicate that type ii calcimimetics, in the presence of low concentrations of calcium, can up - regulate vdr mrna . Rats injected with a calcimimetic (r-568, 1 mg / kg intravenously 3 and 6 h before euthanasia) exhibited an increase in vdr mrna expression compared with controls (p <0.001), with a marked increase observed when r-568 and calcitriol were coadministered (figure 1). Rats were treated with r-568 (1 mg / kg intravenously 3 and 6 h before euthanasia; n = 22), calcitriol (10 pmol / kg intraperitoneally every 30 min for 5.5 h before euthanasia; n = 22), or a combination of r-568 and calcitriol (n = 22) before euthanasia and measurement of vdr mrna by quantitative real - time polymerase chain reaction . A control group (n = 20) received no treatment . Mean serum concentrations of ionized calcium and pth are also included . P <0.01 versus control; p <0.05 versus calcimimetic; p <0.05 versus control . Data are mean standard error . Adapted with permission from rodriguez et al . . These data indicate that, in addition to regulating pth levels and restoring car density, calcimimetics can up - regulate vdr expression in parathyroid cells . This effect of calcimimetics on vdr expression is of clinical importance because it may result in an additive interaction between calcimimetic and vitamin d sterol therapy . Although parathyroid cells are relatively quiescent under normal conditions, the mineral and hormonal imbalances characteristic of shpt, particularly low serum calcium and high serum phosphorus, drive cell proliferation leading to polyclonal and oligoclonal cell expansion . The importance of maintaining calcium homeostasis is evidenced in weanling rats fed a low - calcium diet for 10 days, in which the number of proliferating cell nuclear antigen (pcna)-positive parathyroid cells was increased sixfold compared with controls . Calcimimetics, in the presence of low serum calcium, can hold proliferation in check, as evidenced by decreased parathyroid cell proliferation in uraemic rats following treatment and their ability to cause regression of advanced hyperplasia in some experimental animal models . Long - term administration of the calcimimetic cinacalcet significantly reduced the number of proliferating (pcna - positive) cells and glandular hyperplasia, as measured by weight, in 5/6 nephrectomized rats . This effect was parathyroid tissue - specific and was due to a direct effect on cell growth, rather than to induction of apoptosis . Taken together, the mechanistic data presented here, and reviewed in greater detail in another article in this supplement (riccardi and martin), suggest that calcimimetics have the potential to retard shpt disease progression . Preclinical studies in uraemic rats indicate that calcimimetics can modify the defining parameters in shpt by significantly lowering pth and blood - ionized calcium in a dose - dependent manner without inducing significant changes in serum phosphate . One of the primary treatment goals of shpt is to restore mineral and hormone balance . The national kidney foundation kidney disease outcomes quality initiative (kdoqi) has established recommendations regarding levels of serum calcium, phosphorus, calcium phosphorus product (ca p), and intact pth (ipth) for patients based on their degree of kidney function . As kidney function decreases, mineral homeostasis is increasingly perturbed, resulting in increased pth, serum phosphorus, and ca p. as this dysregulation progresses, the response to standard therapy worsens . Consequently, pth can become high enough to increase mortality and impede adequate control of phosphate [1921]. These findings underscore the need for new or adjunct treatments with improved efficacy over current modalities that can stabilize disease progression during a long - term course when initiated early, but can also maintain effectiveness in patients with more severe disease . Because of the ability of calcimimetics, demonstrated in animal studies, to modulate disease - related processes through multiple mechanisms of action implicated in the development of disease, this therapeutic modality appears to be well suited for use in combination with moderate doses of vitamin d as the primary mode of treatment in shpt management . As described earlier, calcimimetics up - regulate car and vdr expression and inhibit parathyroid cell proliferation and hyperplasia . These multiple mechanisms of action may allow calcimimetics to (1) maintain effectiveness during long - term therapy; (2) maintain enhanced efficacy in patients with severe disease despite decreased car and vdr expression and (3) halt development of severe disease if initiated early in patients with mild or moderate shpt . Phase 3 clinical trials in shpt patients with advanced disease have shown that cinacalcet decreased pth, as well as serum calcium, phosphorus and ca p . Analysis of the data from these trials has been performed to investigate the efficacy of cinacalcet in the treatment of both early- and later - stage shpt and to investigate the potential of long - term cinacalcet therapy to stabilize disease progression . Results of these analyses, presented below, suggest that calcimimetics have the ability to address these unmet therapeutic needs . A post - hoc analysis of the combined results of three 26-week, placebo - controlled, phase 3 studies evaluating the efficacy and safety of a once - daily dose of cinacalcet for the treatment of shpt demonstrated the impact of therapy initiated in early or advanced disease . These studies included adult patients on maintenance dialysis who had ipth levels 300 pg / ml despite standard treatment with active vitamin d and phosphate binders . Patients received standard therapy in combination with cinacalcet (n = 546) or placebo (n = 408). Patients included in the analysis were divided into eight subgroups according to their baseline ipth level, which was used as a marker of disease severity (300 to> 1000 pg / ml). Findings from this analysis indicated that the use of cinacalcet (30 mg titrated up to 180 mg) significantly decreased both ipth and ca p levels regardless of disease severity at baseline, compared with standard therapy (figure 2). The progressive nature of shpt, even when patients receive standard therapy, is evidenced by positive changes from baseline in ipth seen in these patients (figure 2a). Furthermore, the effect of cinacalcet on ca p (figure 2b) and serum phosphorus levels was more pronounced in patients with more severe disease, suggesting a more significant impact of cinacalcet on bone turnover in these patients . Percentage change in (a) ipth and (b) ca p by disease severity . Patients were treated with cinacalcet (beginning at 30 mg with possible titration up to 180 mg; n = 546) or placebo (n = 408) for 26 weeks . Patients were divided into groups according to baseline ipth levels (pg / ml). However, although these data demonstrate that cinacalcet is effective in reducing serum ipth and ca p, even in patients with severe disease, these patients remain less likely to achieve their kdoqi targets than patients with less severe disease . After 6 months of treatment with cinacalcet, 76% of patients with a baseline ipth of 300500 pg / ml achieved a serum ipth level of <300 pg / ml compared with 55% of patients with a baseline ipth of 500800 pg / ml and 16% of patients with a baseline ipth of> 800 pg / ml . Similarly, 54% of patients with a baseline ipth of 300500 pg / ml achieved an ipth of <300 pg / ml and their ca p target (<55 mg / dl), compared with 36% and 9% of patients in the 500800 and> 800 mg / ml groups, respectively . These data suggest that early treatment of shpt provides the best opportunity to maintain control of serum pth, calcium, phosphorus and ca p. treatment with cinacalcet for up to 1 year resulted in increased attainment of kdoqi targets compared with standard therapy . After 12 months, 66% of patients on dialysis receiving cinacalcet achieved an ipth of 300 pg / ml compared with 15% in the placebo group . Similarly, 55% of patients receiving cinacalcet achieved their ca p target (<55 mg / dl) compared with 43% in the placebo group . Furthermore, the proportion of patients able to achieve both an ipth of 300 pg / ml and their ca p target was considerably higher in the cinacalcet group than in the placebo group; 42% of patients in the cinacalcet group achieved both targets simultaneously, whereas only 8% of patients in the placebo group reached both targets (figure 3a). Proportion of patients achieving serum ipth 300 pg / ml and ca p <55 mg / dl during treatment with either cinacalcet (n = 122) or placebo (n = 126). (a) achievement of serum ipth 300 pg / ml and ca p <55 mg / dl after 1 year . (b) maintenance of serum ipth 300 pg / ml after 6 and 12 months of treatment . (c) maintenance of combined serum ipth and ca p target after 6 and 12 months of treatment . Adapted with permission from frazo et al . . In this study, treatment with cinacalcet not only improved the ability of patients to achieve their kdoqi targets, but also their ability to maintain these targets . Seventy - one percent of cinacalcet - treated patients were able to achieve an ipth of 300 pg / ml after 6 months of cinacalcet treatment . Of the group who achieved an ipth of 300 pg / ml at 6 months, 82% also maintained an ipth of 300 pg / ml after 1 year . In contrast, in the standard care group, 13% of patients achieved an ipth concentration of 300 pg / ml, with 53% of this group continuing to maintain an ipth 300 pg / ml after 1 year (figure 3b). Cinacalcet also improved maintenance of the combined ipth and ca p targets . In the cinacalcet group, 52% of patients reached the combined target after 6 months compared with 7% in the standard care group . Of these patients, 59% in the cinacalcet group maintained the combined target after 1 year compared with just 22% in the standard care group (figure 3c). These data demonstrate that cinacalcet effectively reduces and maintains the four critical disease biomarkers associated with shpt in ckd patients . Cinacalcet, therefore, reduces the shifting in and out of targets often observed in patients using standard therapy . Open - label extension studies have evaluated the ability of cinacalcet to stabilize disease in shpt patients for up to 4 years [2628]. These analyses were based on a series of double - blind randomized clinical trials in which patients received standard care treatment in combination with either placebo or cinacalcet for 26 or 52 weeks, after which patients could receive open - label cinacalcet treatment . In one such open - label extension study, serum ipth was found to progressively increase in patients treated with standard care for 52 weeks . After open - label cinacalcet treatment was started, serum ipth was rapidly reduced to a level matching that of cinacalcet - treated patients . (n = 59) had achieved an ipth of 300 pg / ml at each study visit . Serum ca p did not increase during this period . In a separate study, a limited number of patients (n = 21) taking cinacalcet were followed up for as long as 4 years . A substantial upward trend in ipth was noted in control patients who received standard therapy during the first year of treatment in this study, before switching to cinacalcet therapy, with levels increasing from approximately 600850 pg / ml . In these patients, cinacalcet reduced and maintained ipth levels throughout the follow - up period (figure 4). After 4 years, the mean serum ipth was 396 pg / ml, 65% of patients achieved a serum ipth of 300 pg / ml, and the mean serum ca p was 50.5 mg / dl . These findings again highlight the progressive nature of shpt in patients receiving standard therapy as first - line treatment, as well as the inability of traditional therapies to effectively control or stabilize disease in both the short and long term . These and other clinical observations discussed in this section are also consistent with preclinical evidence supporting the car as a therapeutic target to optimize the achievement of treatment goals and slow or prevent disease progression . Patients received double - blind treatment with either cinacalcet (n = 9) or placebo (n = 12) for 1 year followed by a 3-year open - label extension treatment with cinacalcet . Calcimimetics are highly specific therapeutic agents that effectively lower concentrations of plasma pth and partially correct disturbances in mineral metabolism that are characteristic of shpt . The mechanism of action involves allosteric modification of the car, rendering it more sensitive to calcium signalling, thereby helping counteract the decreased car expression and prevent and halt the progression of parathyroid hyperplasia . In addition to effects on car signalling, new experimental studies indicate that calcimimetics may act to increase vdr expression . These studies raise the possibility that treatment with calcimimetics might improve the sensitivity of the parathyroid glands to calcitriol and vitamin d analogues, allowing reduced doses to be used and thus minimizing the risk of hypercalcaemia and hyperphosphataemia . Preclinical mechanistic studies with calcimimetics have suggested that this modality may arrest disease progression, even in patients with advanced disease at treatment onset, and stabilize shpt, thereby allowing improved maintenance of kdoqi targets ., cinacalcet was found to be effective for the reduction and long - term maintenance of ipth, calcium and phosphorus levels in patients with shpt . Cinacalcet has been shown to allow more patients to achieve and maintain kdoqi targets compared with standard therapy, and extended use (up to 4 years) has been shown to be effective and well tolerated.
Many neurodegenerative disorders are characterized by their presence in neural tissue of aberrant protein aggregates (see table 1). In general, these aggregates arise after the modification of a native protein . That modification could result in a conformational change of the native protein that promotes the aberrant aggregation . The most studied model of this mechanism has been the prion protein where a change from an alpha helix to beta sheet structure facilitates the polymerization of a protein with a different conformation that appears to have a cytotoxic effect . In a similar way, conformational changes between a native protein and its aberrant protein counterpart with capacity for self - assembly have been studied in many neurodegenerative diseases . Among the most common techniques used for these analyses are x - ray diffraction, nuclear magnetic resonance, circular dichroism, or fourier - transformed infrared spectroscopy . Similarly, to the case of prion protein, in some disorders a change from alpha helix to beta sheet conformation has been suggested to cause protein aggregation (table 1), probably because in alpha helix, intramolecular hydrogen bonds could occur whereas intramolecular hydrogen bonds are facilitated in beta sheet conformation, facilitating protein aggregation . However, there is one case, the formation of aberrant aggregates of tau, where the aggregated protein contains also a high proportion of alpha helix structure . Although, in some cases, like that of prion protein, the formation of aberrant aggregates of protein could result in a toxic effect in the affected neurons, in other cases, like huntingtin aggregates, the formation of the aberrant aggregates could be a survival response of the affected neurons . In other neurodegenerative diseases, it will be of interest to know if protein aggregation is synonymous of cell toxicity or not . Protein conformation could also play a role in a possible toxic mechanism . In this way, a protein with a high proportion of alpha helix and hydrophobic regions could be inserted in cell membrane promoting toxic effects . Additionally, the presence of aberrant polymers could affect the protein degradation cell machinery (the proteasome complex), decreasing its activity and promoting a toxic effect . Recently, some good reviews have been published on protein aggregation and neurodegenerative disorders [6, 7]. In this review we will mainly focus on those aggregates assembled from tau protein, aggregates that could be present in the neurological disorders known as tauopathies (for a review see) (table 1). It has been described that large amounts of native or unmodified tau protein were enough to promote tau assembly into fibrillar polymers resembling those found in ad [912]. Thus, obviously, an increase in tau concentration will favour the formation of tau polymers . Recently, it has been reported that not all the brain areas have a similar amount of tau protein . Thus, it suggests a different probability in the formation of tau polymers in different brain regions . The amount of tau will be the consequence of its synthesis and its degradation . Changes in transcription have been indicated for other proteins related to neurodegenerative disorders, where a tata binding protein may play a role . Tau degradation may take place through the proteasome complex [15, 16] and it has been suggested that such degradation could be regulated by posttranslational modifications occurring in tau molecule, like its phosphorylation . Also, tau degradation by other proteases could be regulated by its level of phosphorylation . It should be also indicated that in some cases like parkinson's disease or lafora disease, mutations in the e3 ubiquitin ligases like parkin or malin will result in the appearance of aberrant protein aggregates . A conformational change, that could be followed by antibodies that react with tau molecule after that conformational change [2124] has been also suggested to be required for the transition tau monomer - tau polymer . Also, it has been suggested that different posttranslational modifications like phosphorylation, glycation, or truncation, may play a role in the formation of tau polymers . Due to the alternative splicing of its heterogenous (or nuclear) rna, different tau isoforms could be expressed and, therefore, different tau aggregates with different tau isoforms in different tauopathies could occur, but we will not discuss this point here . For further information see ., it has been suggested that removal of the amino and/or carboxy terminal regions, leaving the tubulin binding region will facilitate tau polymerization [21, 22, 28]. Some work has been done in vitro and in vivo [30, 31] about a possible role of tau phosphorylation on tau assembly, suggesting that in some conditions tau phosphorylation may increase the capacity of tau for its self - assembly . Not only an increase in serine / threonine phosphorylation of tau could regulate its aggregation but also an increase in tau tyrosine phosphorylation may increase the formation of tau aggregates . This assembly process may involve the formation of oligomers, filaments, and aggregates of filaments (tangle - like structures). In the formation of these aggregates of filaments, glycation may play a role . The possible relation between phosphorylation and tau aggregation has been studied in transgenic mice expressing human tau bearing some mutations found in human fronto - temporal dementia linked to chromosome 17 (ftdp-17). In this mouse, tau phosphorylation mainly occurs by gsk3 . When a specific inhibitor of this kinase, lithium, was given to the transgenic mice no tau phosphorylation was found, and in addition no aggregation of the protein was detected suggesting a correlation between tau phosphorylation and aggregation in this model . This result was supported by an additional experiment using another mouse also expressing human tau with a ftdp-17 mutation . Alternatively, protein chaperones, acting on tau or in phosphotau, could modify the level of tau aggregation, examples could be the protein 14 - 3 - 3, musashi-1, or pin-1 [37, 38]. The chaperone associated ubiquitin ligase chip could be able to target phosphotau for proteasomal degradation [16, 18]. It has been described that tau binding to microtubules is regulated by the level of tau phosphorylation at some specific sites . It is known that hyperphosphorylated tau binds with less affinity to microtubules resulting in the decrease in the interaction with microtubules, in a decrease of microtubule stability, and probably in a microtubule dysfunction inside the cell that could result in a toxic effect . Also, tau phosphorylation could result, as indicated above, in a decrease of its proteolysis . More recently, it has been indicated that expression of a tau mutant (p301l) could result in an increase of its phosphorylation, since once it is phosphorylated, that mutation can prevent the binding of those phosphatases involved in its dephosphorylation . This phosphotau could have a decreased capacity for microtubule binding and it could be toxic for the cell . Additionally, it has been indicated that hyperphosphorylated tau can cause neurodegeneration, in the absence of large tau aggregates . On the other hand, only the overexpression of wild - type human tau in a mouse is sufficient to cause tau phosphorylation, aggregation, and neural toxicity . On the other hand, it has been suggested that tau phosphorylation may represent a protective function in ad . Sometime ago, the development of tau pathology, related with dementia in ad, was clearly described by braak and braak by following the development of neurofibrillary lesions at different stages of the disease . Also, the formation of neurofibrillary (tau aggregates) pathology within those neurons of hippocampus and cerebral cortex affected at different stages was found . These neurons could degenerate yielding extracellular ghost tangles (enft). In the hippocampus, an inverse relation has been found between the number of enft and the number of surviving neurons [4851]. It suggests that neurons that degenerate, have previously developed tau aggregates . On the other hand, it has been indicated that neurons bearing neurofibrillary lesions could survive for a long period of time, and, by comparing with other neurodegenerative disorders, like huntington disease, it can be suggested that tau aggregates could protect against neurodegeneration by sequestering toxic (phospho?) Monomeric tau that could be present in a high amount inside a cell in pathological conditions . Also, it has been suggested, using a transgenic mouse model, that behavioural (memory) deficits could be unrelated to the formation of tau polymers, although, more recently, the discussion of those experiments suggested that hyperphosphorylated, aggregated tau intermediates could be the ones that cause neurodegeneration . In this way, the implication of different types of protein aggregates in neurodegeneration has been extensively discussed [19, 54]. A possibility about the existence of neurotoxic tau intermediate aggregates in human tauopathies is based in the fact that patients with ftdp-17 show an extensive neurodegeneration with a high level of tau phosphorylation but with a low number of tangles . In any case, even if the formation of tau aggregates has a protective function for the neurons, that function is not working well, as described by braak and braak, and afterwards by delacourte et al, indicating a correlation between progression of tau pathology and progression of the disease . This idea is supported by those experiments indicating that neural loss and neurofibrillary tangle number increase in parallel with the progression of the disease . Similar results have been described in other neurological disorders like brain encelphalopathies, where the formation of aberrant polymers are related to the onset of neurodegeneration; whereas this is far from clear in other disorders like huntington disease . Examples of neurological diseases where aberrant protein aggregates are found, and the suggested conformations in the aberrant aggregates are indicated.
Only those contraceptive methods are new in which synthetic steroid is used . Regarding the current status of growth, the world population is doubled every 54 years; however, in poor countries, population is doubled in less than 20 years . To complete and preserve personal health during pregnancy, the optimal use of contraceptive methods is effective, and planning is necessary before pregnancy . The rate of pregnancy in women with a high potency for fertility who use no contraceptive method is 90% during one year . Therefore, conscious and accurate decision making for pregnancy and precise care play an important role in decreasing the maternal mortality . Induced abortion that can be the important complication followed by unwanted pregnancy play a significant role in the incidence of infection, fever, risk of the next premature delivery, low birth weight, and infertility [1, 4]. Pregnancy is a temporary crisis that creates deep mental, physical, and behavioral changes in a woman . Conscious and accurate decision making for pregnancy, continuation, and precise care play an important role in declining the maternal mortality . According to the studies by world health organization, close to one - third of the pregnancies in the third world countries considering the fact that induced abortion may create some significant complications such as infection, septic shock, fever, risk of the next premature delivery, low birth weight, and infertility, it can also cause some anatomic complications resulting from the surgery such as uterus, bladder, and intestinal rupture [1, 4]. Planning for pregnancy can help the safety of childbirth, and unplanned pregnancies increase the maternal mortality . Despite the attempts by the authors in health centers, some unwanted pregnancies occur that can danger the women . Therefore, we decided to evaluate some factors related to unwanted pregnancy in women referred to health centers . It was a cross - sectional study on 400 randomly pregnant women, who were referred to several clinics and health center in ahwaz city during 2010 . Data was collected through interview and filling up a designed questionnaire containing demographic characteristics, fertility, and so forth . The mean age of women with unwanted pregnancy was 27.5 5.7 years, and in women with intended pregnancy was 24.6 4.5 years . The percentage of the older women (35 years) in unwanted pregnancy was 3 times of intended pregnancy which was statistically significant . Most of pregnant women lived in urban area (70.1%), and the percentage of rural women who intended pregnancy was higher (35.6%). The educational level in the subjects was 36% in secondary school and 44% in high school . 37% of subjects with unwanted pregnancy were illiterate and primary school (table 1). The findings have shown that most of pregnant women were housewives (82%), and their husbands had self - employment (40%), and 7.1% of subjects with unwanted pregnancy had unemployed husband . Half of the women with unwanted pregnancy had low economic status, while, in women with intended pregnancy, this rate was only 20% . According to the interviews, good relationship with regarding fertility characteristics, the findings showed that more than half of the women with unwanted pregnancy were in the third trimester 54%, (figure 1). 80% of unwanted pregnancy had more than two times pregnancies, but the percentage in intended pregnancy was 38% . This study showed that more than half of the pregnant women had used one of the contraceptive methods before the recent pregnancy, and 30% had used natural (interrupted) methods . The most percentage of women with unwanted pregnancy used unreliable methods like interrupted method (59.1%). The incidence of pregnancy followed by the consumption of contraceptive pills in women unwanted pregnancy was 16% . This study has shown that 26% of women who wish to become pregnant had more knowledge and performance about pregnancy health 26%, and the low levels of knowledge and performance were more observed in unwanted pregnancy . The incidence of unwanted pregnancy is different in the world, but it has the same undesired outcomes . Our study has shown that prevalence of unwanted pregnancy was 26%, and similar conducted studies indicated that the incidence rate of unplanned pregnancy was 25%, 30%, 43%, and 52% [69]. In this study, relative decrease in the incidence of unwanted pregnancy in our country in comparison with the other countries may be due to the hopeful success of health centers . Considering the point that intended pregnancy which results in a healthy childbirth from a healthy mother as an aim of midwifery science [10, 11], pregnancy must be occurred based on the accurate and conscious decision and according to the physical, mental, economic, social, and cultural status [2, 3]. Alenova in a study after evaluating the activities of consultation clinics guiding women for that the prevention of pregnancy states that prevention of unwanted pregnancy is more necessary in aged women, and it becomes more vital with the increase of age . Mohammadloo in his study declares that more than half of the unwanted pregnancies occur in women more than 30 years . One of the causes of not intending pregnancy is the age of more than 35 years that has been noted in other studies too [6, 8, 13, 14]. In the present study, low education has a close relationship with increased incidence of unwanted pregnancy, and, in a study by bennett et al . Most of the women have the ability to become pregnant for at least three decades in their life, but most of men are potentially fertile all over their life . In our study, the rate of unwanted pregnancy was higher in individuals with more number of children . Also, in similar studies, the increased prevalence of unwanted pregnancy is observed with an increase in the number of children, from 7.9% in childless women to 92.8% in women with 4 children or more . In our study, unwanted pregnancy was more observed in employee women, and some other studies have achieved this result too . Low income, poverty, unemployed husband, and inappropriate job play significant role in the incidence of unplanned pregnancy [7, 1519]. A study in zimbabwe has shown that women with unemployed husbands were more exposed to the unwanted pregnancy . Some studies in africa and new york indicated that unwanted pregnancy more occurred in poor, low income, and homeless women, which necessitates more concern about the poor women . Other studies in thailand and usa have found that one of the related factors with unwanted pregnancy is the relationship with the spouse; also they showed that good relationship with the spouse in women with unwanted pregnancy was less than intended cases . A study from london has found that planned pregnancy was more observed in couple with more strengthened union in marriage . It seems that couples' relationship is an important and positive factor which affects the increase of their cooperation in regarding the fertility health . It was indicated in this study that, in 20% of women with unwanted pregnancy, no contraceptive method was used, and more than half of them used unreliable methods of which failure was more observed in unwanted pregnancies . A study in egypt states that 47% of pregnant women with unplanned pregnancy do not use adequate prevention, and 28.8% encounter with the failure of their contraceptive method . In a study in china, it has been indicated that the failure of the contraceptive method has been the main cause of unwanted pregnancy . It seems that of the most important educational needs and the most effective attempts to prevent unwanted pregnancy are creating motivations in families, providing necessary facilities, and helping them in selection and accurate use of different methods by holding educational classes in health centers . Therefore, introducing accurate information about the contraceptive methods acquires a profound understanding, and using this information is very important in planning for the pregnancy . Considering the lack of safe methods, educational programs, introducing adequate contraceptive options, and consultation before pregnancy 76% of world population live in developing countries, 85% of births, 95% of neonatal mortality, and 99% of maternal mortality occur in these countries . So, regulating reproduction and adequate planning for pregnancy is significant in these countries . In conclusion, more attempts must be taken to decrease complication of pregnancies such as unwanted cases . In the recent years, it has been proved that, in addition, the population control and educational program for contraceptive methods are important and necessary in preventing unwanted pregnancy . Because in iran abortion is illegal consider in the muslim religious, unwanted pregnant women cannot do abortion except if physician has diagnosis that mother has complication to continue of her pregnancy or there is intrauterine growth retardation in the first trimester.
At the end of past millennium, flexible bronchoscopy was regarded as one of the most frequently performed procedures by the physicians of multiple disciplines to inspect the airway . In our institute, the fibreoptic bronchoscopies are performed by either the pulmonologists or the anaesthetist . At many places, fibreoptic bronchoscopies are still performed after topical anaesthesia only, without any sedation . This simplified approach is safe and results in decreased expenditures . In a retrospective study done by colt et al ., intravenous sedation was reported in 50% of the procedures; however, this technique requires the use of adequate anaesthetic resources and is associated with a low but real morbidity and mortality . The intravenous sedation limits dynamic analysis of the airways such as vocal cords, presence of local or diffuse malacia or effects of voluntary cough . Therefore, a risk benefit approach comparing the same procedure performed under sedation and under local anaesthesia alone is indicated . There are several techniques for anaesthetising the vocal cords and tracheobronchial tree, each with its own potential advantages and disadvantages . Topical lignocaine applied by the spray as you go technique with direct instillation of 4% solutions is used by a few bronchoscopists, while the others use transcricoid route for local anaesthesia of the vocal cords and tracheobronchial mucosa . We have compared a transcricoid injection of local anaesthesia with the spray as you go technique without sedation in patients posted for elective fibreoptic bronchoscopies requiring only bronchoalveolar aspirate for diagnosis and not biopsies of any kind . It is a very safe technique which can be performed with or without conscious sedation . After obtaining institutional ethical committee approval, 60 patients in the age group 2070 years of either sex, undergoing elective fibreoptic bronchoscopies for diagnostic bronchoalveolar aspirate, were included in this study . The patients of the specified age group coming to the bronchoscopy suit, requiring only diagnostic bronchoalveolar lavage over a period of 6 months, were selected and alternatively divided into two groups of 30 each . Group i patients were given a single transcricoid injection of lignocaine, while in group ii patients lignocaine was used as spray as the bronchoscopist entered inside, after the lignocaine sensitivity test was done in all the patients . If any contraindication for transcricoid injection was present, like any local pathology, then these patients were included in the other group . After taking an informed written consent and lignocaine sensitivity test was done, the patients were alternatively assigned to different groups . All the patients were given injection atropine 0.6 mg intramuscularly, 20 min prior to the procedure . Nebulisation with 3 ml of 4% lignocaine was done in all the patients for 15 min before starting the procedure . The blood pressure, pulse and oxygen saturation (spo2) were recorded before the procedure in both the groups . The patency of the nostril was checked, and in the more patent nostril, 2 ml of 2% lignocaine gel was applied in all the patients . Group i patients received transcricoid injection of 3 ml of 4% lignocaine solution given as a bolus through a 21-g hypodermic needle in the sitting position after confirming its position by aspirating air under aseptic conditions . In group ii patients, 2 ml of 4% lignocaine was instilled on to the vocal cords under direct vision after insertion of the bronchoscope . Further boluses of lignocaine were instilled through the bronchoscope if local anaesthesia was thought to be inadequate in both the groups . The assistant as well as the bronchoscopist could not be blinded to the local anaesthetic techniques . A single endobronchial procedure was selected and the bronchoscopist was also not changed to allow a fair comparison of the two techniques studied . The pulse rate and systolic blood pressure were recorded before the procedure and 5 min after the bronchoscopy . The time from the nasal insertion of bronchoscope to reach the carina was recorded in both the groups . A bout of coughing was considered as a single episode of cough . The total dose of lignocaine used in both the groups was also noted . Record of any complication like bleeding from the transcricoid site was made, as well as any other complication if detected was observed and noted . Thirty min after the procedure, an assistant who was unaware of the patients group was asked to assess any discomfort to the patients, using a 10-cm visual analogue scale (vas). The vas score of 0 was considered as no discomfort, 1 as mild, 2 as moderate discomfort and 3 or more as severe discomfort . The data were analysed using chi - square test and the p values were calculated . Group i was transcricoid group and group ii patients received spray as you go technique . The age of patients in group i (51.6614.08 years) was comparable with that in group ii patients (48.2613.32, p = ns). As shown in table 1, mean basal values of systolic blood pressure in both the groups were comparable before starting the procedure . In group ii, the systolic blood pressure increased significantly from the baseline when measured 5 min after the procedure (p<0.02). Similarly, as shown in table 2, the pulse rate was comparable in both the groups before starting the procedure (p = ns), but it increased significantly in group ii when measured 5 min after the procedure . The total dose of lignocaine used in group ii (372.6624.90 mg) was significantly higher than that used in group i (3149.32 mg, p<0.001) as shown in figure 1 . The number of coughs in group i (40.98) was significantly lesser than in group ii (4.91.24, p<0.05), as shown in figure 2 . The mean time to reach the carina was significantly shorter in group i (57.3312.98 sec) compared to group ii (79.3322.35 sec, p<0.02). Systolic blood pressure before and 5 min after the procedure pulse rate before and 5 min after the procedure dose of lignocaine used plotted against the no . Of patients cough episodes plotted against the no . Of patients as shown in table 3, the values of vas score were comparable after 30 min of the procedure in both the groups . There were no cases of haematoma or subcutaneous emphysema when the neck was examined after the procedure in the transcricoid group . Lignocaine is the most commonly used local anaesthetic agent for fibreoptic bronchoscopy and has a wide margin of safety . It has been suggested that the total dose should be limited to 300400 mg as absorption of lignocaine from the respiratory mucosa is known to be rapid . The risk of more serious side effects increases when blood concentrations exceed 5 mg / l, with seizures and hallucinations occurring at concentrations of 812 mg / l and cardiorespiratory arrest at 2025 mg / l . The peak concentration is influenced by dose per unit weight administered and not by the factors considered likely to influence mucosal absorption from the bronchial tree, such as sputum production, airflow obstruction or cigarette smoking . A major proportion of the total dose of lignocaine is required to anaesthetise the nose, pharynx and larynx, with only a small proportion needed for the bronchial tree . Although most clinical studies have reported non - toxic blood lignocaine concentration associated with bronchoscopy, several have reported concentration in the toxic range (> 5 mg / l). The control of coughing is of paramount importance for the quality of a bronchoscopy as this facilitates ease of viewing the bronchial tree and obtaining good biopsy samples . Activation of the cough centre in the brain stem causes the respiratory muscles to induce cough, the bronchial smooth muscle to cause bronchoconstriction and subsequently the airway submucosal glands to secrete mucus . Various local anaesthetic techniques can be used to anaesthetise the respiratory mucosa for fibreoptic bronchoscopy . It would be safe and not unpleasant for the patient and would at the same time provide acceptable conditions for the bronchoscopist . In our institute these patients are malnourished and have deranged liver function tests as well as the pulmonary function tests, hence doing fibreoptic bronchoscopy under local anaesthesia only without sedation is a routine . The present study was designed to compare the techniques of applying topical anaesthesia to the respiratory mucosa for elective diagnostic fibreoptic bronchoscopy . The bronchoscopist was not changed, to allow a fair comparison of the two techniques studied . Although the current british thoracic society guidelines provide a consensus statement on the current evidence base without specific guidance on drugs or techniques and without defining methods of sedation, the guidelines recommend offering sedation to all, except where there are contraindications . If a centre has experience of performing unsedated diagnostic flexible bronchoscopy, it is reported that patient co - operation is not improved with sedation . Performed a double - blinded, placebo - controlled trial in 100 patients in a centre that normally performs unsedated bronchoscopy, and could not demonstrate improved patient tolerance, comfort and co - operation with lorazepam . Tried to identify the common fears of patients undergoing fibreoptic bronchoscopy and also determine whether any factors might contribute to reducing these fears . It was found that doctors were more likely to explain the indication for bronchoscopy than how it would be performed . They concluded that provision of detailed information about sensations that are likely to be experienced in bronchoscopy could be used to allay some of the common fears . Improved preparation of patients with lower education, inferior health status and asthma may lead to decreased pain during fibreoptic bronchoscopy . Although sedation is associated with major complications, sedative drugs are often given immediately before fibreoptic bronchoscopy in the belief that patient's comfort is improved . Compared two such regimens with placebo and concluded that routine sedation has little part to play in patients undergoing a single diagnostic procedure . The present study compared two techniques of anaesthetising the respiratory mucosa for diagnostic bronchoscopy without sedation . The transcricoid method was more effective than the spray as you go technique . No complication was associated with transcricoid injection and minor bleeding associated with the technique did not interfere with the bronchoscopy . They recommended transcricoid technique as a safe method of inducing effective local anaesthesia that is well tolerated by the patient . In our study, we nebulised all the patients with 3 ml of 4% lignocaine for 15 min before the procedure . The systolic blood pressure and pulse rate were compared between the two groups before the procedure and 5 min after the procedure . The systolic blood pressure and the pulse rate were comparable in both the groups before the procedure, but the systolic blood pressure increased significantly in group ii after the procedure (p<0.02). Similarly, the pulse rate also increased significantly after the procedure in group ii (p<0.001). The vas score was similar in both the groups, 30 min after the procedure (p = ns). The incidence of side effects was negligible in both the groups . The dose of lignocaine used in group ii (372.6624.90 mg) was significantly higher than that used in group i (3149.32 mg, p<0.001). The number of coughs in group i (40.98) was also significantly lower than in group ii (4.91.24, p<0.05). The time to reach the carina was also significantly lesser in group i (57.3312.98 sec) compared to group ii (79.3322.35 sec, p<0.02) as the transcricoid injection given probably brought about excellent relaxation of the vocal cords, making the introduction of the bronchoscope smooth . Diagnostic fibreoptic bronchoscopy without sedation with transcricoid injection of lignocaine can be recommended as a safe method of anaesthetising the respiratory mucosa, which is well tolerated by the patients with negligible side effects and provides acceptable conditions for the bronchoscopist as compared to the spray as you go technique.
Charcot - marie - tooth disease (cmt) is the most common hereditary motor and sensory neuropathy, and cmt type 1a (cmt1a) neuropathy, which is the most common type of cmt, is subject to a gene dosage effect.1 the primary genetic cause of cmt1a is a duplication of pmp22 resulting from the unequal crossover between two homologous repetitive elements that flank the 1.4-mb region of chromosome 17p12.2 a new mechanism responsible for the outbreak of cmt1a - which is related to a nonrecurrent rearrangement has recently been reported.3 cmt1a is also caused by a point mutation in pmp22.4 abnormal axon - schwann cell interactions cause abnormalities in axonal structure and function, but the exact pathogenic mechanism causing cmt1a is unknown.5 one obvious possibility is that the weakness and sensory loss are the result of axonal degeneration; secondary axonal degeneration is common in cmt1a patients, and its degree determines the patient's functional disability.6 this finding is consistent with the electrophysiological and clinical findings in patients with cmt1a and in several animal models of demyelinating neuropathy.7,8 axonal dysfunction induced by demyelinating schwann cells can also occur without axonal degeneration.9 the relationship between motor nerve conduction velocities (mncvs) and disease disabilities has been the subject of much debate.10 - 14 most cmt1a research groups have found a relatively weak correlation between the severity of muscle weakness and nerve conduction velocities,10 - 12 although some have noted that patients with slower velocities developed more weakness.13,14 moreover, longitudinal studies have shown that the velocities remained unchanged over decades, whereas the compound muscle action potential (cmap) amplitudes decreased.15,16 however, the relationship between mncvs, cmaps, and the disease severity has not been investigated in koreans . Therefore, we determined whether there is a correlation between clinical disabilities and electrophysiological values in korean cmt1a patients with pmp22 duplication . We enrolled 167 cmt1a patients (93 males, 74 females) with pmp22 duplication who took part in nerve conduction studies at 20 university hospitals in korea . This study was approved by the institutional review board of the ethics committee of the ewha womans university hospital . The patients' clinical information was obtained and analyzed, including assessment of the gender ratio, age at onset, disease duration, motor and sensory impairment, deep tendon reflex, and muscle atrophy . The age at onset was determined by asking the patients when their symptoms - such as distal muscle weakness, foot deformity and/or sensory change - first appeared . The impairment and progression of cmt was evaluated in these patients using two scales: the functional disability scale (fds)13 and the cmt neuropathy score (cmtns).17 disease severity was quantified for each patient using a 9-point fds based on the following criteria: 0, normal; 1, normal but with cramps and fatigability; 2, unable to run; 3, difficulty walking but still able to walk unaided; 4, walking with a cane; 5, walking with crutches; 6, walking with a walking frame; 7, wheelchair bound; and 8, bedridden . In addition, we determined the cmtns based on the motor and sensory symptoms, and according to the pain and vibration, muscle strength, and neurophysiologic test results . The scores of this test were used to divide the patients into two groups: mild (0<cmtns10) and moderate or severe (cmtns11). The mncvs and sensory nerve conduction velocities (sncvs) for the median, ulnar, peroneal, tibial, and sural nerves were determined in all 167 patients . The mncvs of the median and ulnar nerves were determined by stimulating at the elbow and wrist, respectfully, while cmaps were recorded over the abductor pollicis brevis and the adductor digiti quinti, respectively . The mncvs of the peroneal and tibial nerves were determined by stimulating at the knee and ankle while recording the cmaps over the extensor digitorum brevis and adductor hallucis, respectively . The sncvs and sensory nerve action potentials (snaps) were obtained over a finger - wrist segment from the median and ulnar nerves, and they were also recorded for the sural nerves . The terminal latency indices (tlis) that adjust the distal motor latency for the terminal distance and the proximal mncv were calculated . The calculated tli was compared to the tli of the normal population as reported previously.18 tlis were calculated for the median nerves as follows: tli = terminal distance (mm)/[mncv (ms)distal motor latency (ms)]. 11.0 (spss, chicago, il, usa). Percentages and means were compared by the chi - square test and student's t - test, respectively . Correlations were determined using pearson's correlation coefficient (r), and probability values of p<0.05 were considered to be indicative of statistical significance . We enrolled 167 cmt1a patients (93 males, 74 females) with pmp22 duplication who took part in nerve conduction studies at 20 university hospitals in korea . This study was approved by the institutional review board of the ethics committee of the ewha womans university hospital . The patients' clinical information was obtained and analyzed, including assessment of the gender ratio, age at onset, disease duration, motor and sensory impairment, deep tendon reflex, and muscle atrophy . The age at onset was determined by asking the patients when their symptoms - such as distal muscle weakness, foot deformity and/or sensory change - first appeared . The impairment and progression of cmt was evaluated in these patients using two scales: the functional disability scale (fds)13 and the cmt neuropathy score (cmtns).17 disease severity was quantified for each patient using a 9-point fds based on the following criteria: 0, normal; 1, normal but with cramps and fatigability; 2, unable to run; 3, difficulty walking but still able to walk unaided; 4, walking with a cane; 5, walking with crutches; 6, walking with a walking frame; 7, wheelchair bound; and 8, bedridden . In addition, we determined the cmtns based on the motor and sensory symptoms, and according to the pain and vibration, muscle strength, and neurophysiologic test results . The scores of this test were used to divide the patients into two groups: mild (0<cmtns10) and moderate or severe (cmtns11). The mncvs and sensory nerve conduction velocities (sncvs) for the median, ulnar, peroneal, tibial, and sural nerves were determined in all 167 patients . The mncvs of the median and ulnar nerves were determined by stimulating at the elbow and wrist, respectfully, while cmaps were recorded over the abductor pollicis brevis and the adductor digiti quinti, respectively . The mncvs of the peroneal and tibial nerves were determined by stimulating at the knee and ankle while recording the cmaps over the extensor digitorum brevis and adductor hallucis, respectively . The sncvs and sensory nerve action potentials (snaps) were obtained over a finger - wrist segment from the median and ulnar nerves, and they were also recorded for the sural nerves . The terminal latency indices (tlis) that adjust the distal motor latency for the terminal distance and the proximal mncv were calculated . The calculated tli was compared to the tli of the normal population as reported previously.18 tlis were calculated for the median nerves as follows: tli = terminal distance (mm)/[mncv (ms)distal motor latency (ms)]. 11.0 (spss, chicago, il, usa). Percentages and means were compared by the chi - square test and student's t - test, respectively . Correlations were determined using pearson's correlation coefficient (r), and probability values of p<0.05 were considered to be indicative of statistical significance . The mean age at examination was 36.9 years (range, 5 - 75 ye - ars) and the mean age at onset was 15.5 years (range, 2 - 45 years). The mean disease duration at the time of examination was 22.3 years (range, 0 - 57 years). The disease had manifested before the second decade of life in 69% of the patients . Twelve individuals (7%) with pmp22 duplication did not complain of any clinical symptoms . Muscle weakness and atrophy started predominantly in the distal legs, with these symptoms being noted to a lesser extent in the distal upper limbs . The paresis in the distal regions of the lower limbs varied from asymptomatic to severe weakness . Pinprick sensory loss was found in 87 (52%) patients, and proprioception loss was found in 112 (67%). Heel gait defects occurred more commonly than toe gait defects (p<0.001), and high - arched foot deformities were common (89%). Both the mncv and sncv of the median nerve were uniformly reduced in the cmt1a patients . The cmap of the median nerve was significantly correlated with the disease duration (fig . 1a; p<0.001) and the cmap of the ulnar nerve (fig . 1b; p<0.01). However, no correlations were found between the age at onset and the cmaps in the median and ulnar nerves (fig . The tli of the patients' median nerve (0.310.10, meansd; range, 0.13 - 0.72) did not differ significantly from the normal values seen in healthy control subjects (0.310.04; range, 0.23 - 0.43). These results suggest that demyelination in cmt1a patients is uniformly distributed between the proximal and distal portions of the nerves . The characteristics of the mild (0<cmtns10) and the moderate or severe (cmtns11) cmtns groups are listed in table 1 . The gender ratio was similar in the two groups, and the mean age at onset did not differ significantly between them . However, the disease duration at the time of examination was significantly longer in the moderate or severe cmtns group (p<0.001). The patients in the moderate or severe cmtns group were more severely affected than those of the mild cmtns group by muscle weakness (p<0.001), muscle atrophy (p<0.001), sensory loss (p<0.001), the frequency of areflexia (upper, p<0.001; lower, p<0.01), heel and toe gait defects (p<0.001), and foot deformity (p<0.05). The amplitudes of the evoked median nerve motor responses were more severely affected in the moderate or severe cmtns group than in the mild cmtns group . In contrast, neither the mncvs nor the sncvs differed significantly between the two groups . The cmaps of the median (p<0.001), ulnar (p<0.001), peroneal (p<0.05), and tibial (p<0.001) nerves were significantly correlated with the cmtns, and the cmaps of the median (p<0.001), ulnar (p<0.001), peroneal (p<0.05), and tibial (p<0.001) nerves were correlated with the fds score (table 2). In contrast, the mncv of the median nerve was only correlated with the cmtns (p<0.05). The snaps and sncvs of the median, ulnar, and sural nerves the mean age at examination was 36.9 years (range, 5 - 75 ye - ars) and the mean age at onset was 15.5 years (range, 2 - 45 years). The mean disease duration at the time of examination was 22.3 years (range, 0 - 57 years). The disease had manifested before the second decade of life in 69% of the patients . Twelve individuals (7%) with pmp22 duplication did not complain of any clinical symptoms . Muscle weakness and atrophy started predominantly in the distal legs, with these symptoms being noted to a lesser extent in the distal upper limbs . The paresis in the distal regions of the lower limbs varied from asymptomatic to severe weakness . Pinprick sensory loss was found in 87 (52%) patients, and proprioception loss was found in 112 (67%). Heel gait defects occurred more commonly than toe gait defects (p<0.001), and high - arched foot deformities were common (89%). Both the mncv and sncv of the median nerve were uniformly reduced in the cmt1a patients . The cmap of the median nerve was significantly correlated with the disease duration (fig . 1a; p<0.001) and the cmap of the ulnar nerve (fig . 1b; p<0.01). However, no correlations were found between the age at onset and the cmaps in the median and ulnar nerves (fig . The tli of the patients' median nerve (0.310.10, meansd; range, 0.13 - 0.72) did not differ significantly from the normal values seen in healthy control subjects (0.310.04; range, 0.23 - 0.43). These results suggest that demyelination in cmt1a patients is uniformly distributed between the proximal and distal portions of the nerves . The characteristics of the mild (0<cmtns10) and the moderate or severe (cmtns11) cmtns groups are listed in table 1 . The gender ratio was similar in the two groups, and the mean age at onset did not differ significantly between them . However, the disease duration at the time of examination was significantly longer in the moderate or severe cmtns group (p<0.001). The patients in the moderate or severe cmtns group were more severely affected than those of the mild cmtns group by muscle weakness (p<0.001), muscle atrophy (p<0.001), sensory loss (p<0.001), the frequency of areflexia (upper, p<0.001; lower, p<0.01), heel and toe gait defects (p<0.001), and foot deformity (p<0.05). The amplitudes of the evoked median nerve motor responses were more severely affected in the moderate or severe cmtns group than in the mild cmtns group . In contrast, neither the mncvs nor the sncvs differed significantly between the two groups . The cmaps of the median (p<0.001), ulnar (p<0.001), peroneal (p<0.05), and tibial (p<0.001) nerves were significantly correlated with the cmtns, and the cmaps of the median (p<0.001), ulnar (p<0.001), peroneal (p<0.05), and tibial (p<0.001) nerves were correlated with the fds score (table 2). In contrast, the mncv of the median nerve was only correlated with the cmtns (p<0.05). The snaps and sncvs of the median, ulnar, and sural nerves the present study investigated the correlations between disease disabilities and the electrophysiological findings in korean cmt1a patients with pmp22 duplication, and found that the disabilities were related to the cmaps rather than to the ncvs . It has been reported that cmt symptoms progress due to axonal degeneration and loss,5,19 with axonal loss being the major cause of impairment and disability in patients with cmt1a.20 the accentuation of muscle wasting and sensory impairment in the distal extremities was correlated with the corresponding reduction in the cmaps and snaps but not with the decrease in mncv or sncv.10 - 12 our results support the hypothesis that the disease - related disabilities are correlated with the decreased nerve action potentials rather than the conduction velocities . It is important to determine how the demyelination of sch - wann cells causes axonal injury, but this remains elusive.21 many hypotheses have been postulated.22 - 25 for example, local biochemical changes in axons, such as decreased neurofilament phosphorylation, increased neurofilament density, and decreased axonal transport due to demyelinating schwann cells, could lead to axonal dysfunction and eventual axonal loss.9 some longitudinal cmt studies have revealed that nerve conduction velocities remain unchanged for more than 10 years, but that the cmaps progressively decrease over the same period.16,26 in the present study we also found an absence of velocity changes over a 10-year period in nine patients despite gradually increasing clinical severities . These results suggest that the degree of change in cmap amplitude is useful for predicting the clinical severity of the disease . As a marker of axonal loss, we also found that the clinical manifestations of muscle atrophy were also strongly correlated with the cmaps and axonal loss, which became more pronounced as the disease progressed . The disease severity varies considerably among patients with cmt1a.27 attempts to identify the mitigating factors are underway, and these efforts could lead to effective therapeutic approaches for reducing the expression of pmp22.28 pmp22 duplication mainly induces a demyelinating phenotype, while the features of axonal pathology appear concomitantly with advancing age.6 the patients with pmp22 duplication show a great variability of disease severity, ranging from asymptomatic individuals to wheelchair - bound patients . These results suggest that other molecular, endogenous, and environmental factors (e.g., genetic factors related to the pmp22 expression) influence the progression of this disease . In the present study, the age at onset was the first decade in 44% of the cmt1a patients and before 20 years of age in 69% . The age at onset as an indicator of the cmt disease severity has been the subject of debate.11,12,26 although birouk et al.13 reported an association between the age at onset and the severity of the disorder, others have suggested that the age at onset is not a reliable indicator of disease severity; the latter is consistent with our findings.11,26 instead, one of the most important disease - modifying factors in our study appeared to be the disease duration . A longitudinal study found that axonal involvement progressed in a time - dependent manner, and that axonal degeneration proceeded concomitantly with advancing disease.10,16 the age - dependent reduction in the cmaps, and the increase in axonal degeneration and clinical disability observed in our study were also in good agreement with previous reports.15,29 furthermore, the median nerve tli did not differ between korean cmt1a patients and healthy controls . Despite the distal location of the cmt symptoms, the nerve conduction velocities slowed uniformly along the proximal and distal segments, which implies a uniform pathological process of demyelination . In conclusion, a significant correlation was found between the clinical - electrophysiological manifestations and the physical disabilities among the korean cmt1a patients in this study . Although our study was cross - sectional in design and involved only a single race, we propose that the clinical disease severity in patients with cmt1a is determined by the extent of axonal dysfunction.
A negative balance between the formation and resorption of bone mass can lead to the development of osteoporosis . Moreover, osteoporosisis characterized by skeletal fragility resulting from reduced bone mass and disrupted bone microarchitecture . Osteoporosis increases the risk of fracture and thus has been considered a major public health concern . Traditionally, the development of osteoporosis is related to several risk factors, such as aging, immobility, hypertension, antihypertensive agents, hyperparathyroidism, menopause, diabetes mellitus, corticosteroid usage, low calcium intake, vitamin d deficiency, and genetic vulnerability . Vertebral fracture is the most common osteoporotic fracture, but only from one - third to one quarter of the patients with vertebral fracture can be clinically identified . Furthermore, it is reported that asymptomatic vertebral fractures are associated with future hip fracture by threefold and other nonvertebral fracture by twofold . Hip fracture is the second common osteoporotic fracture and it may incur substantial healthcare costs resulted from disability . Furthermore, the effect of hip fracture on mortality increase can adversely extend up to 10 years or more . The mortality following a hip fracture generally increases with the increment of age and is greater in men, but the sex difference declines after age 80 . Although the rates of hip fracture have declined in the west, the rate is increasing in the developing world . It is estimated that> 50% of hip fractures worldwide will occur in asia by 2050 . The functional outcome and increased mortality of osteoporotic fracture are heterogeneous and depend on age, activity of daily living prior to fracture, pre - fracture comorbidities, and the cognition . Hepatitis c virus infection (hcv) is a global health problem estimated to affect 170 million people worldwide . Hepatitis c virus infection is a hepatotropic virus that mainly causes inflammation and fibrosis of the liver . It is reported that 20% of hcv - infected patients will progress to liver cirrhosis . However, hcv can also cause several extrahepatic manifestations, such as diabetes mellitus, rheumatic disorders, lymphoproliferative disease, cardiovascular events, and cognitive impairment . Although the role of osteoporosis as a sequence of cirrhosis or advanced liver disease has been thoroughly documented, the effect of hcv exposure on bone mineral density in the absence of advanced liver disease remains debated . Some scholars have proposed that chronic hcv infection without liver cirrhosis contributes to reduced bone mineral density, whereas other scholars have asserted the opposite . To assess the association between hcv exposure and subsequent development of osteoporosis, we conducted a nationwide population - based cohort study by analyzing data from a nationwide medical database, the national health insurance research database (nhird). The national health insurance (nhi) program, initiated on march 1, 1995, provides comprehensive coverage for the medical care of taiwan residents . At the end of 2014, 23.75 million people (99.9% of the population) were enrolled in the program . In cooperation with the bureau of national health insurance (bnhi), the national health research institutes established several data sets for public use, including the longitudinal health insurance database 2000 (lhid2000), a cohort data set comprising 1,000,000 randomly selected cases from the registry of nhi beneficiaries in 2000 . To maintain confidentiality, personal information, such as patient identification numbers and sensitive personal data, are encrypted in the database, and international classification of diseases, ninth edition, clinical modification (icd-9-cm) codes are used for disease classification . This study was approved to fulfill the condition for exemption by the institutional review board (irb) of china medical university (cmuh-104-rec2 - 115). The irb also specifically waived the consent requirement . From 2000 to 2011, patients aged 20 years and older with diagnosed hcv infection (icd-9-cm codes 070.41, 070.44, 070.51, and 070.54) identified from the lhid2000 comprised the hcv infection cohort . Patients with a history of osteoporosis (icd-9-cm codes 733.0 and 733.1) and hepatitis b virus (hbv) infection (icd-9-cm codes 070.20, 070.22, 070.30, and 070.32) diagnosed before the index date, those with missing information, and those younger than 20 years were excluded . Using 1:m case - control studies is to increase the power and to control possible confounding . Based on the statistical efficiency does not gain much when m> 4, we constructed a 1:4 matched cohort study . For each hcv case, 4 insurers with no history of hcv exposure, hbv infection, and osteoporosis were assigned to a comparison cohort and frequency matched with the hcv exposure cohorts according to age (every 5-year span), sex, and index year . Individuals were excluded from the comparison cohort using the same criteria used for the hcv exposure cohort . The primary endpoint in this study each participant was followed from the index date until the endpoint, withdrawal from the nhi program, or december 31, 2011 . The baseline characteristics of participant comorbidities were also analyzed; the comorbidities included diabetes (icd-9-cm code 250), hypertension (icd-9-cm codes 401405), hyperlipidemia (icd-9-cm code 272), heart failure (icd-9-cm code 428), stroke (icd-9-cm codes 430438), obesity (icd-9-cm code 278), and cirrhosis (icd-9-cm codes 571.2, 571.5, and 571.6). The chi - square test and t test for categorical and continuous variables, respectively, were first used to compare the distributions of age, sex, and baseline comorbidities between the hcv exposure and the comparison cohorts . The incidence densities of osteoporosis were estimated in person - years for the various risk factors . To estimate the hazard ratios (hrs) and 95% confidence intervals (cis) of osteoporosis, multivariable models were simultaneously adjusted for age, sex, and the comorbidities of diabetes, hypertension, hyperlipidemia, heart failure, stroke, and cirrhosis . Meier estimates were plotted to illustrate the cumulative incidence of osteoporosis, and the log - rank test was performed to examine the difference between the hcv exposure and the comparison cohorts . All statistical analyses were performed using the sas package (version 9.4 for windows; sas institute, inc, cary, nc). The national health insurance (nhi) program, initiated on march 1, 1995, provides comprehensive coverage for the medical care of taiwan residents . At the end of 2014, 23.75 million people (99.9% of the population) were enrolled in the program . In cooperation with the bureau of national health insurance (bnhi), the national health research institutes established several data sets for public use, including the longitudinal health insurance database 2000 (lhid2000), a cohort data set comprising 1,000,000 randomly selected cases from the registry of nhi beneficiaries in 2000 . To maintain confidentiality, personal information, such as patient identification numbers and sensitive personal data, are encrypted in the database, and international classification of diseases, ninth edition, clinical modification (icd-9-cm) codes are used for disease classification . This study was approved to fulfill the condition for exemption by the institutional review board (irb) of china medical university (cmuh-104-rec2 - 115). From 2000 to 2011, patients aged 20 years and older with diagnosed hcv infection (icd-9-cm codes 070.41, 070.44, 070.51, and 070.54) identified from the lhid2000 comprised the hcv infection cohort . The date for hcv exposure coding patients with a history of osteoporosis (icd-9-cm codes 733.0 and 733.1) and hepatitis b virus (hbv) infection (icd-9-cm codes 070.20, 070.22, 070.30, and 070.32) diagnosed before the index date, those with missing information, and those younger than 20 years were excluded . Using 1:m case - control studies is to increase the power and to control possible confounding . Based on the statistical efficiency does not gain much when m> 4, we constructed a 1:4 matched cohort study . For each hcv case, 4 insurers with no history of hcv exposure, hbv infection, and osteoporosis were assigned to a comparison cohort and frequency matched with the hcv exposure cohorts according to age (every 5-year span), sex, and index year . Individuals were excluded from the comparison cohort using the same criteria used for the hcv exposure cohort . Each participant was followed from the index date until the endpoint, withdrawal from the nhi program, or december 31, 2011 . The baseline characteristics of participant comorbidities were also analyzed; the comorbidities included diabetes (icd-9-cm code 250), hypertension (icd-9-cm codes 401405), hyperlipidemia (icd-9-cm code 272), heart failure (icd-9-cm code 428), stroke (icd-9-cm codes 430438), obesity (icd-9-cm code 278), and cirrhosis (icd-9-cm codes 571.2, 571.5, and 571.6). The chi - square test and t test for categorical and continuous variables, respectively, were first used to compare the distributions of age, sex, and baseline comorbidities between the hcv exposure and the comparison cohorts . The incidence densities of osteoporosis were estimated in person - years for the various risk factors . To estimate the hazard ratios (hrs) and 95% confidence intervals (cis) of osteoporosis, multivariable models were simultaneously adjusted for age, sex, and the comorbidities of diabetes, hypertension, hyperlipidemia, heart failure, stroke, and cirrhosis . Meier estimates were plotted to illustrate the cumulative incidence of osteoporosis, and the log - rank test was performed to examine the difference between the hcv exposure and the comparison cohorts . All statistical analyses were performed using the sas package (version 9.4 for windows; sas institute, inc, cary, nc). Of the 51,535 sampled patients, 41,228 and 10,307 were categorized as the comparison and hcv exposure cohorts, respectively (table 1). Most patients were aged 50 years (61.7%), and 54.6% of the patients were women . The mean age was 54.1 15.3 years in the hcv exposure cohort and 53.7 15.5 years in the comparison cohort . Regarding baseline characteristics, the hcv exposure cohort exhibited a higher prevalence of diabetes, hypertension, hyperlipidemia, heart failure, stroke, obesity, and cirrhosis than did the comparison cohort . During the mean follow - up periods of 5.44 and 6.01 years for the hcv exposure and comparison cohorts, respectively, the kaplan meier curve revealed that the cumulative incidence of osteoporosis was higher in the hcv exposure cohort than in the comparison cohort (figure 1, log - rank test p <0.001). Demographic characteristics and comorbidities in cohorts with and without hcv exposure cumulative incidence comparison of osteoporosis for patients with (dashed line) or without (solid line) hcv exposure.hcv = hepatitis c virus . The overall incidence of osteoporosis in the hcv exposure cohort was higher than that in the comparison cohort (8.27 vs 6.19 per 1,000 person - years; crude hr = 1.33, 95% ci = 1.201.47) (table 2). After we adjusted for factors such as age, sex, and comorbidities, namely diabetes, hypertension, hyperlipidemia, heart failure, stroke, and cirrhosis, the risk of developing osteoporosis was significantly higher in the hcv exposure cohort than in the comparison cohort (adjusted hr [ahr] = 1.35; 95% ci = 1.211.51). Compared with patients aged 49 years, the risk of developing osteoporosis was 4.05-fold higher in those aged 50 to 64 years (95% ci = 3.8454.760) and 8.82-fold higher in those aged 65 years or older (95% ci = 7.4810.40). In the multivariate model, the risk for osteoporosis was 3.10-fold higher for women than for men (95% ci = 2.813.43) and was higher for patients with the comorbidities of hypertension (ahr = 1.19, 95% ci = 1.071.31), hyperlipidemia (ahr = 1.17, 95% ci = 1.051.29), and heart failure (ahr = 1.23, 95% ci = 1.021.49). The incidence and hazard ratio for osteoporosis and osteoporosis - associated risk factor the incidence of osteoporosis increased with age, was higher in women than in men, and increased with comorbidity in both cohorts (table 3). The overall risk of osteoporosis related to several variables including age, sex, and presence of comorbidities was compared in the hcv exposure cohort and the comparison cohort . The risk of osteoporosis in patients exposed to hcv in all stratifications was higher than that in the comparison cohorts . However, the risk of osteoporosis contributed by hcv decreased with age (aged 49: ahr = 1.79, 95% ci = 1.322.43; aged 5064: ahr = 1.36, 95% ci = 1.141.62; aged 65: ahr = 1.23, 95% ci = 1.051.44) and the presence of comorbidity (no comorbidity: ahr = 1.54, 95% ci = 1.261.89; comorbidity: ahr = 1.27, 95% ci = 1.121.43). Incidence of osteoporosis by age, sex, and comorbidity and cox model measured hazards ratio for patients with hcv infection compared those without hcv exposure the patients exposed to hcv exhibited a 1.38-fold (95% ci = 1.241.55) higher risk of developing osteoporosis compared with the patients who were not exposed to hcv (table 4). The risk of osteoporotic fracture did not differ significantly between patients exposed to hcv and the comparison cohorts (ahr = 0.80, 95% ci = 0.441.45). Comparisons of hazard ratios between patients with and without hcv exposure for different outcomes osteoporosis (or osteoporotic fracture) consistent with the literature proposing that hcv seroprevalence peaks after age 55 and that women are predisposed to hcv infection, our results revealed that most patients (61.7%) were aged 50 years and that 54.6% of the patients were women . The mean age in the hcv exposure cohort was 54.1 15.3 years . Compared with patients who were not exposed to hcv, patients who were exposed to hcv tended to have more comorbidities, including diabetes, hypertension, hyperlipidemia, heart failure, stroke, obesity, and cirrhosis . Our results revealed that the hcv exposure cohort had more comorbidities; however, the risk of osteoporosis remained higher in the hcv exposure cohort after adjusting for age, sex, and the comorbidities of diabetes, hypertension, hyperlipidemia, heart failure, stroke, and cirrhosis . The mechanism affecting the pathophysiology that causes comorbidities in patients exposed to hcv may include hcv - associated insulin resistance and atherosclerosis . Hcv may cause diabetes mellitus by directly interfering with insulin signaling and inducing insulin resistance in hepatocytes; hcv - infected hepatocytes can secrete mediators that induce extrahepatic insulin resistance, notably in the skeletal muscle . Hepatitis c virus may increase the risk of cardiovascular and cerebrovascular events through atherosclerosis induced by systemic inflammation, chronic endothelial injury, or direct infection of the arterial wall . Our study revealed that the osteoporosis incidence increased with age and was higher in women than in men . The association between osteoporosis and aging has been confirmed in the literature, and the gene expression of the rennin the decreasing rate of bone mineral density is swifter in the early postmenopausal period, which typically begins after age 50, and women aged 40 to 59 years have the highest risk of developing osteoporosis . Consistent with our result that women are predisposed to osteoporosis, the us preventive services task force indicated that as many as 1 in 2 postmenopausal women and 1 in 5 men are at risk for osteoporosis - related fracture . Moreover, in the present study, osteoporosis was associated with hypertension, hyperlipidemia, and heart failure . Both osteoporosis and hypertension are common among the aging population and may share similar etiologies, such as low calcium intake and levels, vitamin d and vitamin k deficiency, high salt consumption, imbalanced nitric oxide levels, and antihypertensive agents that exert detrimental effects on the skeletal metabolism, strength, and density . Hyperlipidemia can reduce bone formation and promote bone loss by causing the products of lipid oxidation to accumulate in the subendothelial spaces of the vasculature and bone . Moreover, hyperlipidemia can induce secondary hyperparathyroidism, thereby impairing bone regeneration and mechanical strength . Heart failure and osteoporosis share several risk factors, such as aging, smoking, and postmenopausal and antihypertensive agents; heart failure can also accelerate bone loss by inducing hyperaldosteronism and secondary hyperparathyroidism . However, the effect of obesity on bone metabolism is controversial . Obesity is traditionally regarded as a protective factor for osteoporosis, conferring a positive mechanical loading on bone formation . For example, both osteoblasts and adipocytes are derived from a common mesenchymal stem cell and agents inhibiting adipogenesis - stimulated osteoblast differentiation and vice versa . Furthermore, the reduced bone formation caused by aging is usually accompanied by adipogenesis in bone marrow cavities . Moreover, elevated oxidative stress is common in people with obesity and osteoporosis . To our knowledge, this is the first population - based study to assess the relation between hcv exposure and the incidences of osteoporosis and osteoporotic fracture . Our statistical analyses benefited from the use of a nationwide database and the 12-year observation of participants selected from a representative cohort comprising 1,000,000 residents covered by the nhi program . Hansen et al conducted a large - scale population - based study to explore the association between hcv exposure and all - site fracture, but omitted discussing osteoporosis development . They concluded that the risk of fracture equally increased in patients exposed to hcv (chronic or cleared infections), and the major determinants of fracture in such patients are lifestyle - related factors, such as alcohol and drug abuse, which substantially increases fracture risk . By contrast, our epidemiological study demonstrated that hcv exposure increases the risk of osteoporosis and the detrimental effect of hcv on osteoporotic fracture was not obvious . Several pathogenic mechanisms are involved in bone mineral density loss in patients exposed to hcv . First, fibronectin can infiltrate the bone matrix to enhance matrix mineralization, reducing its production by the liver . Second, insulin - like growth factor 1, involved in osteoblast differentiation and proliferation, is produced by the liver and is reduced in patients with chronic liver disease . Third, the receptor activator ratio of nuclear factor kappa ligand and osteoprotegerin is higher in patients with chronic liver disease, enhancing bone resorption . Fourth, interleukin-6 is increased by chronic hcv infection and can activate osteoclasts to increase bone resorption . By contrast, the association between hcv and osteoporosis may be due to shared risk factors since the prevalence of important osteoporosis risk factors was higher in the hcv exposed patients as compared to the patients without hcv exposure . However, it is reasonable to conclude that the increased risk of osteoporosis observed in hcv exposed patients was more likely to be due to the effect of their hcv status since the possible confounding effect of osteoporosis risk factors has been significantly minimized in our study . Moreover, the osteoporosis risk contributed by hcv decreased with the increment of age may be due to the absence or low prevalence of osteoporosis - associated comorbidities in the younger patients . The results of our subgroup analyses in which we excluded patients with important comorbidities at baseline also confirmed the validity of our results . This finding, coupled with the results of the subgroup analyses, affirms the possible causal association between hcv and osteoporosis, and suggested that hcv may be a possible risk factor for osteoporosis . Nevertheless, hcv may be less influential than other risk factors, such as hypertension, hyperlipidemia, and heart failure, in contributing to the development of osteoporosis . First, the large - scale national database provided statistical benefits to our longitudinal study to evaluate the association between hcv and osteoporosis . Second, the recruited subjects were a stable population and 99% of the residents in taiwan have been covered by the nhi program . First, we could not ascertain the patients viremic status, such as the viral loads or the genotypes of the hcv . Nevertheless, all patients in our case cohort exhibited positive anti - hcv antibodies, which mean recent or past hcv exposure . Moreover, the effect of antiviral therapy on the stage of liver fibrosis or the grade of liver inflammation could not be ascertained in our study . Therefore, we could not prove the ameliorating effect of antiviral therapy on bone mineral density through the arrest of liver cirrhosis even though the association between osteoporotic fracture and cirrhosis has been thoroughly documented . . However, to enhance the accuracy of diagnosis, we only recruited patients who received medical care for osteoporosis> 3 separate visits . Moreover, the bnhi organizes regular audits performed by medical experts to ensure the accuracy of insurance claim codes in taiwan . Finally, we cannot clarify the temporal association between hcv exposure and osteoporosis since the date of hcv exposure could not be ascertained . In conclusion, this nationwide population - based cohort study concludes that hcv exposure increases the risk of developing subsequent osteoporosis, but no detrimental effect on osteoporotic fracture was observed . Furthermore, hcv may be less influential than other risk factors, such as hypertension, hyperlipidemia, and heart failure, in contributing to the development of osteoporosis.
Each disaster leaves an imprint on the affected population, a singular signature . A critical unmet need in the field of disaster mental / behavioral health is the capability to tailor mental health and psychosocial support (mhpss) to the unique constellation of psychological risk factors operating within each disaster event . We have developed and introduced trauma signature (tsig) analysis to the fields of disaster mental / behavioral health and disaster public health in response to this identified need . We define tsig analysis in the following manner: trauma signature (tsig) analysis is an evidence - based method that examines the interrelationship between population exposure to a disaster, extreme event, or complex emergency and the interrelated physical and psychological consequences for the purpose of providing timely, actionable guidance for effective mental health and psychosocial support (mhpss)or disaster behavioral health (dbh) * support that is organically tailored and targeted to the defining features of the event . According to shultz et al . 2013 (in press), tsig examines the extent to which disaster survivors were exposed to empirically - documented risk factors for psychological distress and mental health disorders . Grounded on the disaster ecology model, tsig is premised on the assumption that each disaster exposes the affected population to a novel pattern of traumatizing hazards, loss, and change . This singular signature of exposure risks is a predictor (or series of predictors) of the psychosocial and mental health consequences . Disaster - specific analysis is important because, as kessler and team have documented across a spectrum of international disasters, secondary stressors unique to a particular disaster situation have more impact than the disasters themselves in determining the prevalence of post - disaster mental disorders . Too frequently, mhpss response to disasters is unguided, uncoordinated, and unscientific . For example, in our tsig analysis of the 2010 haiti earthquake it was evident that mhpss was never prioritized . The disaster behavioral health response that was cobbled together in haiti evidenced many serious deficits (table 1) including: self - deployment and arrival on - scene of many uninvited personnel, lack of coordination among response teams, provision of non - evidence - informed services loosely labeled as psychosocial, provision of services such as critical incident stress debriefing (cisd) that are known to be ineffective and potentially harmful for survivors, provision of services that were not culturally - adapted or language - appropriate for haiti, premature cessation of many services, and minimal or no program evaluation . From the perspective of responders, there was a lack of pre - deployment guidance about the specific stressors that would be encountered, and lack of on - scene psychological support, leading to high rates of traumatization among response personnel . Disaster - specific risk factors can be identified from disaster situation reports issued in the early aftermath allowing mhpss response to be tailored and timed to the defining features of the event . It is time to infuse science into mhpss response; we need evidence - based support and intervention . Optimal mhpss response that incorporates tsig analysis can be summarized as a sequence of six steps (see also figure 1): 1) initial tsig analysis to guide response based on the defining features of the disaster or potentially - traumatizing event (pte), 2) tsig - guided preparation of responders for what to expect and deployment of appropriate response assets, 3) delivery and evaluation of evidence - informed early interventions, 4) implementation of onsite validated disaster behavioral health needs assessment, 5) identification and intervention with individuals at high risk for disaster - related psychological distress, impairment, or psychopathology, and 6) continuous monitoring of responder and survivor mental health throughout recovery with comprehensive (end - to - end) evaluation of the mhpss response . Optimal mental health and psychosocial support (mhpss): six sequential steps while the components that are uniquely contributed by tsig analysis are primarily incorporated into steps 1 and 2, in order to achieve optimal mhpss response, significant quality improvement is needed and desirable for steps 36 . An electronic appendix, entitled operationalizing tsig (www.landesbioscience.com/journals/disasterhealth/commart-sup.pdf) provides details for each of the six steps in the categories of description, rationale, operationalization, unmet needs, research questions, and opportunities . The initial tsig analysis is the first step of six that comprise an optimal evidence - based mhpss response (summarized in figure 1). As illustrated in figure 2, initial tsig analysis consists of these elements: 1) characterization of the affected communities (geographic scope, numbers affected, demographics); 2) real - time collection and synthesis of information from disaster situation reports (sitreps) issued regularly in the early days and weeks post - impact; 3) identification and data collection from disaster monitoring and scientific resources specific to the disaster event (e.g., united states geological survey (usgs) earthquake data on the haiti earthquake, national hurricane center (nhc) data on superstorm sandy); 4) consultation with disaster sciences subject matter experts (e.g., usgs geophysicists for haiti earthquake, nhc meteorologists for superstorm sandy); 5) construction of a hazard profile based on open - source data developed in the preceding steps; 6) review and integration of the scientific literature on evidence - based risk factors for psychological distress and psychopathology for persons exposed to the event - specific hazards; 7) incorporation of information on disaster stressors identified anecdotally in media and social media accounts of the event; 8) creation of an event - specific stressor / risk factor matrix that is cross - referenced with the evidence - based literature (stressors are enumerated by disaster phase within the categories of exposure to hazard, loss, and change), ccounts, cross - referenced with the evidence - based literature; and 9) generation of a tsig summary for the disaster based on the estimated psychological severity of exposures to hazards, loss, and change . Figure 2 . Initial post - impact trauma signature analysis several key points in this process require more explanation . Initial tsig analysis is triggered when a disaster alert or warning is issued, or when disaster strikes without warning . Local disaster response is activated immediately and higher - level response (regional, national, international) is brought into play as needed depending upon the scale of the event . For major events, publicly - accessible disaster situation reports (sitreps) are generated and hosted on websites such as the global disaster alert and coordination system (gdacs), united nations office for the coordination of humanitarian assistance, and reliefweb within the early hours or days, hazard profile . To create the hazard profile that provides a scientifically - sound description of the event, tsig begins with review of disaster data including sitreps from governmental and ngo agencies and available data from disaster monitoring and scientific resources . Subject matter experts (smes) may be contacted to assure that the event is correctly described from a disaster sciences perspective . This step describes the physical forces of harm that impact the human population in harm s way in order to characterize exposure to hazard . Disaster epidemiologic data are also collected on numbers of deaths, injuries, displaced, affected . The next step involves matching the scientific event description to the ever - expanding literature on psychological risk factors (for distress, disorder, and psychiatric diagnosis). This is summarized in a stressor matrix: risk factors are arrayed by disaster phase for categories of exposure to hazard, loss, and change . News media and social media feeds may also be incorporated at this stage to enrich the list of stressors . Trauma signature summary . The final step in the initial tsig analysis involves the construction of a summary table of salient psychological risk factors, grouped into categories of exposure to hazard, loss, and change with an estimate of the exposure severity for each risk factor . To demonstrate the feasibility of conducting tsig analysis, we developed a series of disaster case studies, beginning with post hoc analyses of historical disasters and progressing to real - time analyses of unfolding events . Our first tsig case study was performed on data from the 2010 haiti earthquake which we described as a potent example of the rare catastrophic event where all major risk factors for psychological distress and impairment are prominent and compounding . Our tsig case studies have been wide - ranging, including examples of natural, human - generated, and hybrid disasters, and complex emergencies . In addition to tsig analyses for the 2010 haiti earthquake, we performed analyses on other natural disaster events including the 2011 us, super tornado outbreak, 2009 and 2011 river floods in the upper midwest, and 2012 superstorm sandy . In the realm of hybrid (interacting natural and human - generated components), we carefully analyzed the 2011 great east japan disaster . The 2010 deepwater horizon oil spill provided an application of tsig for an anthropogenic (human - generated) event . In the realm of complex emergencies / humanitarian crises, we have examined patterns of internal displacement in colombia and the russia - georgia conflict in south ossetia . With each case study, the research literature database of evidence - based research studies is expanded . The case studies have provided an open invitation for colleagues with interest in a particular disaster event to join with us both in authorship and in advancing the tsig methodology . Tsig developers and co - authors have presented a series of invited papers, workshops, and institutes to introduce the methodology and actively seek feedback . Currently, an international cadre of co - authors and co - developers spans five continents . However, at present we are soliciting additional disaster case studies, consulting with subject matter experts, creating the structure for the literature database, and launching an internet - based delphi process in 2013 to develop expert consensus regarding the methodology . We will then seek opportunities to partner with lead response agencies in real - world, real - time applications of the tsig process . Once refined, our intention is to develop a practical system that can help infuse evidence - based science into the decision - making process for matching mhpss response to the defining features of the disaster event . Tsig is designed to contribute to the creation of mhpss response that increasingly approaches the attributes of optimal response outlined in table 2.
Cerebrovascular diseases (cvds) are associated with balance disturbances, dependency in performing daily living activities, and limited ambulation . As life expectancy increases spasticity is a speed - dependent resistance to the passive motion of muscles and tendons, developing secondary to enhanced stretch reflexes in patients with upper motor neuron lesions . Spasticity is a major problem in rehabilitation programs, and it hampers patient progress and may trigger severe complications2, 3 . Quantitative assessment of spasticity is critical when gauging responses to medical treatment and physical therapy, and when making a prognosis . At present, rather limited clinical scoring systems of spasticity, and biomechanical and electrophysiological data, are used . Electroneuromyographic evaluation of the spinal reflex organization allows detailed follow - up of physiological changes . This measure, and also the f reflex, assessed using various techniques, are employed to quantitate and explore spasticity4 . Transcutaneous electrical nerve stimulation (tens) is a rehabilitative form of physical therapy used to treat pain . Various types of nerve fiber are stimulated at different frequencies, wavelengths, and amplitudes . Tens delays the h reflex through the 1a fibers, which mediate presynaptic inhibition8 . In the present study, we evaluated the utility of tens for patients with spasticity due to cvd, who were undergoing rehabilitation, was evaluated using clinical scoring systems and electrophysiological assessment . This study also aimed to explore whether tens affects spasticity electrophysiologically, and the extent of the effects on clinical features . Patients hospitalized in ankara physical therapy and rehabilitation training and research hospital, who were participating in a rehabilitation program for hemiplegia with spasticity in the lower extremity, were recruited . Patients who had received any treatment for spasticity, who lacked spasticity, who had a history of any systemic disease that might cause peripheral neuropathy, or symptoms of radiculopathy in the lower extremities, who had suffered strokes less than 30 days prior, who had a prior history of cerebrovascular disease, who had upper motor neuron damage on the non - hemiplegic side, who could not co - operate, or who were obese, were excluded . Aphasic and geriatric patients were also excluded; because their co - operation was lacking . Patients with spasticity of at least ashworth grade 1 in the lower extremities were included . The active motor power and spasticity of all patients reflex, and superficial and deep sensorial examinations were performed . The time taken to walk 10 m by patients who could walk was measured before and after tens . After a clinical evaluation, emg examinations were initially performed on healthy controls and, later, on the affected sides of patients . All electrophysiological work was performed in a calm, intermediately lit room with patients in the supine position . Their feet extended from the examination bed, commencing at the ankle joints, each of which was in the 90 neutral position; the knee joint was at 180 of extension . All patients were asked to relax their muscles as much as possible during the electrophysiological study . The h reflexes and m responses of the gastrocnemius - soleus muscle were electrophysiologically recorded on both the healthy and hemiplegic sides . Subsequently, a single 30 min session of tens was applied to the hemiplegic lower extremity . Ten minutes later, the h reflexes and m responses were re - assessed and compared with the pre - tens data . A nihon kohden neuropack four - channel emg platform was used for measurements in the single - channel mode . The active electrode was placed on the gastrocnemius - soleus muscle, in a slightly medial position, 14 cm proximal to the junction of the achilles tendon and the heel . The reference electrode was placed at the junction of the tendon and the heel, and a velcro ground electrode was placed between the active electrode and the stimulator . The latter electrode was placed on the fossae popliteal, to stimulate the tibial nerve, and the cathode was placed across the active electrode . The stimulus intensity was increased in steps of 2 ma, commencing at zero, and the combined action potentials of the soleus muscle were recorded . The peak - to - peak amplitudes and latencies of the h and m responses were measured . The latencies were assessed at the point of separation of each response from the baseline . The sensitivity, low frequency filter, and high frequency filter were 2 mv / dv, 200 hz, and 3 khz, respectively . Stimuli were created randomly, and delivered to the popliteal fossa with the posterior tibial nerve lying proximal to the cathode . A rectangular flow 1 ms in duration, delivered every 2 s, served as the stimulus . The sweep speed and sensitivity were 10 ms and 2 mv / dv, respectively . Subsequently, the largest h wave was determined by varying the strength of the stimulus with the location of the stimulator unchanged . After all electrophysiological work was completed, and all wave amplitudes calculated, the h / m maximum amplitude ratio was calculated for each patient by dividing the maximal amplitude of the h reflex by that of the m responses from both the healthy and affected sides . In addition, to evaluate alpha motor neuron excitability and the extent of spasticity, the slopes of the rising sides of the h and m amplitude - stimulus severity slopes were calculated using excel and the h slope, m slope, and h slope / m slope values were obtained . Tens was performed in the supine position with the feet hanging in a neutral manner . Electrodes were placed bilaterally on the tibialis nerve, between the tendon of the muscle and the medial malleolus; thus the gastrocnemius muscle was included . The recording electrodes used in the electrophysiological study were not removed . A systems 200e tens platform was used . This equipment has two output channels the flow intensity in each channel can be adjusted independently . The frequency was 50 hz and the pulse width was 100 ms in normal stimulation . The current intensity was not allowed to exceed a mean of 50 ma and no contractions were noted . Electrophysiological data were collected before and after tens, the same as for the patient group . Controls were positioned the same as patients, and tens was applied for 30 min . Electrophysiological data were gathered from only the right lower extremities of the controls and the pre- and post - tens values were compared . The, wilcoxon signed - rank, and kruskal - wallis tests, were used, as appropriate, to evaluate the experimental parameters . The patient group with hemiplegia secondary to cvd numbered 27 [12 females (44.4%) and 15 males (55.6%)]. A total of 24 healthy subjects [10 females (41.7%) and 14 males (58.3%)] served as controls . The mean ages of the patients and controls were 60.9312.8 and 49.886.85 years, respectively . The mean heights of the patients and controls were 160.858.99 and 165.38 + 8.56 cm, respectively . The mean age, but fourteen (51.9%) and 13 (48.l%) patients respectively had left- and right - side hemiplegia . Thromboembolic and hemorrhagic stroke were the principal causes of hemiplegia in 21 (77.8%) and 6 (22.2%) patients, respectively . Superficial sensory nerve examinations revealed hypoesthesia in 10 patients (37%), but 17 were normal (63%). The deep tendon reflexes (dtrs) on the hemiplegic sides were normal in the upper extremity of one patient (3.7%) but hyperactive in 26 (96.3%); the lower extremity on the affected side was hyperactive in all patients . On the healthy sides, the dtrs were normative in both the upper and lower extremities of 26 (96.3%) patients, and hyperactive in one (3.7%). No significant difference in the mean pre- and post - tens lower - extremity dtrs on the hemiplegic side was found (p>0.05). The values of the spasticity parameters before and after tens differed significantly in the patient group (table 1table 1.change in the spasticity between pre- and post - tensashworth 1ashworth 2ashworth 3pre - tens10 (37%)13 (48.2%)4 (14.8%)post - tens14 (51.9%)11 (40.7%)2 (7.4%) all p values <0.05). Fourteen patients were able to walk, either with support or independently, but 13 were not . After tens, 11 patients exhibited increased walking speeds and 3 reduced speeds . When patient hemiplegic and healthy lower extremities were electrophysiologically compared, the mean maxima of the h reflex, the h / m ratio, the h slope, and h slope / m slope ratio, were significantly greater on the affected side . In addition, the mean m response amplitude was significantly different (greater on the healthy side). The differences in the h / m maximum and h slope / m slope ratios were particularly marked (table 2table 2.electrophysiological variables of the healthy and affected sides of the patientsvariablehealthy sideaffected sideh max amp (mv)1,669.851559.792,899.701,698.69h max lat (msec)31.212.1831.082.28 m max amp (mv)8,735.892,820.527,439.892,825.16 m max lat (msec)4.900.444.930.40h / m max amp (mv)0.190.160.420.22h slope0.010.0090.030.22 m slope0.030.0060.030.006h / m slope0.40.320.910.43). The h maximum amplitude values were higher on the spastic hemiplegic side than on the healthy side . The h / m ratio, h slope, and h slope / m slope ratio were higher on the affected side . However, the mean m amplitude was higher on the normal than on the hemiplegic side . The mean h reflex amplitude, the h / m amplitude ratio, the h maximum latency, the h slope, and the h / m slope ratio, differed significantly pre- and post - tens on the hemiplegic side . The post - tens electrophysiological changes in the control group were similar to those in the patient group . The h maximum amplitude, the h / m ratio, the m maximum amplitude, and the m slope ratio, decreased, whereas the h and m maximum latencies increased, with statistical significance . The duration of disease was positively associated with the mean h maximum amplitude, the h / m ratio, and the h / m slope ratio; however, no association was statistically significant . The h maximum latency was lowest in patients with disease durations greater than 100 days . Higher mean h maximum amplitudes, h / m ratios, and h slopes evident as spasticity worsened . However, only the mean h slope differed significantly among the spasticity subgroups, with significant differences being found between subgroups i and iii, and ii and iii, in parallel with worsening of spasticity . Tens significantly reduced spasticity scores and increased the walking speed . On the spastic side, tens significantly reduced the m amplitude and increased the h reflex amplitude, the h / m maximum amplitude ratio, the h slope, and the h slope / m slope ratio . Also, the mean patient h reflex amplitude, the h / m ratio, the h slope, and the h slope / m slope ratio, decreased significantly, and the h reflex maximum latency increased, after tens . In controls, tens significantly decreased the h maximum amplitude, the h / m ratio, the m maximum amplitude, and the m slope ratio, and lengthened the h and m maximum latencies . The h maximum latency was the only variable affected by lesional duration . The mean h maximum amplitude, the h / m ratio, and the mean h slope increased as spasticity worsened on the ashworth scale . Explored changes in the spasticity of patients with medulla spinalis injuries after tens, and found there were short - term reductions in spasticity9 . Robinson et al . Applied 20 min electrical stimulation to the quadriceps muscles of patients with medulla spinalis injuries and reported a significant decrease in leg spasticity10 . Cho et al . Found that a single session of high - frequency tens significantly improved the spasticity (for less than 1 day) of chronic stroke patients . Similarly, tens reduced the spasticity of stroke patients, and repeat tens reduced the hyperactive tension reflexes of the plantar flexors, and the passive resistance to plantar flexor movement11 . In a study that included acute stroke patients, tens applied to acupuncture points for 3 weeks decreased plantar flexor spasticity and increased dorsiflexion of the ankle joint . In chronic stroke patients, however, ankle joint dorsoflexion was strengthened by stimulation, and muscle co - contraction was decreased . In addition repeated skin stimulation was advocated, to increase the number of cortical fields represented12 . In a study that included chronic stroke patients, addition of tens to a 20-session rehabilitation program decreased spasticity by 30% and markedly improved muscle strength13 . Park et al . Showed that balance, walking, and the functional activity of chronic stroke patients improved after 30 tens sessions (each 30 min in duration), and a combination of exercise and tens increased the activation of pathways associated with proprioception and balance14 . In the present study, a significant decrease in spasticity and an increase in walking speed were evident after acute hemiplegic the patients in a rehabilitation program received a single session of tens . Fisher reported that the m amplitudes of patients with first - motor neuron diseases decreased, after tens, compared to the amplitudes of polyneuropathic patients15 . Angel and hoffman, who first showed that the h / m ratio was increased in spastic patients, reported mean ratios of 9.17 and 0.48 in the extremities of control and spastic groups, respectively . The excitability of alpha motor neurons increased when various physiopathological mechanisms were in play; therefore, the responses to stimuli carried by 1a afferent fibers increased16 . Garcia - mullin and mayer reported mean h / m ratios of 0.33 and 0.46 on the healthy and paretic sides, respectively, of spastic hemiplegic stroke patients in whom the disease duration was longer than 90 days17 . Bakhtiary showed that spasticity was reduced by electrical stimulation, but no significant change in the h reflex amplitude was evident . Also, the h / m maximum ratio did not change, and this was attributed to muscle weakness caused by stimulation18 . Gaft et al . Noted significant decreases in both spasticity and the h reflex amplitude after stimulation . In the present study, significant differences were found in the m response and h reflex amplitude between the healthy and affected sides of spastic the hemiplegic stroke patients . The mean maximum amplitudes of the h reflexes of the triceps surae were 1,669 and 2,899 mv on the healthy and paretic sides, respectively, and the difference was significant . The difference in the h / m ratio between the healthy and plegic sides was also significant19 . Huang et al . Reported that the h / m maximum and h2/h1 ratios were not adequate for defining the extent of spasticity as assessed by the ashworth scale . The mean h / m maximum and h2/h1 values increased in stroke patients, compared to healthy controls, but the m maximum ratio did not significantly differ between the groups . The h2/h1 and m maximum ratios did not significantly correlate with other measures of spasticity . In contrast to other previous studies, huang et al . Concluded that the mas value correlated with the h / m ratio20 . In the present study, we noted a decrease in the m amplitude on the spastic side was observed, whereas the h reflex amplitude and the h / m maximum amplitude ratio, increased . Higashi et al . Found that the change in the h slope / m slope ratio was more significant on the spastic than the healthy side, and no significant difference in the h / m maxima of the two sides was evident . As the h slope / m slope graph is bell - shaped, such data are more compatible with the brunnstrom stage of hemiplegic patients than are other indicators . In conclusion, the h slope/ m slope ratio is primarily useful for objectively evaluating the extent of spasticity, and the excitability of the motor neuron pool of the spastic sides of hemiplegic patients21 . Measured the excitability of the motor neuron pool and compared the h / m ratios, the h maximum amplitude, and the h slope / m slope ratio, in spastic and normal individuals, and found there were marked differences between the two groups . They concluded, the h slope / m slope ratio was particularly useful for evaluating motor neuron pool excitability22 . In the present study, steeper h slope and greater h slope / m slope ratio on the spastic (compared to healthy) side of hemiplegic patients prior to tens were observed . One study evaluating the electrophysiological components of the h reflex and f wave compared the pre- and post - tens h reflexes, and f wave latencies and amplitudes, and reported the reduction in spasticity was associated with electrophysiologically proven decreases in the h reflex amplitude, the f wave amplitude, and the h / m and f / m ratios23 . In another study, tens was applied segmentally and heterosegmentally to the median nerve of the wrist, and to the common peroneal nerve (placebo). The h reflex latencies increased in 75% of the patients in the experimental groups, but the h amplitude did not change significantly . Aydin et al . Compared tens with baclofen, the commonest drug prescribed to treat spasticity in patients with medulla spinalis injuries . Significant improvements in all parameters, except pain, were evident in the tens group, and they were associated with decreases in the h amplitude and h / m ratio, and the m response . The electrophysiological and clinical changes did not differ significantly between the tens and baclofen group25 . Tekeoglu et al . Also reported that tens reduced spasticity in stroke patients, and improved their capacity to engage in activities of daily living, concluding that tens effectively improved motor function26 . In the present study, a single session of tens resulted in significant clinical inhibition of spasticity; however, lower extremity strength did not improve . On the spastic side, tens significantly decreased the mean h reflex amplitude, the h / m ratio, the h slope, and the h slope / m slope ratio; and increased the h reflex maximum latency . This study had some limitation walking speed was not measured after several tens applications . In conclusion, tens for hemiplegic patients with spastic lower extremities due to cvd markedly improved clinical parameters and significantly changed electrophysiological variables . The results of this study suggest that tens is effective when used to manage spasticity.
Proteinase - activated receptors (pars) belong to a family of g - protein - coupled receptors with seven transmembrane domains activated via proteolytic cleavage by serine proteinases . A total of four pars have been identified and cloned . Among them, par-1 [2, 3], par-3, and par-4 are targets for thrombin, trypsin, and cathepsin g, whereas par-2 is resistant to thrombin but can be activated by trypsin, mast cell tryptase [6, 7], neutrophil elastase, and insect - derived proteinase . Pars are expressed by various cells involved in inflammatory and immunological responses, such as vascular endothelial cells, epithelial cells, mast cells, t cells, monocyte, eosinophils, and neutrophils [10, 11]. In these cells, activation of pars affects their main functions such as proliferation, degranulation, and release of inflammatory mediators [10, 11]. In our previous study, we have showed the expression of par-1, par-2, and par-3 on t cells, and thrombin-, trypsin-, and tryptase - induced interleukin (il-6) release from t cells . It has also been reported that cytoplasmic free calcium and phospholipase c and protein kinase c activation are increased in t - leukemic cell lines following stimulation with thrombin or the thrombin receptor agonist peptide . Thrombin and thrombin receptor agonist also enhanced cd69 expression and il-2 productions by cross - linking t cell receptors in both jurkat t cells and peripheral blood lymphocytes . We, therefore, anticipated that thrombin, trypsin, and tryptase might induce tnf release from t cells through pars . Tnf is a major proinflammatory cytokine that is thought to be important in the pathogenesis of asthma, food allergy, ocular allergy, and atopic dermatitis . It has been reported that the increased number of tnf expressing cells and levels of tnf is observed in the bronchoalveolar lavage (bal) and in the airways of asthmatics . Inhaled tnf increases airway responsiveness to methacholine in asthmatic subjects associated with a sputum neutrophilia . Since pars, tnf, and t cells all play roles in inflammation, we believe, there must be some linkages between them . The aim of the present study is to investigate roles of thrombin, tryptase, trypsin, elastase, and agonist peptides of pars in the secretion of tnf from purified human t cells and subtypes of t cells . Human thrombin, trypsin (specific activity: 10,000 baee u / mg protein), soybean trypsin inhibitor (sbti), and bovine serum albumin (bsa, fraction v) were purchased from sigma (st louis, mo, usa). Recombinant hirudin and human neutrophil elastase (specific activity: 20 meo - suc - ala - ala - pro - val - pna u / mg protein) were obtained from calbiochem (san diego, ca, usa). Recombinant human lung tryptase (specific activity: 1,000 n cbz - l - lysine thiobenzyl ester u / mg protein) was from promega (madison, wi, usa). Agonist peptides of pars, and their reverse forms, and par-2 antagonist peptide fsllry - nh2 were synthesized in cl bio - scientific inc . The sequences of the active and reverse peptides were par-1, sfllr - nh2 and rllfs - nh2, tfllrn - nh2 and nrllft - nh2; par-2, sligkv - nh2 and vkgils - nh2 as well as trans cinnamoyl (tc)-ligrlo - nh2 and tc - olrgil - nh2; par-3, tfrgap - nh2 and pagrft - nh2 . Rpmi 1640 and newborn calf serum (ncs) were obtained from gibco (carlsbad, ca, usa). Pe - conjugated mouse anti - human cd3 monoclonal antibody, pe - conjugated goat - anti rabbit igg, and tnf opteia elisa kits were purchased from bd pharmingen (san jose, ca, usa). Trizol reagent and sybr green i stain were purchased from invitrogen (carlsbad, ca, usa). Cellular activation of signaling kits for extracellular signal - regulated kinase (erk), 2-(2-diamino)-3-methoxyphenyl-4h-1-benzopyran-4-one (pd98059), akt, pi3k, and p38 2-(4-morpholinyl)-8-phenyl-4h-1-benzopyran-4-one (ly294002) was purchased from cell signaling technology (beverly, ma, usa). Exscript rt reagent kit and sybr premix ex taq (perfect real time) were obtained from takara (dalian, china). Rabbit anti - human par-1 and rabbit anti - huamn par-2 polyclonal antibodies were purchased from santa cruz biotechnology (santa cruz, ca, usa). Fitc - conjugated mouse anti - human cd4 monoclonal, pe - conjugated mouse anti - human cd8 monoclonal, percp - cy5.5-conjugated mouse anti - human tnf monoclonal, fitc - conjugated mouse anti - human ifn monoclonal, pe - conjugated mouse anti - human il-4 monoclonal, apc - conjugated mouse anti - human cd25 monoclonal, and apc - conjugated mouse anti - human il-17 monoclonal antibodies were purchased from ebioscience . All other reagents were of analytic grade and obtained from sigma (st louis, mo, usa). Human t cells were isolated from peripheral blood mononuclear cells (pbmcs) by a macs system with t cell isolation kit i according to the manufacturer's protocol . In brief, pbmcs were isolated from fresh blood donated by healthy volunteers, 100 ml from each individual per visit . The informed consent from each volunteer and agreement with the ethical committee of the first affiliated hospital of nanjing medical university were obtained . After being separated from red blood cells by ficoll - paque density gradient, pbmcs were collected and incubated with microbead - linked anti - cd3 monoclonal antibody for 15 min at 8c . Cd3 + t cells were separated from other cells by passing through a magnetic cell separation system . For purity analysis, the cells were resuspended in pbs and incubated with pe - conjugated monoclonal antibody against human cd3 for 1 h. the purity of t cells was consistently more than 95% and cell viability was more than 98% . The purified cd3 + t cells were then used for the further cell challenge tests . T cells were cultured in 24-well culture plates at a density of 5 10cells / well in rpmi 1640 medium containing 10% ncs at 37c for 2 h with 5% co2, respectively . The culture supernatants were then removed and cells were washed twice with fresh serum - free rpmi 1640 medium at 300 g for 10 min . For challenge experiments, cells were exposed to various doses of thrombin (0.013.0 g / ml, 1 u = 0.5 g, 1 u / ml = 5.6 nm, u = nih unit), trypsin (0.010.3 g / ml, 1 g / ml = 42 nm), tryptase (0.252.0 g / ml, 1 g / ml = 7.4 nm), and elastase (0.010.3 g / ml, 1 g / ml = 34 nm, 1 u / ml = 1700 nm) with or without their inhibitors; and to agonist peptides of par-1, par-2 and par-3 (all at 0.1100 m) and their reverse peptides, respectively, for 16 h before the culture, supernatants were harvested and stored at 40c till use . Quantitative expression of tnf mrnas in t cells was determined by real - time pcr following the manufacture's protocol . Briefly, after synthesizing cdna from the total rna by using exscripttm rt reagent kit, real - time pcr was performed by using sybr premix ex taq on the abi prism 7000 sequence detection system (perkin elmer applied systems, foster city, ca, usa). Each reaction contains 12.5 l of 2 sybr green master mix, 1 l of 10 m of primers, 1 l of the cdna, to a total volume of 25 l . The thermal cycling conditions included an initial denaturation step at 50c for 2 min, 95c for 10 min; 40 cycles at 95c for 15 s, annealing temperatures at 60c for 30 s, and extension at 72c for 30 s. the sequences of pcr primers for tnf and -actin were 5-ccccagggacctctctctaatc-3 (forward) and 5-ggtttgctacaacatgggctaca-3 (reverse); 5-aggggccggactcgtcatact-3 (forward), and 5-ggcggcaacaccatgtaccct-3 (reverse), respectively . Consequently, at the end of the pcr cycles, specificities of the amplification products were controlled by dissociation curve analysis . Expression of mrna in each sample was finally determined after correction with -actin expression . The gene specific threshold cycle (ct) for each sample (ct) was corrected by subtracting the ct for the housekeeping gene -actin . Untreated controls were chosen as the reference samples, and the ct for all experimental samples was subtracted by the ct for the control samples (ct). T cells were preincubated with 50 m of pd98059, 20 m of ly294002, or medium alone for 30 min before adding thrombin 3.0 g / ml, trypsin 0.3 g / ml, or medium alone for 30 min, 2 h, or 6 h. the cells were lysed in a buffer containing 20 mm of tris - hcl (ph 7.4), 137 mm of nacl, 10% glycerol, 1% triton x-100, 2 mm of edta, 25 mm of -glycerophosphate, 2 mm of sodium pyrophosphate, and 0.5 mm of dithiothreitol at 4c for 30 min . Cell debris was removed by centrifugation of the lysate at 12,000 g for 10 min . The supernatants were mixed with equal volumes of 2x sodium dodecyl sulphate (sds) sample buffer and heated to 100c for 10 min . An equal volume of sample was fractionated by sds - page on a 10% acrylamide gel and transferred onto polyvinylidene difluoride (pvdf) membranes with a bio - rad transfer system, according to the manufacturer's instructions . After blocking nonspecific binding sites with 5% bsa in tbst (50 mm of tris, 0.15 m of nacl, 0.1% tween 20, ph 7.6) for 1 h, membranes were probed with phospho - erk1/2, phospho - akt, phospho - p38, or phospho - pi3k antibodies at 4c overnight, followed by incubation with hrp - conjugated secondary antibodies . Immunoreactive bands were visualized by using enhanced chemiluminescence reagents according to the manufacturer's protocol . Densitometry analysis of immunoblots was carried out using quantity one software (bio - rad, usa). The levels of tnf in culture supernatants were measured with opteia elisa kits according to the manufacturer's instructions . The plates were read on a plate reader (molecular devices, menlo park, ca) with the softmax data analysis program . The minimum detectable concentration of tnf was 2.2 pg / ml . To test the par1 and 2 expressions after treatment of trypsin and thrombin, isolated t cells were pelleted by centrifugation at 450 g for 10 min after cells were stimulated with thrombin 3.0 g / ml, trypsin 0.3 g / ml, or medium alone for 16 h. for par1 and par2 staining, cells were incubated with rabbit anti - human par1 or par2 antibodies at 37c for 1 h. after washing, cells were incubated with pe - conjugated goat anti - rabbit igg antibody 37c for 45 min . After washing, cells were analyzed on a fluorescence - activated cell sorting (facs) arial flow cytometer with celldevia software (bd biosciences, usa). To test the secretion of tnf from subtypes of t cells, isolated t cells were pelleted by centrifugation at 450 g for 10 min and then fixed and permeabilized by using a cell fixation / permeabilization kit (bd pharmingen). Briefly, thoroughly resuspended cells were added in 100 l of bd cytofix / cytoperm solution and incubated for 20 min at 4c . Cells were then incubated with fluorescence labeled anti - human cd4, cd8, cd25, tnf, ifn, il-4, and il-17 monoclonal antibodies or isotope control, respectively (at a final concentration of 4 g / ml) at 4c for 30 min . After washing, cells were analyzed on a fluorescence - activated cell sorting (facs) arial flow cytometer with celldevia software (bd biosciences, usa). The purity of t cells was consistently more than 95% (date was shown in supplementary material, figure s1). It has been shown that thrombin, trypsin, and tryptase can induce proinflammatory cytokine il-6 release from t cells, but little is known of serine proteinase - induced tnf release from t cells . Here, we showed that thrombin at concentrations of 1.0 and 3.0 g / ml provoked tnf release from t cells following 16 h incubation period in a dose - dependent manner . Approximately up to 2.5-fold increase in tnf release was observed when t cells were incubated with thrombin for 16 h. at 6 h following incubation, data (not shown) on both basal and induced tnf release from t cells were inconsistent . This is most likely due to the limitation of the assay sensitivity and relatively low secretion of tnf . Par-1 agonist peptides, sfllr - nh2 at the concentration of 100 m and tfllrn - nh2 at the concentration of 5 m, induced a significant release of tnf at 16 h following incubation . However, rllfs - nh2, a reverse peptide of sfllr - nh2, and nrllft - nh2, a reverse peptide of tfllrn - nh2, had little effect on release of tnf from t cells (figure 1(a)). Hirudin, a specific thrombin inhibitor, was able to inhibit thrombin - induced secretion of tnf . Approximately up to 82.4% inhibition of thrombin - induced secretion of tnf was observed when 3.0 g / ml of thrombin and 10 u / ml of hirudin were added to t cells for 16 h. hirudin alone at the concentrations tested had little effect on tnf secretion from t cells . Sch 79797, a par-1 antagonist at the concentration of 1 m, inhibited 89% thrombin - induced tnf release from t cells (figure 1(a)). Similarly, trypsin at the concentration of 0.3 g / ml induced 2.3-fold increase in tnf release from t cells at 16 h (figure 1(b)). However, tryptase at the concentrations up to 2 g / ml and elastase at the concentrations up to 6 u / ml had little effect on tnf release from t cells (data not shown). Inhibitors of trypsin, sbti at the concentrations of 10 and 30 g / ml, eliminated 0.3 g / ml trypsin - induced tnf release by a value up to 94.8 and 94.2%, respectively . Sbti alone at the concentrations tested had little effect on tnf secretion from t cells . Sch 79797, a par-1 antagonist at the concentration of 1 m, inhibited 96.8% trypsin - induced tnf release from t cells (figure 1(b)). Sligkv, an agonist peptide of par-2 and tfrgap - nh2, an agonist peptide of par-3 at the concentrations up to 100 m, did not appear to have any effect on tnf release from t cells (data not shown). In order to confirm the findings above, we investigated the influence of the serine proteinases on the expression of tnf mrna in t cells . It was found that the expression of tnf mrna was upregulated when t cells were incubated with thrombin at 1 and 3 g / ml for 2 and 6 h. the maximum enhanced expression of tnf mrna was 4.2-fold over baseline control (figure 2(a)) after 6 h incubation . Hirudin, a specific thrombin inhibitor at the concentration of 3 u / ml, completely abolished thrombin - induced upregulated expression of tnf mrna after 6 h incubation (figure 2(b)). Trypsin at the concentration of 0.3 g / ml also induced increased expression of tnf mrna by a value up to approximately 4.0-fold in t cells (figure 2(a)), which was completely blocked by sbti (figure 2(b)). Similarly, sch 79797 at the concentration of 1 m inhibited both thrombin- and trypsin - induced upregulated expression of tnf mrna in t cells by a value up to 72 and 72.5%, respectively (figure 2(b)). Sfllr - nh2 at the concentration of 100 m and tfllrn - nh2 at the concentration of 5 m significantly increase the expression of tnf mrna at 2 and 6 h following incubation (figure 2(a)). But rllfs - nh2, a reverse peptide of sfllr - nh2, and nrllft - nh2, a reverse peptide of tfllrn - nh2, had little effect on expression of tnf mrna in t cells (data not shown). At the same time, neither thrombin nor trypsin showed obvious effect on the expression of par-1 and par-2 (data not shown). It is wellknown that there are numerous subtypes of t cells and each of them has distinctive functions . We, therefore, investigated subtypes of t cells by flow cytometer analysis in order to determine the subtypes that upregulate tnf in response to trypsin or thrombin . The results showed that trypsin and thrombin induced upregulated expression of tnf in cd4 + t cells, but not cd8 + t cells, following 16 h incubation period . Among cd4 + t cells, trypsin and thrombin enhanced tnf expression in il-4 + or cd25 + t cells, but not in ifn+ or il-17 + t cells . Sch 79797 was able to inhibit enhanced tnf expression induced by trypsin and thrombin (figures 3(a) and 3(b)). In order to examine signal transduction pathways of thrombin and trypsin, t cells were preincubated with pd98059, ly294002, or medium alone for 30 min before adding thrombin 3.0 g / ml, trypsin 0.3 g / ml, or medium alone for 16 h. following 16 h incubation period, pd98059 an inhibitor of mapk pathway, and ly294002, an inhibitor of pi3k, completely blocked thrombin- and trypsin - induced release of tnf (figure 4(a)). Furthermore, pd98059 inhibited thrombin- and trypsin - induced upregulation of expression of tnf mrna by a value up to 91.2 and 98.6%, and ly294002 eliminated thrombin- and trypsin - induced expression of tnf mrna by 95.5 and 83.2% in t cells following 6 h incubation (figure 4(b)). 3 g / ml) induced enhanced phosphorylation of erk1/2 in t cells following 0.5, 2, and 6 h incubation periods . However, thrombin and trypsin did not significantly affect phosphorylation of p38 in t cells following 0.5, 2, and 6 h incubation periods (date was shown in supplementary material, figure s2). Pd98059 was able to completely block thrombin- and trypsin - induced phosphorylation of erk1/2 when it was preincubated with t cells for 30 min . Thrombin at a concentration of 3 g / ml and trypsin at a concentration of 0.3 g / ml induced significantly increased phosphorylation of akt in t cells following 0.5, 2, and 6 h incubation periods . However, thrombin and trypsin did not significantly affect phosphorylation of pi3k in t cells following 0.5, 2, and 6 h incubation periods (date was shown in supplementary material, figure s3). Ly294002 was able to block thrombin- and trypsin - induced phosphorylation of akt when it was incubated with t cells for 30 min . We discovered in the present study that serine proteinases thrombin and trypsin, but not tryptase induced tnf release from human t cells . Since tnf is a potent proinflammatory cytokine, our observation is likely to add some novel information for, understanding of actions of serine proteinases in causing inflammation . As little as 1.0 g / ml of thrombin was able to induce significant tnf release from t cells, suggesting this proteinase is a potent secretagogue of tnf . This concentration of thrombin should be easily achieved in blood, particularly when the processes of platelet aggregation and coagulation are initiated . Inhibition of thrombin - induced tnf release by a specific inhibitor of thrombin and hirudin indicates that action of thrombin on t cells was dependent on the enzymatic activity of this serine proteinase . There are 3 receptors for thrombin on cells, including par-1, par-3, and par-4 [2, 3]. Since par-1 agonist peptides sfllr - nh2, and tfllrn - nh2, but not par-3 agonist peptide tfrgap - nh2 were capable of stimulating tnf release, a par-1 antagonist sch 79797 almost completely abolished thrombin - induced tnf release from t cells, and purified human t cells do not express par-4; the action of thrombin on t cells is most likely through activation of par-1 . Our previous report which found thrombin - induced il-6 secretion from human peripheral blood t cells may support our current findings . While little information is available on induction of tnf release from t cells by trypsin, the ability of trypsin to stimulate il-6 secretion from t cells may support the anticipation that trypsin is capable of inducing cytokine release from t cells . As little as 0.3 g / ml of trypsin was able to provoke tnf secretion from t cells proved that it is a potent stimulus of tnf release . As for thrombin, inhibitor of trypsin sbti was able to inhibit trypsin - induced tnf release from t cells, indicating that an intact catalytic site is required for the serine proteinase to stimulate tnf release . Since par-1 is one of three receptors of trypsin, par-1 agonist peptides sfllr - nh2 and tfllrn - nh2 are capable of stimulating tnf release from t cells, and sch 79797 almost completely abolished trypsin - induced tnf release from t cells, the action of trypsin on t cells is most likely through activation of par-1 . Since par-2 agonist peptide sligkv - nh2 and tryptase are not capable of stimulating tnf release from t cells, the action of trypsin on t cells is not likely through activation of par-2 . Trypsin- and thrombin - induced upregulated expression of tnf was observed in cd4 +, il-4 + or cd25 + t cells, indicating that il-4 +, and cd25 + t cells are major sources of tnf . While little information on the relationship between cd25 + t cells and tnf is available, a study which found that the percentage of cd4(+)cd25(+) t cells were significantly high, but the percentage of foxp3(+) cells were low in allergic rhinitis patients, and that il-4, il-5, and tnf levels in nasal lavage fluids were high indicates that the increased tnf release may be from cd4(+)cd25(+), nonregulatory t cells . We believe that the current study is the first work that demonstrates coexpression of cd25 and tnf in the subtype of cd4(+) t cells . Similarly, we clearly found that il-4 + t cells express enhanced tnf, though little information on co - expression of il-4 and tnf in t cells is available . This finding implicates that trypsin and thrombin may be involved in the inflammation through induction of tnf release from il-4 + or cd25 + t cells . It was demonstrated that nickel - specific cd4 + t cell lines and th17 cells corelease il-17 and tnf, but trypsin- and thrombin - induced tnf release appears not from il-17 + t cells as tnf expression in il-17 + t cells was not upregulated by these two proteinases . Mapk / erk pathway is the signaling pathway that is most likely involved in the thrombin- and trypsin - induced tnf release from highly purified t cells, as pd98059, an inhibitor of mapk / erk pathway, almost completely blocked thrombin- and trypsin - provoked phophorylation of erk and tnf release . While little information on signaling pathways associated with par-1 signaling in purified t cells is available, the previous reports that par-1 agonists activated mapk / erk and p38 mapk signaling pathways in dermal and cardiac fibroblasts may support our current observation that mapk / erk pathway is the signaling pathway that is most likely involved in the thrombin- and trypsin - induced tnf release . In addition, pi3k / akt signaling pathway seems also to be involved in thrombin and trypsin induced tnf secretion, as ly294002 an inhibitor of pi3k / akt signaling pathway partially diminished thrombin and trypsin induced tnf secretion and completely abolished thrombin and trypsin provoked phosphorylation of akt . This finding is in the same line with the report, which showed that thrombin stimulated enhance pi3k activity in hamster embryonic fibroblasts, but different from our previous report, which showed that thrombin did not enhanced pi3k activity in human dermal fibroblasts . The discrepancy between these studies may be due to the difference in cell origin and species . Tnf is a member of a growing family of peptide mediators comprising at least 19 cytokines, including lymphotoxin-, fas ligand, and cd40 ligand . The family is now considered as central mediators of a broad range of biological activities in protective immune responses against a variety of infectious pathogens . On the other hand, tnf also exerts host - damaging effects in sepsis and autoimmune disease [30, 31]. These findings indicate that tnf is one of key mediators of inflammation; therefore, our current study is of importance in understanding tnf - related inflammation and the mechanism of proteinase - induced cytokine production in t cells . In conclusion, it is discovered in the present study that serine proteinases thrombin and trypsin are potent stimuli of tnf secretion from highly purified t cells . Their actions on t cells depend on their enzymatic activities and are likely through activation of par-1 . Stimulation of tnf secretion from t cells by serine proteinases further proved that these proteinases are actively involved in the pathogenesis of inflammation and regulation of immune response in man.
Relapsing chronic inflammation found in the gut of individuals affected by inflammatory bowel diseases (ibd) is a result of several overlapping factors, including dysregulation of the immune response to the enteric microbiota, genetic susceptibility, and environmental factors [1, 2]. Reactive oxygen (ros) and nitrogen (rns) species generated by inflammatory cells during an immune response create oxidative stress and are considered as important factors contributing to the pathogenesis of ibd . Lymphocytes, neutrophils, and macrophages activated during the gut inflammation produce high amounts of ros / rns destroying surrounding tissue . Although oxidative stress is a major factor in the inflamed tissue leading towards necrosis, dna damage, and carcinogenesis, certain amounts of ros and other free radicals have an indispensable role in regulating different cell signalling pathways . In addition, some immune cells, namely, the macrophages and neutrophils, use ros to combat the microorganisms responsible for the infection . Current therapies of ibd, including immunosuppressive drugs, antibiotics, and biological drugs, are efficient in controlling the course of the disease . However, for many affected individuals, conventional therapy becomes an inadequate choice for long - term treatment because of significant side effects and risk factors, such as increased cancer risk, development of tuberculosis, and heart failure [57]. In recent years, hyperbaric oxygen (hbo2) therapy has been introduced as a possible additional treatment for ibd patients, especially in the case of refractory disease when the standard therapy is ineffective . Hbo2 involves exposure to 100% oxygen under pressure greater than 1 atmosphere of absolute pressure (atm). It is a well - established procedure frequently applied in the medical practice, especially effective in treating wounds of various aetiologies [811]. Hbo2 increases blood and tissue oxygen saturation resulting also in enhanced production of ros and rns . Previous studies have verified that the clinical efficacy of hbo2 derives from modulation of intracellular transduction cascades, leading to synthesis of growth factors which promote wound healing, neoangiogenesis, and ameliorates postischemic and postinflammatory injuries . Additional investigations on the mechanism underlying hbo2-induced wound healing have revealed a central role of hypoxia inducible factor-1 alpha (hif-1) as transcriptional regulator of genes involved in angiogenesis, energy metabolism, and cell proliferation [9, 14, 15]. A further important function attributed to the hif-1 is modulation of the immune responses, including the helper t - cell differentiation towards regulatory (treg) versus th17 phenotype [16, 17] and its strong anti - inflammatory activity in the gastrointestinal mucosa and hypoxic epithelium as a result of the transactivation of specific genes encoding for barrier - protective elements such as mucins [1820]. Hypoxia and ros / rns can induce stabilization of hif-1 leading to the activation of the hypoxia signal transduction pathway . Increased oxidative stress in the gut mucosa has been verified in humans suffering from ulcerative colitis [22, 23], as well as in experimentally induced colitis in animals [24, 25]. A recent study revealed increased activity of antioxidative enzymes and reduced oxidative stress in the inflamed gut mucosa following hbo2 exposure; however, specific mechanisms inducing activation of antioxidative enzymes in the inflamed colonic tissue upon hbo2 remain unknown . Since there is evidence that the intracellular redox status is in a close correlation with the inflammatory microenvironment, and it can also be changed by hbo2, the aim of this study was to investigate the effects of hbo2 on the mrna expression of hif-1, proinflammatory cytokines, and antioxidative enzymes in the gut and peripheral lymphoid organs of balb / c mice with dss - induced colitis . An additional aim was to assess the activity of antioxidative enzymes and whether hif-1 gene expression regulation during the gut inflammation and hbo2 treatment correlates with the changes in antioxidative and proinflammatory gene expression . Our findings reveal that hbo2 treatment may effectively modulate the intestinal milieu in inflammatory conditions involving hif-1-mediated regulation of antioxidative gene expression . Balb / c mice, obtained from charles river (calco, italy), were bred at the animal facility of the medical faculty osijek (croatia). Mice were provided with standard rodent chow (mucedola, settimo milanese, italy) and water ad libitum . The experimental facility was maintained at 22 2c, 55 5% humidity, and 12-hour light / dark cycle . All procedures involving live animals were conducted in accordance with the european guidelines for the care and use of laboratory animals (directive 86/609/eec) and were approved by the local ethical committee (faculty of medicine, university of osijek) and croatian ministry of agriculture . For each experiment male mice at the age of 1012 weeks were randomized into 4 groups (n = 4 - 5 mice / group / experiment): control mice (ctrl), control mice undergoing hbo2 (ctrl + hbo2), mice receiving dextran sodium sulphate (dss), and dss treated mice undergoing hbo2 (dss + hbo2). The average body weight of the mice at the time of inclusion into the study was 22.8 0.4 g. colitis was induced by 5% (w / v) of dss (mr 36.00050.000, mp biomedicals, illkrich, france) in drinking water ad libitum for 7 consecutive days [26, 27]. The hbo2 treatment was initiated at day 1 and was administered twice a day, 12 hours apart, until the end of experiment (the last session was applied in the morning of day 8; 15 sessions in total). During one hbo2 session, mice were exposed to 100% o2 for 60 minutes at 2.4 bars with addition of 15 minutes for gradual compression and decompression . Mice were sacrificed by cervical dislocation on day 8, after the morning hbo2 session . Disease activity index (dai) was assessed by daily measurement and scoring of animal body weight loss, stool consistency, and the presence of occult or gross blood per rectum . Measurements were performed at the same time each day until the end of experiment (day 8). Dai was determined as a sum of body weight loss score (0, none; 1, 15% loss; 2, 510%; 3, 1015%; 4,> 15%), stool consistency score (0, normal; 2, loose stool; 4, diarrhea), and score of occult / gross bleeding (0, normal; 2, occult bleeding; 4, gross hematochezia). Mice were sacrificed by cervical dislocation on day 8 following the last morning hbo2 treatment . Colons, mesenteric lymph nodes (mln), and spleens were collected for further analysis . Colonic tissue was removed immediately after the animals were sacrificed, washed in pbs, and fixed in 4% paraformaldehyde . After 72 hours fixed tissue was embedded in paraffin and cut into a series of 6 m thick sections . Histological evaluation was preformed according to modified geboes score as follows: grade 0 (structural (architectural changes)): 0: no abnormality, 1: mild abnormality, 2: mild or moderate diffuse or multifocal abnormalities, 3: severe diffuse or multifocal abnormalities . 2: mild or moderate diffuse or multifocal abnormalities, 3: severe diffuse or multifocal abnormalities . Grade 1 (chronic inflammatory infiltrate): 0: no increase, 1: mild but unequivocal increase, 2: moderate increase, 3: marked increase . 1: mild but unequivocal increase, 2: moderate increase, grade 2 (lamina propria leukocytes): 0: no increase, 1: mild but unequivocal increase, 2: moderate increase, 3: marked increase . 1: mild but unequivocal increase, 2: moderate increase, grade 3 (intraepithelial neutrophils): 0: none, 1: <5% crypts involved, 2: <50% crypts involved, 3:> 50% crypts involved . 1: <5% crypts involved, 2: <50% crypts involved, 3:> 50% crypts involved . Grade 4 (crypt destruction): 0: none, 1: probable, local excess of neutrophils in part of crypt, 2: probable, marked attenuation . 1: probable, local excess of neutrophils in part of crypt, 2: probable, marked attenuation . Grade 0 (structural (architectural changes)): 3: unequivocal crypt destruction . 3: unequivocal crypt destruction . Grade 5 (erosion or ulceration): 0: no erosion, ulceration, or granulation tissue, 1: recovering epithelium + adjacent inflammation, 2: probable erosion focally stripped, 3: unequivocal erosion, 4: ulcer or granulation tissue.slides were analysed using light microscopy at magnifications 40x, 100x, 200x, and 400x and photographed at 200x (olympus bx50 microscope, olympus c-5050 digital camera, and quickphoto pro imaging software (promicra s.r.o ., prague, czech republic)). 0: no erosion, ulceration, or granulation tissue, 1: recovering epithelium + adjacent inflammation, 2: probable erosion focally stripped, 3: unequivocal erosion, 4: ulcer or granulation tissue . After isolation, the colon was cleaned of intestinal content and freed of fat tissue, washed in dmem (sigma aldrich, steinheim, germany), cut into 5 cm long pieces, and, while shaken at 100 rpm, incubated at 37c for 20 minutes in dmem with 25 mm edta . The tissue was then thoroughly washed in pbs buffer for at least 5 times, cut into 2 mm long strips, and digested in dmem containing 5 u / ml dnase (roche, mannheim, germany) and collagenase ii (gibco, paisley, uk) at 37c for 20 min . Following this, supernatant was removed, and the previous step was repeated until complete digestion of the tissue was achieved . The supernatant was then filtered through a 100 m sized filter; dmem + 2% fbs (sigma aldrich, steinheim, germany) was added and centrifuged at 800 g for 10 min at room temperature . Cells were resuspended in 5 ml of 40% percoll (ge healthcare, uppsala, uk), overlaid on 4 ml of 80% percoll, and centrifuged at 900 g/20 min/4c . Lymphocytes were isolated from the mesenteric lymph nodes (mln) and spleen by teasing apart the organs between the frosted ends of two microscopic slides . The cells were incubated with a mixture of pe anti - cd4 (clone gk1.5, exbio antibodies), fitc anti - b220 (clone ra3 - 6b2, obtained from the american type culture collection and conjugated with fitc using standard procedures), and apc anti - cd3 antibodies (clone 145 - 2c11, exbio antibodies) and the other panel with percp anti - cd45 (clone 30-f11, bd biosciences), fitc anti - gr-1 (clone rb6 - 8c5, bd biosciences), and pe anti - f4/80 (clone bm8, stemcell technologies inc .) Or pe anti - cd4 (clone mem-241, exbio antibodies) and percp anti - cd8 (clone mem-31, exbio antibodies) antibodies . Dead cells were excluded based on 7-aminoactinomycin d (7-aad) (applichem, darmstadt, germany) staining . At least 20,000 live cells were collected by a bd facs canto ii cytometer (facs canto ii, becton dickinson, san jose, ca, usa) and analysed using the flowlogic software (inivai technologies, mentone, australia). To assess hydrogen peroxide (h2o2) and peroxynitrite (onoo) level, 10 lymphocytes isolated from mln and spleens were incubated for 30 min on + 4c with 10 m dichlorofluorescein diacetate (dcf - da) (biomol, hamburg, germany), washed for 5 min at 400 g on + 4c, and analysed with the facs canto ii . Following this, cells were stimulated with 100 nm phorbol 12-myristate 13-acetate (pma, calbiochem, darmstadt, germany), incubated for 30 min, and analysed for the second time . At each measurement minimum of 10,000 target cells colon, mln, and spleen samples were isolated, snap frozen in liquid nitrogen, and stored at 80c till analysis . Rna purity and concentration was assessed by nanophotometer p - class p330 - 30 (implen, munich, germany). In order to purify rna from all polysaccharides, including dss, an additional purification step using 8 m licl was performed, followed by the standard genomic dna purification step using deoxyribonuclease i kit (sigma aldrich, st louis, mo, usa). One microgram of rna was used for cdna synthesis by high capacity cdna kit with rnase inhibitor (applied biosystems, foster city, ca, usa). Real - time pcr was performed on cfx96 system (bio rad, singapore) to assess relative expression of catalase (cat), glutathione peroxidase 1 (gpx1), superoxide dismutase 1 (sod1), hif-1, il-1, il-2, and il-6 . Except for the primers for il-6 gene published by jeong et al ., all other primers were custom made using primer 3 software . Messenger rna expression was determined using ssofast evagreen supermix (bio rad, singapore). Tissue powder was additionally homogenized in 100 mm phosphate buffer solution (ph 7.0) containing 1 mm edta (1: 10, w / v) using ultra turrax t10 homogenizer (ika, staufen, germany) while kept on ice . Tissue homogenates were sonicated for 30 seconds on ice in three 10 seconds intervals and then centrifuged at 20,000 g for 15 minutes at 4c . Cat, gpx, and sod enzyme activities were determined using a lambda 25 uv - vis spectrophotometer equipped with uv winlab 6.0 software package (perkin elmer for the better, massachusetts, usa). Catalase (ec 1.11.1.6) activity was estimated spectrophotometrically using h2o2 as a substrate . The reaction mixture consisted of 10 mm h2o2 in 50 mm phosphate buffer ph 7.0 . Changes in absorbance of the reaction mixture were measured at 240 nm during 2 minutes after the sample addition . One unit of activity corresponds to the loss of 1 mol of h2o2 per minute . Cat activity was calculated using molar extinction coefficient (= 0.04 mm cm) and expressed as u mg protein . To assess glutathione peroxidase (ec 1.11.1.9) activity a modified method described by wendel using h2o2 as a substrate was employed . Gpx activity was determined indirectly by measuring the rate of nadph oxidation to nadp+, accompanied by a decrease in absorbance at 340 nm . The assay mixture consisted of 50 mm phosphate buffer with 0.4 mm edta and 1 mm sodium azide (ph 7.0), 0.12 mm nadph, 3.2 units of gr, 1 mm glutathione, and 0.0007% (w / w) h2o2 in a total volume of 1.55 ml . One unit catalyses the oxidation by h2o2 of 1.0 mole of reduced glutathione to oxidized glutathione per minute at ph 7.0 and 25c . Gpx activity was calculated using molar extinction coefficient for nadph (= 6.220 mm cm) and expressed as u mg protein . Superoxide dismutase (ec 1.15.1.1 .) Activity was determined using cytochrome c (0.05 mm) as an inhibitory molecule in pbs buffer saline with 0.1 mm edta in system xanthine (1 mm)/xanthine oxidase (50 u) by flohe method . Total soluble protein concentration in protein extracts was determined by bradford reagent (sigma aldrich, steinheim, germany) following manufacturer's protocol and using bovine serum albumin as a standard . Were tested by one - way anova or kruskal - wallis test followed by the holm - sidak / tukey or dunn's post hoc multiple comparison procedure, respectively (sigma plot 11.0, sigmastat inc ., the student t - test and mann - whitney u statistic were used to compare the differences between the two groups in the case of normally distributed variables and variables that violated assumption of normality, respectively . Spearman's correlations were calculated where appropriate (sigma plot 11.0, sigmastat inc . ). Dai results were analysed by two - way anova and bonferroni post hoc test (graphpad prism 5.0, graphpad software, inc ., la jolla, ca, usa). A p value of <0.05 was considered statistically significant for all procedures . All data are presented as mean values standard error of mean (s.e.m . ). In order to determine the effects of hbo2 on the course of acute colitis, balb / c mice were exposed to 5% dss in the drinking water ad libitum and daily monitored for body weight, stool consistency, and occult / gross rectal bleeding to calculate dai . In this study the dss treatment induced substantial weight loss, rectal bleeding, loose stool, and colon shortening resulting in significantly higher dai compared to the control (ctrl) group, starting from day 3 until the end of the experiment (p <0.01; figure 1(a)). Mice that received dss and underwent hbo2 treatment (dss + hbo2 group) also presented with significantly higher dai compared to the ctrl group; however, in this group of mice hbo2 treatment significantly reduced dai compared to the dss mice, starting from day 5 throughout day 8 (days 5 and 8 p <0.01; days 6 and 7 p <0.05; figure 1(a)). In addition, average colon length in the dss group of mice was significantly shorter (8.14 0.23 cm) compared to the ctrl group (13.88 0.45 cm; p <0.0001), while this effect was significantly ameliorated by hbo2 treatment in the dss + hbo2 group (11.34 0.38 cm) compared to the dss group (p = 0.0001, figure 1(b)). Dss induced colitis resulted in significant body mass loss compared to ctrl group, irrespective of hbo2 treatment (11.89 0.03% and 8.24 0.01% in the dss and dss + hbo2 group, resp . ; ctrl and ctrl + hbo2 gained body mass during the experiment, 8.25 0.2% and 0.33 0.01%, respectively . Histological assessment of the colon revealed severe inflammation and ulceration extending into the deep portions of the mucosa with loss of crypts and with increased number of lamina propria leukocytes in dss group of mice . We also found structural changes of mucosa in the colonic tissue of dss + hbo2 mice but with reduced infiltration of inflammatory cells and decreased crypt distortion . When compared to the dss + hbo2 group, the dss group had significantly higher total histological score as well as individual scores (see modified geboes score in section 2.3, figure 1(d), p <0.001), except for the intraepithelial neutrophil infiltration score (p = 0.104). Distribution of inflammatory cells among the peripheral lymphoid organs, including gr-1 leukocytes (monocytes and neutrophils), f4/80 leukocytes (monocytes), cd3 t lymphocytes, and b220 b lymphocytes (figure 2), was assessed at the end of the experiment . In the mln, frequencies of gr-1 cells did not differ among the experimental groups, while dss induced a significant increase in f4/80 and decrease in cd3 cell frequencies (p <0.05 and p = 0.032, resp . ; figure 2(b)). These findings in the dss group were accompanied by a b - cell increase which was not statistically significant . Hbo2 alone had no effect on the cell frequencies in mln of control mice, whereas it substantially ameliorated these changes in mice with dss - induced colitis (dss + hbo2 group) but without reaching statistical significance . In the spleen, gr-1 cell frequencies were significantly decreased in the dss group compared to ctrl (p = 0.006) and ctrl + hbo2 groups (p <0.001; figure 2(a)). Hbo2 treatment reversed gr-1 cell frequencies to control values in the dss + hbo2 group (p = 0.056; figure 2(a)). In addition, the dss mice showed reduced frequencies of b220 lymphocytes compared to the ctrl group (p = 0.016), and hbo2 treatment abolished these effects in the dss + hbo2 group (p = 0.034; figure 2(c)). In addition, our study revealed that dss - induced immune responses in the mln and the spleen were significantly dampened by hbo2 treatment . Frequency of cd4 cells among the colon lamina propria lymphocytes of dss and dss + hbo2 groups was significantly increased compared to the ctrl group (p = 0.015 and p = 0.047, resp . ), while the frequency of cd8 cells was significantly increased only in the dss group when compared to the ctrl group (p = 0.011; figure 3). Inflammatory conditions have been known to include the enhanced production of ros and other oxidative mediators that may affect transcriptional regulation via hif-1. To investigate the role of hif-1 in the regulation of the antioxidative response / capacity during dss - induced colitis and hbo2 treatment, hif-1, cat, gpx1, and sod1 mrna expressions were determined using quantitative pcr method . Mrna expression was significantly changed by the hbo2 treatment and the inflammatory microenvironment in the gut mucosa . Dss - induced colitis resulted in significant upregulation of hif-1 gene in colonic mucosa (p = 0.008 for dss group compared to ctrl), and the hbo2 treatment further increased hif-1 mrna expression in the dss + hbo2 group (p = 0.028 compared to ctrl; figure 3(a)). In addition, the activity of hif-1 protein was indirectly confirmed by measuring mrna expression of well - established hif-1 target genes, vegf and pgk1 . Both genes showed strong positive correlation to the hif-1 mrna (supplementary figure 1) (see supplementary material available online at http://dx.doi.org/10.1155/2016/7141430). There was also a tendency for upregulation of hif-1 gene in mln and spleens of the dss group and its downregulation via hbo2 in the dss + hbo2 group (figure 4(a)); however, these changes did not reach statistical significance . Inflammation during dss - induced colitis and the hbo2 treatment also induced significant changes in mrna expression of target antioxidative genes . Dss - treated mice presented with significant downregulation of the cat gene in the colon compared to the ctrl group (p = 0.031), while there was a significant upregulation of cat gene in the spleen of the dss + hbo2 mice compared to the ctrl group (p = 0.026; figure 4(b)). In the colon, gpx1 mrna expression was increased in the dss (p = 0.034) and the dss + hbo2 (p = 0.003) group compared to ctrl group . The upregulation was even greater in mice with dss - induced colitis that underwent the hbo2 treatment (dss + hbo2 group; figure 4(b)). Sod1 mrna expression was significantly reduced in the colon of the dss + hbo2 group compared to ctrl (p = 0.008) and ctrl + hbo2 (p = 0.007) groups . Similar changes in sod1 gene expression were also found in the mln of the dss + hbo2 group (p = 0.025 compared to ctrl; figure 4(b)). To summarize, colitis resulted in gpx1 gene upregulation and cat gene downregulation, while hbo2 downregulated sod1 and further upregulated gpx1 in a tissue - specific manner . To examine the possible role of hif-1 in transcriptional control of antioxidative genes in the colon, the results revealed a strong negative correlation between hif-1 and sod1 (r = 0.651, p = 0.001) and a positive correlation of hif-1 to the gpx1 gene (r = 0.750, p <0.001), while there was no significant correlation between the hif-1 and the cat gene in the colonic tissue (figure 4(c)). The early phase of inflammation is mediated by several proinflammatory mediators, which prompted us to assess how hbo2 treatment affects their production . We found that gut mucosa inflammation was accompanied with a significant increase in il-1 and il-6 gene expression . In the case of il-6 gene, this was significant for dss and dss + hbo2 groups compared to the ctrl + hbo2 group (p = 0.024 and p = 0.021, resp . ; furthermore, il-6 gene was significantly upregulated in the mln of the dss group (p = 0.001 compared to ctrl), and hbo2 reduced its expression almost to control values in the dss + hbo2 group (p = 0.016 compared to dss; figure 5(a)). Il-1 mrna expression in the colonic mucosa was significantly increased during inflammation in dss group compared to ctrl (p = 0.014) and ctrl + hbo2 groups (p = 0.041). Il-1 mrna levels in the colon of the dss + hbo2 group did not significantly differ from the control groups, suggesting that hbo2 treatment blocked the increase of il-1 gene expression in the inflamed mucosa . Il-2 gene was significantly upregulated in the mln of the dss group compared to the ctrl group (p = 0.003), and hbo2 treatment resulted in its significant downregulation in the dss + hbo2 group (p = 0.032). Similarly, il-2 gene was significantly downregulated in the spleen of mice from the dss + hbo2 group compared to the ctrl group (p = 0.025). In addition, there was a strong positive correlation between hif-1 and il-6 gene (r = 0.749, p <0.001; figure 5(b)), while there was no correlation between hif-1 and il-1 or il-2 genes in the colonic tissue (figure 5(b)). In addition to their mrna expression, we also tested the enzymatic activity of antioxidative enzymes . We found that both the inflammation and the hbo2 treatment per se were able to change the activity of antioxidative enzymes in the colonic mucosa and the peripheral lymphoid organs (mln and spleen). In spleen hbo2 treatment per se induced significant increase of sod activity (p = 0.040 compared to ctrl). Mice with dss - induced colitis presented with significantly increased activity of sod (p = 0.012 compared to ctrl) in the colon and reduced cat activity in mln and spleen (p = 0.023 and p = 0.032, resp .) Compared to ctrl group . Treatment did not change sod activity in the inflamed colonic mucosa, which was comparable to the levels found in dss and ctrl + hbo2 groups (significantly increased compared to ctrl, p = 0.010). On the other hand, hbo2 treatment significantly increased cat (p = 0.020) and gpx (p = 0.001) activities in the spleens of the dss + hbo2 . Immune cells at the site of inflammation and in the peripheral lymphoid organs are an important source of ros . Therefore we assessed the basal levels of intracellular h2o2 and onoo and their production upon pma - induced activation in the lymphocytes isolated from mln and spleens of the mice from all experimental groups (figure 6). Basal h2o2 and onoo production in the mln was not significantly different among the groups, except for the lymphocytes from the dss + hbo2 group which presented with a significant increase of h2o2 and onoo levels compared to the ctrl group (p = 0.033). Pma stimulation resulted in increased intracellular h2o2 and onoo production, although statistically significant only for ctrl (p = 0.031) and dss + hbo2 (p = 0.012) groups . In the spleen, hbo2 increased lymphocyte h2o2 and onoo production in ctrl + hbo2 and dss + hbo2 groups (p = 0.004 and p = 0.007 compared to the ctrl; and p = 0.005 and p = 0.009 compared to the dss group). Their production after pma - induced activation was decreased in all experimental groups except the ctrl group; however, this effect reached statistical significance only in the ctrl + hbo2 group (p = 0.018 compared to unstimulated lymphocytes). In the present study, the experimental model of dss - induced colitis in balb / c mice was employed to explore the effects of hbo2 on the antioxidative enzymes, transcription factor hif-1, and proinflammatory cytokine genes during colonic inflammation and their role in modulating the course of the disease via hbo2 treatment . The most important findings are that (a) hbo2 significantly reduces symptoms and severity of dss - induced colitis, as evidenced by clinical appearance, contraction of the immune cell expansion and mobilization, and reversal of il-1, il-2, and il-6 gene expression; (b) hbo2 modulates the expression of antioxidative enzyme genes and enzyme activities during colitis; and (c) hbo2 enhances hif-1 mrna expression in the inflamed colonic tissue which is in a strong correlation with gpx1, sod1, and il-6 mrna expression . Several previous studies in animals and humans demonstrated the positive effects of hbo2 treatment in influencing the severity of colitis and reducing gut mucosa inflammation [27, 34]; however, data on the precise underlying mechanisms are scarce . Considerably more data on the beneficial anti - inflammatory effects of hbo2 are available for other conditions such as septic shock, ischemia / reperfusion injuries, and atherogenesis, where the previous studies reported reduced proinflammatory cytokine expression, suppressed development of th cells, shrinking of spleen and lymph nodes, decreased responses to antigens, and reduced frequencies of circulating leukocytes [3542]. Although this is the first animal study investigating the effects of hbo2 performed on dss - induced colitis in balb / c mice and correlating it with the immune cell frequencies, our results are in line with previous findings on the changes associated with dss - induced acute immune response, as well as on the effects of hbo2 on the antioxidative enzyme activities determined in other animal models, such as tnbs and acetic acid induced colitis in rats [27, 43, 44]. During colitis mice presented with decreased t and b cell frequencies in the spleen and reduced t cell frequencies in the mln, suggesting that lymphocytes are recruited from the peripheral lymphoid organs and probably migrate to the inflamed colonic mucosa . One element of the beneficial effect of hbo2 may be linked to normalized t and b cells frequency in the mln and spleen of mice with dss - induced colitis after hyperbaric treatment (figures 1 and 2). By measuring cd4 and cd8 lymphocyte in colon we confirm our hypothesis of t - cell recruitment from the peripheral lymphoid organs and their migration to the inflamed colonic mucosa, as well as immunomodulatory effect of hbo2 . In our model hbo2 did not affect cell frequencies in the peripheral lymphoid organs of control mice, in contrast to previous findings where hbo2 treatment per se was able to change lymphocyte subset populations in the spleen . For a long time macrophages and neutrophils have been considered as immune cells exclusively producing proinflammatory cytokines, chemokines, and large amounts of ros / rns contributing to aggravated inflammation . We have found decreased spleen gr-1 cell frequencies during colitis and their normalization upon hbo2 treatment (figure 2). In addition, we showed increased mln frequencies of f4/80 cells in dss group, while hbo2 treatment reversed their frequencies almost to control values . These findings indicate that hbo2 can modulate distribution of phagocytes by retaining neutrophils in the spleen and instigating macrophage migration towards the site of inflammation, in agreement with previous findings describing inhibited neutrophil infiltration into the gut of mice with dss - induced colitis . Furthermore, hbo2 treatment alone did not change the expression of proinflammatory cytokines in the colon, mln, or spleen of the control mice; however, dss - induced colitis resulted in a significant il-1 and il-6 gene upregulation in the colonic tissue and il-2 gene upregulation in the mln (figure 5). Consistent with previous studies, hbo2 treatment abolished these effects, further confirming its anti - inflammatory potential [4749]. Several animal studies on the effects of hbo2 on the experimental colitis reported an increased antioxidative capacity and changes in antioxidative enzyme activity [24, 25]. It has been proposed that an optimal hbo2 treatment could generate ros which would function primarily as intermediates in the antioxidative signalling pathways leading to increased expression of antioxidative enzymes, reduced inflammation, and ameliorated colitis symptoms but would not further damage the colonic tissue [50, 51]. . Showed that 24 hours after a two - hour hbo2 treatment at 2 bars in rats oxidative stress is not elevated, as evidenced by assessing ferric reducing antioxidant power ability of plasma (frap) and thiobarbituric acid reactive substances (tbars) level . In addition, a recent study also suggests that ros produced by nadph oxidase complex are important mediators inducing anti - inflammatory response in autoimmune diseases . Data on the cat mrna level during dss - induced colitis and upon hbo2 treatment were not available prior to this study . We found that cat mrna expression is tissue and treatment specific (figure 4). Colitis resulted in a significant downregulation of cat mrna expression in the colonic mucosa, and the hbo2 treatment induced its upregulation in the spleen of dss + hbo2 group of mice . These results were largely in accordance with our finding on enzymatic catalase activity that was decreased in all measured tissues in the dss group and reversed to control values in the spleen of dss + hbo2 group (table 2), as well as with a previous study demonstrating decreased catalase activity in colonic tissue upon dss treatment . This is also in line with a study on skin transplanted balb / c mice where hbo2 treatment increased catalase, gpx, and sod activity in the spleen . Furthermore, upregulation of protein and mrna catalase levels 14 days after hbo2 treatment, but not after 7 days, was also observed in the ulcer tissue of patients with diabetic foot, indicating a time - course for the effect of hbo2 to prevail . We found that gpx1 mrna level was upregulated in the colon of dss treated mice, irrespective of the hbo2 treatment, and there were no significant differences in the gpx1 mrna expression in mln and spleen . In contrast to our findings on mrna expression, colon gpx enzyme activity was slightly reduced in dss + hbo2 group compared to other groups, which is consistent with previous results obtained in acetic acid induced colitis in rats receiving combined hbo2 and ozone treatment . However, other reports indicate decreased gpx and sod activity in the inflamed distal colon mucosa and the plasma of rats with acetic acid induced colitis, and hbo2 normalized gpx but not sod activity in the colon . The observed discrepancies in the results may be related to the differences in experimental models used among the studies . In addition, in our study hbo2 treatment induced enhanced gpx and sod activity in the spleen of dss mice which is in contrast to reduced sod1 mrna expression and might be explained by additional sod2 and sod3 function in regulation of antioxidative capacity . Although intracellular h2o2 and onoo levels were slightly increased during inflammation and hbo2 treatment in the mln and hbo2 per se increased its level in spleen, impaired lymphocyte function was not observed . Intensive research on the beneficial wound healing effects of hbo2 revealed its capacity to induce neovascularization, reduce oedema, decrease leukocyte adhesion, stimulate fibroblast expansion, and inhibit bacterial growth [14, 57]. Some of these processes are transcriptionally regulated by hif-1, namely, the vascular endothelial growth factor (vegf) expression, regulatory t lymphocyte differentiation, and preservation of epithelial thigh junction integrity . In addition, previous studies employing conditional deletion of epithelial hif-1 or pharmacologic activation of hif-1 in a murine model of colitis demonstrated a protective role for hif-1 in colitis . It has also been shown that hif-1 increases expression of barrier - protective genes (multidrug resistance gene-1, intestinal trefoil factor, cd73), decreases tnf mrna expression, and enhances antimicrobial activity by transcribing beta - defensin 1 . In the present study we found increased expression of hif-1 gene in inflamed colonic tissue, and hif-1 gene expression was changed (upregulated) by the inflammation while hbo2 treatment showed a tendency to reverse this increase . These data suggest involvement of different mechanisms controlling hif-1 gene expression at the site of inflammation (colon) and the peripheral lymphoid organs (mln and spleen), responsible for the initiation of the immune response and the t / b - cell expansion and differentiation, respectively . We also demonstrated a strong positive correlation between hif-1 and gpx1 mrna levels in the colon (figure 4(b)). This is in line with in vitro studies where overexpressed hif-1 in colorectal cancer cells resulted in enhanced gpx1 expression through tgf-ri / smad2/erk1/2/hif-1 signalling cascade, suggesting transcriptional regulation of gpx1 by hif-1 . In the present study we found strong negative correlation between hif-1 and sod1 mrna expression this is in accordance with a previous study showing that docosahexaenoic acid downregulates sod1 gene transcription through an hre - mediated mechanism (hre, hypoxia - response element), involving hif signalling in human cancer cells; thus our results indicate similar mechanism involved in sod1 control in the murine colon mucosa in vivo during colitis and hbo2 . Previous studies revealed that hif-1 mediated transcriptional regulation of different proinflammatory cytokines and growth factor genes are tissue and cell specific and include regulation trough alternative splicing, mrna stability, and interactions with other transcription factors like nf-b [6669]. In our study we found a strong correlation between hif-1 and il-6 mrna levels suggesting involvement of hif-1 in transcriptional regulation of il-6 gene during colonic inflammation and hbo2 . In conclusion, our results confirmed that hbo2 exerts an anti - inflammatory effect on dss - induced colitis in mice, and this effect at least involves hif-1 and antioxidative genes expression regulation (as outlined in figure 7). However, further studies are necessary to identify the cells that may contribute to or are influenced by the effects upon hbo2 treatment.
Sexuality in women is a complex issue with physiological, psychological, and cultural components . Determination of sexual partner involvement, intimate relationships, and past abusive relationships is crucial in female sexual dysfunction (fsd) (1, 2). Sexual dysfunction is more prevalent among women compared to men (43% vs. 31%) (3 - 5). In 1960, masters and johnson studied and reported both on healthy sexual function and sexual dysfunction for the first time . They described four phases of the human sexual response cycle, as follows: excitement, plateau, orgasm, and resolution . Nowadays, the accepted classification of fsd consists of disturbances in desire, including hypoactive sexual desire disorders (hsdd), sexual aversion disorders, sexual arousal disorders, orgasmic disorders, and sexual pain disorders, including dyspareunia and vaginismus (3, 6 - 9). Fsd can further be classified as primary or secondary and persistent or situational (10). Intimate sexual function is one of the best predictors of quality of life, and sexual dysfunction causes many problems for couples . Some researchers have found that the frequency of sexual relationship and sexual satisfaction are positively associated with marital satisfaction (11, 12). Among other factors, illicit drug dependency changes . Moreover, opioids act on a variety of neurotransmitters in the brain, including endorphins . Furthermore, morphine has been found to inhibit sexual behavior in a dose - dependent manner through a complex pharmacological domain (13, 14)., most studies on drug abuse and sexual performance have focused on addicted populations, and have usually considered men . Although many studies have looked at the frequency of sexual dysfunction in women, limited research has focused on the impact of addiction on spouse sexual function . To the best of our knowledge, few studies have been conducted on the effect of males opium dependency on their wives sexuality . Thus, the present cross - sectional study aimed to assess the prevalence of fsd among women with addicted partners . We hypothesized that substance dependency in males would affect their wives sexual function . This cross - sectional controlled study was conducted on 340 women of reproductive age (25 - 50 years old) selected through convenience sampling . The case group included 160 women whose husbands were opioid dependent . Some of them were the wives of opioid - dependent males hospitalized in the addiction treatment units of teaching hospitals affiliated with the shiraz university of medical sciences, shiraz, iran . Other women were encountered in centers where opioid - dependent males spouses were referred for group therapies . In contrast, the control group (n = 160) included women whose spouses were not opioid dependent; they were selected from the general population referring to the clinics of the shiraz university of medical sciences . After giving informed consent, the participants were interviewed privately according to the dsm - iv - tr criteria by a senior female medical student who was one of the researchers and had been properly trained regarding the nature of the study . The interview focused on hsdd, sexual arousal disorder, orgasmic disorder, and sexual pain disorder . In addition, the subjects demographic information, such as age, level of education, marital status, and duration of marriage, was obtained using a self - constructed questionnaire designed by the authors . To include the women in the study the exclusion criteria of the study were a history of substance use disorders, gynecological disorders (gyn), and chronic medical disease that has been shown to affect sexual functioning (e.g. Hypertension, diabetes mellitus, chronic kidney disease, cancer, spinal cord injury, lupus, fibromyalgia, chronic pain and chronic depression). It should be noted that the questionnaires were anonymous and participation in the study was voluntary . The chi - square test was used for categorical data, while the t - test was utilized for continuous data . The protocol for this study was approved by the ethics committee of the shiraz university of medical sciences, shiraz, iran . Out of the 340 women who were selected, 5 in the case group and 15 in the control group were excluded due to missing data . The mean ages of the case and the control group were 36.35 and 33.2 years, respectively . In this study, statistical significance was defined as p <0.05 . It should be noted that in terms of demographic characteristics, there was no significant difference between the case and the control group (p> 0.05), so, we could use further statistical methods on the data . Table 2 shows the frequency of sexual dysfunction in the two study groups . According to the results, approximately more than 50% of the participants had problems in at least one domain of sexual function . Moreover, the main fsd observed in the case women was hsdd (75.46.9%) followed by sexual aversion disorder (73.46.2%), sexual arousal disorder (65 .40.9%), orgasmic disorder (59.37.1%), and sexual pain disorder (45.28.5%). Furthermore, 75 out of the 155 women in the case group (46.9%) and 16 out of the 145 women in the control group (10%) had hsdd, and the difference between the two groups was statistically significant (p <0.05). In addition, 73 women in the case group (46.2%) and 20 in the control group (12.6%) had sexual aversion disorder, with a significant difference between the two groups (p <0.05). In this study, this disorder was a significant problem affecting a considerable number of women in both the case and control groups (table 2). This high prevalence may have resulted from various causes . Although studies have shown that many factors, such as age and level of education, affect sexual function (15 - 17), the findings of the current study revealed no relationship between the demographic characteristics, that is, age, duration of marriage, and level of education, and sexual dysfunction . As can be seen in table 2, the frequency of hsdd and aversive sexual desire disorder was more common in the case group than the control group, and the difference was statistically significant (p <0.05). The high frequency of hsdd among the addicts spouses exhibited in this study was in line with the study performed by noori et al . However, noori et al . Included no control groups in their study, and consequently, it was not possible to compare the characteristics of the subjects with those of women whose husbands were not addicted . The findings of the present study showed that the addicts spouses suffered from hsdd significantly more than the control group . Another finding was the higher frequency of orgasmic disorder in the case group; however, this difference was not statistically significant . The higher rate of orgasmic disorder among the addicts spouses obtained in this research was in line with the study by noori et al . In contrast to our research, in one study conducted on 2626 women in iran in 2006, 31.5% of the subjects (759) suffered from sexual disorder, and the most prevalent disorder was orgasmic disorder, followed by desire disorder (19). In the dsm - iv - tr, sexual desire disorders are divided into two classes, as follows: 1) hsdd characterized by a deficiency or lack of sexual fantasies and desire for sexual activity and 2) sexual aversion disorder characterized by an aversion to and avoidance of genital contact with a sexual partner . No single cause of hsdd has been defined; however, physiological, psychological, and sociocultural factors that contribute to female sexual desire may all be important in its development (20, 21). Master and johnson s linear model of sexual response does not always work for females . Some factors, such as emotional intimacy and relationship satisfaction, may change this model . Studies have shown that the motivating factors for female sexual desire are very complicated . Sexual desire and the presence or absence of orgasm could result from multiple cultural and environmental factors, as well as from interpersonal and intrapersonal distresses, and are greatly affected by emotional intimacy (2). Since addiction in the family could be the origin of many stressors and disputes, such stresses and interpersonal turmoil could have a decisive role in decreasing females sexual desire toward their addicted husbands . Researchers have found that sexual response phases in women are a combination of mental and physical responses which overlap with one another (2, 3). In general, women have diverse reasons to initiate or agree to have sex with their partners . Sexual motivation in females is far more complicated than just the presence or absence of sexual desire and is characterized by thinking or fantasizing about sex and longing to have sex . Moreover, the decision to be sexual may originate from a conscious wish for emotional closeness or result from seduction or a suggestion from a partner . Addicted couples often have conflicts over money and drugs; so that love gradually flies out of the window, and most often these couples relationships end at a sad, bitter point (11, 22, 23). Hence, this kind of relationship is expected to have a negative impact on sexuality . Of course, further studies are needed in order for better characterization and understanding of fsd epidemiology . One of the limitations of this study was the difficulty of gaining access to the case group sample and persuading them to cooperate with the researchers . In addition, when they agreed to take part in the study, the interviewer had to meet them out of their group . Moreover, the subjects might have answered the questions conservatively due to the particular nature of the study subject, that is, sexual behavior, in iranian culture . Another limitation of the study was a lack of control of other contributing factors, such as the economic status of the family and sexual disorders and duration of opioid dependency in addicted husbands, which are assumed to have an effect on their wives sexual dysfunctions.
Laparoscopic surgery has gained widespread acceptance in a variety of procedures, ranging from gastric fundoplication to cholecystectomy . Although laparoscopic appendectomy (la) is more expensive than is open appendectomy (oa) due to the costs of the disposable equipment, and can be more technically challenging in children, the overall cost of the operation has been shown to be similar to that of open appendectomy . The real cost savings of la are due to the shorter postoperative length of hospital stay (los) and the infrequent postoperative complications . The ultimate reduction in postoperative hospital costs to make la most cost - effective would be to perform the procedure as same - day surgery or so - called fast - track surgery . Because it has been our observation that many children can be discharged within 24 hours after a laparoscopic appendectomy, we reviewed our records to determine its feasibility . A retrospective chart review was performed on 79 children who underwent a laparoscopic operation for the suspected diagnosis of acute appendicitis over a 3-year period between july 1997 and july 2000 . Prior to the operation, if the diagnosis of acute appendicitis was unclear, the patients were evaluated with further laboratory and diagnostic testing, which included an abdominal computed tomography scan or ultrasound evaluation, or both of these, in select patients . Laboratory information included white blood count and urinalysis, and depending on the circumstances, might also include a beta human chorionic gonadotropin and rapid streptococcal screen, as well as other tests as indicated . All patients underwent the operation while under general anesthesia, and all received a single dose of a broad - spectrum antibiotic preoperatively for wound infection prophylaxis . The standard laparoscopic operative technique was used, with either a 5-mm or 12-mm port introduced at the umbilicus after inducing pneumoperitoneum with carbon dioxide . A 0- or 30-degree laparoscopic telescope was then introduced through this port, and the abdominal and pelvic cavities were directly visualized with the laparoscope to inspect for other possible pathologies . Next, two 5-mm ports were introduced under direct vision, one in the suprapubic region or the left lower quadrant and another in the right iliac fossa or in the right upper quadrant . The appendix was identified and grasped by its distal end to fully expose the entire organ and its attached structures . The appendiceal artery was isolated, ligated, and divided or cauterized . At that point, a staple line was fired across the base of the appendix by using an endoscopic stapler, or endoscopic loops were used to isolate the base of the appendix, and it was divided . If the appendix was particularly enlarged or friable, it was removed from the abdomen through the umbilical incision with the use of an endoscopic bag . All ports were removed under direct visualization, and the fascia was reapproximated to prevent future incisional hernia occurrences . If any evidence of localized or generalized peritonitis was visualized during the operation, patients were sent home on oral antibiotics, usually a 5-day course of amoxicillin plus clavulanate . If the appendix was perforated prior to removal, patients were placed on triple antibiotic coverage intravenously, which included ampicillin, gentamicin, and clindamycin for 5 days while in the hospital . If any evidence of abscess formation or peritoneal soilage was noted, the abdominal cavity was vigorously lavaged with normal saline . Postoperative analgesia was obtained usually with ketorolac tromethamine and acetaminophen . To be discharged home, patients had to be tolerating oral liquids, be afebrile, and free of nausea and vomiting . Seventy - nine children (44 boys and 35 girls) between 2 to 17 years of age (mean, 11 years) underwent la . In 4 (5%) children, all with perforated appendicitis, the la was converted to an open appendectomy, secondary to technical difficulties in completing the operation laparoscopically . At operation, 51 (64.5%) had acute appendicitis, 22 (27.8%) had perforated appendicitis, 4 (5%) had ruptured ovarian cysts, and 2 (2.5%) had no obvious pathology . Total los for all 79 patients was a median of 58 hours, and median postoperative los was 35 hours . Complications included wound infection, 2 (2.5%); abdominal abscess, 4 (5%); drug rash, 2; and epididymoorchitis, 1 . All but one complication (drug rash, 1) occurred in the perforated group . In the 57 (72%) children without perforated appendicitis, the total los was a median of 42 hours, while median postoperative los was only 28 hours . Thirty - two (56%) of the children without perforated appendicitis went home in 24 hours following la . No significant morbidity occurred in the nonperforated group (drug rash, 1; fever> 24 hrs, 3); and no readmissions or reoperations were necessary on follow - up . Although most appendectomies, especially in children, are not done laparoscopically, many studies have shown that laparoscopic appendectomy (la) is at least as good as open appendectomy (oa), with several benefits, including less postoperative pain and shorter lengths of stay (los). The disadvantages of la, which include increased operative time and increased cost of equipment, are easily offset by the decreased postoperative recovery time and the apparent decreased incidence of postoperative complications . Additionally, it has been suggested that with increasing operative experience the operative time required for la will decrease significantly . Lastly, it has been suggested that even if patients are not discharged from the hospital soon enough after la to make a significant difference between the cost of la versus oa, la has a much shorter recovery time and returns patients to a productive lifestyle sooner, thus justifying la . Although most children are not working, the care - givers or parents can return to work sooner, when their child goes back to school . Adult series have documented a decreased incidence of postoperative complications and a decreased incidence of wound infection after la ., in our series, 2 patients had wound infection and 4 patients developed abscesses, which is a similar complication rate to that in other series . In all instances, these complications arose in the perforated group . Although perforation was at first considered a contraindication for la, it has now been utilized successfully in the management of acute appendicitis as well as perforated appendicitis . It has been suggested that thorough lavage of the abdominal cavity after appendectomy can help to decrease the incidence of abscess formation, and this is a practice that we utilize and is facilitated by the use of laparoscopy . Certainly, the laparoscopic approach facilitates the complete irrigation of the abdominal cavity and identification of all loculated collections . An additional benefit of laparoscopic surgery is that it leads to greater accuracy of diagnosis, especially in teenage female patients with suspected appendicitis . In cases such as obesity and mental retardation, the diagnosis of appendicitis laparoscopy can be used to delineate the source of abdominal pain when the diagnosis of appendicitis is suspected but not certain . In 4 of our patients with the presumed diagnosis of appendicitis, intraoperative visualization revealed normal appendices, and ruptured ovarian cysts were identified as the source of their pain . Obviously, all four patients were female . Others have also found an increased preponderance of unclear diagnoses in the female population . In our patients, the appendix was always removed at the time of operation, despite the fact that occasionally (in 4 patients) the gross appearance of the appendix was normal . In the past, it has been unclear whether it would be of any benefit to the patient to remove a healthy organ, but it has been argued that with advances in laparoscopy and its proven benefit, there is no justification for leaving a visually normal appendix in place . Additionally, microscopic evidence of early appendicitis is occasionally seen . In our study, it was noted that 2 visually normal appendices were found to have microscopic evidence of appendicitis . Outpatient surgery has been widely accepted in a variety of procedures, and many are done laparoscopically . The idea that an appendectomy can be done in the pediatric patient as outpatient surgery is not a new one . In 1993, ramesh and gallard suggested early discharge after open appendectomy, even within 24 hours . In another study, velhote et al also found that most children could be sent home within 24 hours after appendectomy . In that study, the appendectomy was performed through a standard gridiron incision of 2 cm or less . Brosseuk and bathe suggested laparoscopic appendectomy as outpatient surgery in 1999, and they advocated early discharge in both perforated and simple appendicitis . The fact that all major complications in our study occurred in perforated cases would argue against these cases being performed as same - day surgery, although others have disputed this approach . In our treatment protocol, perforated appendicitis would not be placed on a fast - track surgery list because these patients usually receive inpatient intravenous antibiotic therapy for 72 hours . We suggest that laparoscopic appendectomy is a safe and effective treatment in the pediatric population, and that in cases of nonperforated appendicitis this may be performed as a fast - track or short - stay procedure . We believe that this not only is more convenient for patients and their families, but it also adds to the overall cost - effectiveness of laparoscopic appendectomy . Our findings are based on a retrospective review of our charts over the past 3 years, and this is an obvious criticism of the study . In no instance was it prospectively decided that the child would be discharged within 24 hours . In fact, a small number of children met exclusion criteria due to delays in discharge not related to their medical condition . We made our best attempt to identify when the order was given for the child to be released rather than the actual time that the child left the hospital . However, the documentation was occasionally unclear, and these children were excluded if it could not be ascertained exactly when the order was given or when they left the hospital . We believe that in cases of simple appendicitis, there is no reason not to dismiss a child within 24 hours if the above - mentioned discharge criteria are met . We anticipate that in the future, a greater percentage of our pediatric patients will be discharged within 24 hours, and we feel that in cases of simple appendicitis, laparoscopic appendectomy can be done as fast - track or short - stay surgery . It may be safely performed as fast - track or same - day surgery, in select children without perforated appendicitis, with a postoperative stay of 24 hours in the majority of such patients.
A major effort in the nucleocytoplasmic transport field has been directed towards the analysis of all members of the importin family, in particular with the aim of identifying specific transport cargoes . This has led to the characterization of a large number of related proteins (both importins and exportins) in all eukaryotic species analyzed . New members have primarily been identified by sequence homology or biochemically via their interaction with the small gtpase ran . Strikingly, the roles of many of these receptors is conserved from yeast to humans . There are 14 putative members of the importin family in the completed saccharomyces cerevisiae genome, nine of which have been shown to function as importins and four as exportins . The genes encoding yeast transport receptors are dispersed throughout the genome and none of them contains introns . Little is known about the gross structure of the genes encoding these nuclear transport receptors, and our knowledge of the chromosomal localization or the organization of the individual genes encoding members of this family is very poor . The relative molecular masses of members of importin--like proteins vary between 90 kda and 130 kda, but all are characterized by an acidic isoelectric point . The overall sequence similarity between various transport receptors is low (less than 20% amino acid identity) and, in many cases, is restricted to the amino - terminal domain . Work mainly on importin has demonstrated that these receptors bind rangtp via the amino - terminal domain and cargo via the carboxy - terminal domain . To permit shuttling through the nuclear pore complex (npc), transport receptors also contain one or multiple binding domains for components of the npcs, called nucleoporins . Truncation studies using importin indicate that the binding site for nucleoporins containing fxfg repeats (in the single - letter amino acid code, where x is, in many cases, a small polar residue or glycine) is located in an amino - terminal / central region of importin (residues 152 - 352). This was recently confirmed in the crystal structure of an amino - terminal fragment of importin in a complex with five fxfg nucleoporin repeats . Additional information comes from the crystal structures of importin and of transportin 1 (also known as karyopherin 2), which were solved either in a complex with rangtp, with a cargo, or in the free form . Overall, the structures of importin--like receptors are characterized by a very similar series of helical heat repeats (19 in importin and 18 in transportin 1; figure 1). Heat repeats are approximately 40 residues in length and are found in many eukaryotic proteins such as the pr65/a subunit of protein phosphatase 2a . The fundamental repeat unit is a right - handed superhelical structure consisting of a hairpin made up of two helices, named a and b, separated by a sharp turn . Each hairpin is connected to the next by a linker region . In transportin 1, almost all linkers contain a third helix, but there are very few linker helices in importin . In both receptors, one turn is extended into a long acidic loop, which has been suggested to be important for rangtp - mediated cargo release . Full - length importin complexed with the importin--binding domain of importin (ibb) forms a snail - like superhelical structure wrapping tightly around the ibb domain . The structure of the uncomplexed amino terminus of importin reveals a different superhelical architecture with a much steeper helical pitch than the cargo - bound or rangtp - bound forms . This suggests that importin undergoes twisted conformational changes in its heat - repeat helix stacking, which could be essential for the regulation of cargo binding and release and/or for protein interactions during the translocation through the npc . Although the sequence homology is limited, it is expected that exportins will fold in a similar way to the reported importin structures . It still remains unclear, however, why rangtp is required for binding of cargo to exportins but causes cargo dissociation from importins . Structure of importin . (a) importin is composed of 19 helical - repeat motifs (heat repeats). Each consists of an a and a b helix connected by a short turn, which in heat-8 is replaced by an acidic loop critical for the regulation of substrate binding and release . The heat repeats 1 - 8 are required for high - affinity binding to rangtp . The importin--binding (ibb) domain of importin interacts mainly with residues located in repeats 7 - 19 of importin . The binding site for nucleoporins of the npc is located between residues 152 and 352, corresponding to repeats 4 - 8 . On the basis of the crystal structure, the a helices of heat repeats 5 and 6 and a region between heat repeats 6 and 7 are thought to be critical for recognition of the fxfg motif . (b) structure of importin bound to the ibb domain of importin (adapted from). Importin (yellow) forms a superhelical structure that wraps like a snail around the ibb domain (blue). The 19 heat repeats share a common core of 21 residues, comprising the a helix with about three turns and the b helix with about four turns . The helices critical for the interaction with fxfg - repeat nucleoporins are in green . Note the acidic loop, which contacts both rangtp and the ibb domain (white arrow). A major function of transport factors of the importin family is to mediate the transport between the nucleus and cytoplasm of macromolecules that contain nuclear import or export signals . To this end,, they can be found in the nucleus, at the npc or in the cytoplasm . At present, very little is known about the tissue distribution of this family of proteins . All members have the ability to recognize and bind specific cargoes, either directly or via adaptor molecules, to bind rangtp and to interact with nucleoporins at the npc . Interactions between the proteins of the importin family and nucleoporin repeats have been shown both in vitro and in vivo . These interactions contribute to the import or export of importin family members and their cargoes through the central transporter of the npc . Import and export are multistep processes that are initiated by the recognition of nuclear localization signals (nlss) and nuclear export signals (ness). The most thoroughly studied import signals are the' classical' and the bipartite nlss, first identified in sv40 large t antigen and nucleoplasmin, respectively . Their transport is mediated by importin, the first - characterized member of this protein family . Additional importin--dependent adaptors in vertebrates include snurportin 1 (involved in import of m3g - capped small nuclear ribonucleoproteins, snrnps) and xrip, (involved in the import of replication protein a, rpa). Importin can also form a complex with another importin--like factor, importin 7, in order to transport the linker histone h1 into the nucleus . In addition, importin is able to interact directly with a large variety of different cargoes, including the t - cell protein tyrosine phosphatase, the human immunodeficency virus (hiv) tat and rev proteins, human t - cell leukemia virus rex protein, ribosomal proteins l23a, s7, and l5, cyclin b1, smad and the parathyroid - hormone - related protein . Table 1 details known importins, exportins and their cargoes and adaptors . Proteins in the importin--like family and their cargoes abbreviations: h.s ., homo sapiens; s.c ., saccharomyces cerevisiae; x.l ., xenopus laevis . Genbank accession numbers are given in square brackets for each protein, and alternative names are in parenthesis . The import of ribosomal proteins seems to rely on at least partially redundant mechanisms . In yeast, / yrb4p has been shown to be an important mediator of ribosomal - protein import, but the related protein kap121p / pse1p can functionally substitute for kap123p in vivo . In mammalian cells, at least four importin--like transport factors are able to mediate import of ribosomal proteins . Interestingly, both the -like importin receptor binding (bib) domain and some ribosomal proteins can be imported by any of the four receptors importin, transportin 1, importin 5 or importin 7 . The yeast importin kap104p mediates nuclear import of the mrna - binding proteins nab2p and nab4p . Its vertebrate homolog, transportin 1 (kap2), also mediates import of the rna - binding proteins hnrnp a1 and hnrnp f, but also of ribosomal proteins . Mammalian transportin - sr mediates nuclear import of a group of abundant arginine / serine - rich proteins, which are essential pre - mrna splicing factors . In yeast, the tata - binding protein (tbp) is imported into the nucleus by kap114p . Exportin 1 (crm1p, xpo1p) has been identified in both yeast and higher eukaryotes as an export receptor for leucine - rich nes - containing proteins . Human exportin 1 has been found to export protein kinase inhibitor (pki) and hiv rev, ib, snurportin 1, htlv rex, the small nuclear rna, cyclin b1 and the transcription factor nf - at4 . Targets for exportin in s. cerevisiae include the transcription factors yap1p, and ace2p, the mitogen - activated protein kinase hog1p, and the heat - shock protein ssb1p . Msn5p was first identified as a yeast exportin that exports the phosphorylated form of the transcription factor pho4p . Interestingly, msn5p was recently shown to function as an import receptor for the trimeric rpa, suggesting that individual members of the importin--like protein family can function both as import and export receptors . The first cellular rna export receptor to be discovered, named exportin - t in higher eukaryotes or los1p in yeast, mediates nuclear export of trnas . Many of the nuclear transport factors identified in s. cerevisiae are not essential for viability, even though they transport essential cargoes . This phenomenon can be explained by the fact that cargoes can use alternative transport pathways . This is probably best exemplified in the import pathway of ribosomal proteins, which is mediated by at least four different import receptors (see above). Nuclear transport mediated by importins and exportins is strongly directional in vivo: importins bind their cargo in the cytoplasm and transfer it to the nucleus, whereas exportins interact with their substrates in the nucleus and mediate their export to the cytoplasm (figure 2). Protein translocation through the npc is thought to occur by an essentially similar mechanism for all importin--related receptors, except for the fact that, in some situations, additional adaptors are required to bridge the cargo - receptor interaction . This is mediated by importin together with its adaptor importin, which binds both the nls - containing cargo and importin in the cytoplasm . After a trimeric importin -importin -nls complex is formed, importin mediates docking at the npc . In the presence of rangdp and free gtp, this trimeric complex translocates through the npc . Translocation is terminated by binding of rangtp to importin, which releases the complex from the npc and dissociates importin from importin (reviewed in). Free importin has a lower affinity for the nls cargo, and release from importin is therefore believed to trigger release of the nls cargo as well . Thereafter, the importin -rangtp complex, and importin bound to its exportin (cas) and rangtp, are re - exported to the cytoplasm for another round of import . A schematic representation of nuclear import and export cycles through the npc . The cargo - loaded importin translocates through the npc into the nucleus, where the cargo is dissociated from the importin by binding of importin to rangtp . The importin - rangtp complex recycles back to the cytoplasm, where rangtp hydrolysis is stimulated by rangap and ranbp1; this frees the importin for the next round of import . Exportins bind their export substrates in the nucleus, forming a trimeric cargo - exportin - rangtp complex . This complex is exported from the nucleus and dissociated in the cytoplasm by hydrolysis of rangtp to rangdp and inorganic phosphate (pi). This releases the export substrate, and the exportin is recycled back into the nucleus . For details, see text . Transport in the reverse direction, mediated by exportins, is regulated in a converse manner (figure 2). A paradigm for transport out of the nucleus is the export of leucine - rich nes - containing proteins . Exportin 1 binds to substrates containing a leucine - rich nes in the nucleus, forming a trimeric complex with rangtp . This complex is then transferred to the cytoplasm by a mechanism involving binding of exportin 1 to the npc . Once in the cytoplasm, gtp hydrolysis results in dissociation of ran from the complex, allowing exportin 1 to release its cargo . Free exportin 1 re - enters the nucleus to bind and export additional cargo molecules . As illustrated in these examples, the rangtp cycle plays a key role in conferring directionality to nucleocytoplasmic transport events and rangtp acts as a marker of the nuclear compartment for both nuclear import and export (figure 2; reviewed in). Remarkably, this model predicts that only a single molecule of gtp is hydrolyzed per import / export cycle; it strictly requires that rangtp is highly enriched in the nucleus . It is thought that a steep rangtp - rangdp gradient is generated by the cellular compartmentalization of regulators of the ran cycle . Specifically, the guanine - nucleotide exchange factor of ran (rangef or rcc1), which regenerates rangtp is nuclear and bound to chromatin . In contrast, the main gtpase - activating protein (rangap), and the ran - binding proteins, ranbp1 and ranbp2, which stimulate gtp hydrolysis by ran, are found in the cytoplasm . This asymmetric distribution predicts that ran is present mainly in the gtp - bound form in the nucleus, whereas ran is immediately converted to a gdp - bound state in the cytoplasm . Recently, it was suggested that in addition to the ran cycle, the npc itself could provide an additional mechanism to ensure transport directionality . Given that several nucleoporins implicated in binding to importins and exportins have distinctive locations in the structure of the npc, the asymmetric design of the npc may also be important to efficiently drive nuclear import and export . Nuclear transport mediated by importins and exportins is strongly directional in vivo: importins bind their cargo in the cytoplasm and transfer it to the nucleus, whereas exportins interact with their substrates in the nucleus and mediate their export to the cytoplasm (figure 2). Protein translocation through the npc is thought to occur by an essentially similar mechanism for all importin--related receptors, except for the fact that, in some situations, additional adaptors are required to bridge the cargo - receptor interaction . This is mediated by importin together with its adaptor importin, which binds both the nls - containing cargo and importin in the cytoplasm . After a trimeric importin -importin -nls complex is formed, importin mediates docking at the npc . In the presence of rangdp and free gtp, translocation is terminated by binding of rangtp to importin, which releases the complex from the npc and dissociates importin from importin (reviewed in). Free importin has a lower affinity for the nls cargo, and release from importin is therefore believed to trigger release of the nls cargo as well . Thereafter, the importin -rangtp complex, and importin bound to its exportin (cas) and rangtp, are re - exported to the cytoplasm for another round of import . A schematic representation of nuclear import and export cycles through the npc . The cargo - loaded importin translocates through the npc into the nucleus, where the cargo is dissociated from the importin by binding of importin to rangtp . The importin - rangtp complex recycles back to the cytoplasm, where rangtp hydrolysis is stimulated by rangap and ranbp1; this frees the importin for the next round of import . Exportins bind their export substrates in the nucleus, forming a trimeric cargo - exportin - rangtp complex . This complex is exported from the nucleus and dissociated in the cytoplasm by hydrolysis of rangtp to rangdp and inorganic phosphate (pi). This releases the export substrate, and the exportin is recycled back into the nucleus . For details, see text . Transport in the reverse direction, mediated by exportins, is regulated in a converse manner (figure 2). A paradigm for transport out of the nucleus is the export of leucine - rich nes - containing proteins . Exportin 1 binds to substrates containing a leucine - rich nes in the nucleus, forming a trimeric complex with rangtp . This complex is then transferred to the cytoplasm by a mechanism involving binding of exportin 1 to the npc . Once in the cytoplasm, gtp hydrolysis results in dissociation of ran from the complex, allowing exportin 1 to release its cargo . Free exportin 1 re - enters the nucleus to bind and export additional cargo molecules . As illustrated in these examples, the rangtp cycle plays a key role in conferring directionality to nucleocytoplasmic transport events and rangtp acts as a marker of the nuclear compartment for both nuclear import and export (figure 2; reviewed in). Remarkably, this model predicts that only a single molecule of gtp is hydrolyzed per import / export cycle; it strictly requires that rangtp is highly enriched in the nucleus . It is thought that a steep rangtp - rangdp gradient is generated by the cellular compartmentalization of regulators of the ran cycle . Specifically, the guanine - nucleotide exchange factor of ran (rangef or rcc1), which regenerates rangtp is nuclear and bound to chromatin . In contrast, the main gtpase - activating protein (rangap), and the ran - binding proteins, ranbp1 and ranbp2, which stimulate gtp hydrolysis by ran, are found in the cytoplasm . This asymmetric distribution predicts that ran is present mainly in the gtp - bound form in the nucleus, whereas ran is immediately converted to a gdp - bound state in the cytoplasm . Recently, it was suggested that in addition to the ran cycle, the npc itself could provide an additional mechanism to ensure transport directionality . Given that several nucleoporins implicated in binding to importins and exportins have distinctive locations in the structure of the npc, the asymmetric design of the npc may also be important to efficiently drive nuclear import and export . Despite the large amount of progress that has been made in the nucleocytoplasmic transport field in recent years, many important questions remain unsolved . Many import and export receptors have now been characterized and their first cargoes have been identified . The further characterization of new transport receptors and adaptors, and the identification of new import and export substrates, will lead to a more complete picture of nucleocytoplasmic transport . A big challenge for the future will be to understand how translocation through the npc occurs, and how nucleocytoplasmic transport is regulated . To gain insight into these questions, a quantitative analysis of interactions between transport receptors and nucleoporins will be required . It will be also interesting to see whether an increasing affinity gradient of receptors for nucleoporins along the npc exists and, if so, whether it makes an important contribution towards the direction of transport . Other issues that remain to be solved include the structural differences between members of the importin and exportin family . How does rangtp dissociate import complexes in the nucleus but promote binding of export cargoes to exportins? A key mechanistic topic that is poorly understood is the export of rnas and rnps . Although a trna - export factor has been identified, the mechanism of rrna or mrna export is still poorly understood . Several proteins in s. cerevisiae, such as mex67p and yra1p, and their metazoan counterparts tap and aly, have been indicated to play an important role in mrna export . Mex67p / tap does not belong to the family of importin--like proteins, suggesting that there are alternative translocation pathways through the npc.
Musculoskeletal conditions, or disorders of the muscles and bones, are the most common causes of chronic disability in the world . In the us alone, disease of the musculoskeletal system was the primary diagnosis during 83,228,000 office visits in 2009 (8.0% of total visits). A stroke can also cause many of the same physical deficiencies seen in musculoskeletal conditions . Approximately 2.6% of all americans over the age of 20 have had a stroke, and each year 795,000 more people suffer a stroke (1 every 40 seconds). The most common non - medication treatment for musculoskeletal conditions and stroke is rehabilitation . For patients, rehabilitation is a tedious chore that involves many months of treatment, resulting in low compliance . Rehabilitation regimens combine home - based exercises with therapist - monitored sessions; during the latter, the therapist assesses the patient's capabilities and adjusts the exercise tasks accordingly . Previous research on upper body rehabilitation has shown that positive functional outcomes are achieved from programs that emphasize task - oriented, repetitive training exercises combined with biofeedback, . Thus, virtual reality (vr) rehabilitation systems have been developed that can repetitively simulate these task - oriented training exercises, . Vr rehabilitation has been shown to be successful for improving upper body function in stroke patients, most likely because the interesting and engaging virtual tasks encourage increased repetition . Some vr rehabilitation programs have also been designed to be used without the supervision of a physiotherapist . However a previous study demonstrated that subjects trained with an adaptive vr rehabilitation system had improved upper body functionality when compared to subjects trained with conventional rehabilitation . Robotic rehabilitation systems, designed to address the limitations of traditional physiotherapy, have been developed primarily for upper extremity rehabilitation of stroke patients . These robots generally consist of an arm attachment with multiple actuated degrees of freedom that is affixed to the patient and guides them through different exercise tasks through the use of an immersive visual environment . Studies have shown that patients achieved significant motor recovery and improved upper extremity functionality after participating in robotic rehabilitation . Some shortcomings of the current rehabilitation robots are their limited availability and portability, and their high cost ($50,000 usd); as a result, few patients can benefit from robotic rehabilitation . Pneumatic actuators have previously been used in upper body robotic rehabilitation systems because they are lightweight, strong, and mechanically compliant to human anatomy . In terms of control strategies, impedance control and admittance control have been used to ensure the safety of the subjects while they interact with the robot . In these control laws, the controller uses both the position of the robot's joints and the force produced by the actuators in order to limit the amount of force applied to the subject . In terms of efficacy, previous research has suggested that pneumatically actuated rehabilitation robots can be used by patients with movement disabilities or stroke to improve their motor function . The primary drawbacks of pneumatic actuators are that they are difficult to control due to non - linear behavior during actuation, and that they require an external supply of compressed air . The overall objective of this research was to create an adaptive home - use robotic rehabilitation system for the upper body . The specific goals for the system were to collect data from the subject in real - time using simple and portable sensors, to create a customized exercise task for the subject by adapting a standard exercise task, and to build a simple and non - rigid wearable robot that will provide subject - specific resistance training . This paper presents results from comparison testing between the customized exercise tasks and the standard exercise tasks, as well as preliminary testing of a pneumatically actuated prototype for the wearable robot . A microsoft \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sensor and an electromyograph (emg) system were combined with custom software written in c++ in order to collect data from the subject in real time . Data was obtained from the microsoft \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} using official microsoft software (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sdk version 1.6, microsoft, redmond, wa, usa). The 3d joint position data (mediolateral, anteroposterior, and vertical axes) for 20 joints per subject was acquired at the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} native sampling frequency of 30 hz . The data was subsequently smoothed using the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} smoothing algorithm and adjusted based on the height and angle of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sensor . Simultaneously, a wireless surface emg system (cometa, milan, italy) was used to collect electrical signals at 1000 hz from two electrodes, one on the subject's biceps muscle and one on the lateral head of the subject's triceps muscle . The emg signals' linear envelopes were processed in real - time using a previously described method \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm bandpass~filter}=10~{\rm hz}\hbox{--}500~{\rm hz};~{\rm lowpass~filter}=30~{\rm hz})$\end{document}. The emg data were normalized to the subject's maximum voluntary contraction (% mvc). A 3d visual environment (ve) previously created using custom software in c++ and opengl was modified to display the data from the sensors as well as the exercise task (see section iii). 1, part a) where the joint center data collected from the subject by the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} was displayed as a skeleton figure . The subject navigated the ve by using leg gestures to indicate directional arrows that rotated and translated the environment . Separate from the ve, an additional window displayed the color video data from the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} as well as the real - time emg data (fig . 1, part b). This window was used to ensure that the subject remained positioned in front of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sensor and that the emg sensors were functioning properly . 1.visual environment: a) virtual room, b) video and emg data . Visual environment: a) virtual room, b) video and emg data . 1) and the subject used his right hand to follow the sphere as closely as possible as it moved around the ve in a repeating cyclical 2d pattern . The 2d pattern was projected in the 3d visual environment as perpendicular to the subject . From the subject's view, the pattern was constrained to an orthogonal plane directly in front of his viewpoint . The distance to this plane was held constant . As the subject turned and moved within the environment, the pattern turned at the same rate and moved with the subject . In this way, the 2d pattern always appeared in front and perpendicular to the subject's viewpoint . Biofeedback was used to aid the subject's accuracy; the sphere changed color and the volume of music playing in the background increased as the subject's hand approached the center of the sphere . For data, the 3d position of the sphere, the 3d position of the subject's hand, and the subject's biceps and triceps emg signals were recorded simultaneously during the exercise tasks . The protocol for creating the customized exercise tasks, described previously by barzilay and wolf, consisted of generating an inverse model of the subject, which was modeled by a neural network . The subject first completed 10 full cycles of a visual follow training exercise task, or approximately 3 minutes of training . The subject's performance during this training exercise (in terms of the 3d position of the hand, the 3d velocity of the hand, and the biceps and triceps emg signals) for all 10 cycles was averaged together and smoothed to create one full cycle of the subject's performance for each of the 8 measured variables . The averaged 3d position and 3d velocity (6 variables) of the sphere during the training exercise task were used as targets to train the neural network . After the neural network was trained, it was used to simulate the subject's individual performance during a standard exercise task . Averaged emg data from 10 healthy subjects performing the standard exercise task were used as the additional inputs to the neural network . The wearable robot prototype consisted of the test platform, the pneumatics assembly (and associated electronic components), and the control architecture . A test platform was built to model the human arm and elbow joint; in this way, the basic prototype and the control algorithms for the wearable robot could be developed and validated in a safe environment without a risk of injury . The platform was designed to replicate the mass properties \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm total~mass}=1.6~{\rm kg})$\end{document}, inertial properties \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm i}=0.1~{\rm kg}{^{\ast}}{\rm m}^{2})$\end{document}, and dimensions of the human arm . The test platform consisted of two \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $7\times 7\times 30~{\rm cm}$\end{document} aluminum cages (simulating the upper arm and forearm segments) connected together by two ball bearing assemblies to simulate an elbow joint (fig . The upper arm cage was bolted to a wooden base while the forearm cage was allowed to swing freely up to 45\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} in either direction about the elbow joint . In future iterations, the primary requirements for the assembly were that the components be both non - rigid and highly compliant as the robot will eventually be worn on the subject's arm . As such, mckibben - type pneumatic artificial muscles (pams) were chosen as the actuation components; as an additional benefit, these actuators closely replicate the behavior of natural muscles . Two festo dmsp-10 - 120 pams (festo, denkendorf, germany) were attached to the upper arm cage of the test platform to replicate the biceps and triceps muscles (fig . The muscles were rigidly anchored to the base of the upper arm cage and then connected to the forearm cage at the elbow joint with non - elastic metal wire (fig ., actuation of the upper arm cage was accomplished by transforming the force from the antagonistic pams into torque about the joint, as in the human arm . Each pam was independently controlled by using two 2/2 normally closed festo mhj-10 pneumatic valves (festo, denkendorf, germany), one for inlet and one for outlet . Motorola mpx5700 pressure sensors (motorola solutions, schaumburg, il, usa) measured pressure in the pams and output an analog signal to the microcontroller . Finally, an austriamicrosystems as5145 magnetic encoder (ams ag, unterpremstatten, austria) was used to determine the position of the elbow joint, and an arduino uno board (sparkfun, boulder, ca, usa) was used as the microcontroller for the entire prototype . In order to identify the robot's passive dynamic properties, an impulse torque disturbance was applied to the joint and the angular position was recorded with respect to time . The system was modeled as a standard 2nd order linear system, with the following equation for the angular position:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\theta\left(s\right)=\thinspace{{m_{in}(s)}\over{is^{2}+2crs+2kr}}\quad=\quad{{^{\textstyle m_{in}(s)}\!\bigl/\!_{\textstyle i}\over{s^{2}+\left(^{\textstyle 2cr}\!\bigl/\!_{\textstyle i}\right)s+{^{\textstyle 2kr}\!\bigl/\!_{\textstyle i}}}}}\eqno{\hbox{(1)}}$$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $c$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $k$\end{document} are the damping and spring coefficients of the moveable parts of the robot prototype and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $r$\end{document} is the distance from the center of the joint to the attachments of the non - elastic metal wire (fig . 2). The response in time to an impulse in the form of\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$m_{in}\left(t\right)=t\delta (t)\eqno{\hbox{(2)}}$$\end{document} therefore leads to the following equation (in the time domain via the inverse laplace transform):\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\theta\left(t\right)=l^{-1}\left\{{{^{\textstyle t}\!\bigl/\!_{\textstyle i}}\over{s^{2}+\left(^{\textstyle 2cr}\!\bigl/\!_{\textstyle i}\right)s+{^{\textstyle 2kr}\!\bigl/\!_{\textstyle i}}}}\right\}\!.\eqno{\hbox{(3)}}$$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{d}$\end{document}, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{n}$\end{document}, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\zeta$\end{document} were defined as follows:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\eqalignno{\omega_{n}^{2}=&\,{^{\textstyle \rm 2kr}\!\bigl/\!_{\textstyle \rm i}},\quad{2\zeta\omega}_{n}^{2}={^{\textstyle \rm 2cr}\!\bigl/\! {\textstyle \rm i}},\;\;{\rm and}\cr\omega_{d}=&\,\omega_{n}\sqrt{1-\zeta^{2}}.&{\hbox{(4)}}}$$\end{document} as such, the resultant response was \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\theta\left(t\right)=\thinspace{{t}\over{i\omega_{d}}}{\rm sin}(\omega_{d}t)e^{-\omega_{n}\zeta t}\!.\eqno{\hbox{(5)}}$$\end{document} the damping coefficient \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\zeta$\end{document} was assumed to be relatively small \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $(\zeta\leq 0.1)$\end{document} and therefore the difference between \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{d}$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{n}$\end{document} was neglected . The final approximated function was \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\theta\left(t\right)=\thinspace a\cdot{\rm sin}(\omega_{n}t)e^{-\omega_{n}\zeta t}.\eqno{\hbox{(6)}}$$\end{document} the control architecture for the robot was based on pressure control of the pams . The pams were controlled through pulse - width modulation (pwm) of the pressure valves . A simplified dynamic equation for the robot was used for analysis due to the complexity of modeling the highly non - linear behavior of the pams . The dynamic equation used for the control architecture was \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$i{\mathddot{\theta}}=\tau_{dist}+\thinspace\tau_{cont},\eqno{\hbox{(7)}}$$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\tau_{dist}$\end{document} is the disturbance torque due to dampening, friction, gravity, spring constant, and so on, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\tau_{cont}$\end{document} is the control torque applied by the pams and controlled using the sensors, microprocessor, and valves . The control torque applied by the pams was modeled as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$\tau_{control}=\left(f_{left}-f_{right}\right)\cdot r\eqno{\hbox{(8)}}$$\end{document} and a simple proportional loop was implemented (fig . The pwm parameters that determined the duty cycle for the valves were chosen so as not to exceed the limits of the valves, which had a minimum switching time of 1 msec (1000 hz). A 16 msec (60 hz) pwm period (divided into 10% duty cycles) and a 600 hz switching frequency were selected, which resulted in a minimum switching time of 1.6 msec for each 10% duty cycle . In terms of the control rule for the pams, when the pressure error was positive \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm reference}>{\rm measured})$\end{document}, the inlet valve duty cycle was determined as:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $$duty\thinspace cycle = k_{pressure}\cdot\delta pressure\eqno{\hbox{(9)}}$$\end{document} and the outlet duty cycle remained closed (0%). Two different experiments were performed for the assessment of the subject data collection and visual environment . The first experiment focused on the accuracy of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sensor, while the second experiment focused on a comparison of the customized exercise task . 10 young, healthy subjects \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm age}\! :27.1~{\rm years}\pm{2.9},~{\rm height}\! :128.6~{\rm cm}\pm{7.9},~{\rm male}={6})$\end{document} with no history of upper body impairment volunteered to participate in this study . The subjects wore minimal clothing on their upper bodies to allow for placement of 25 reflective markers on their torsos and arms . Each subject completed three trials of two different upper body exercises: 1) a range - of - motion (rom) exercise where the subject slowly raised both of his arms, elbow joints extended, from the sides of his torso to 45\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} above the transverse plane and back down, three repetitions per trial, and 2) a visual follow task, where the subject used his right hand to follow a moving sphere flying in a 3d figure 8 pattern in the ve for a total of 45 seconds . Data was collected during these exercise tasks using both the robotic rehabilitation system and a 10 camera opto - electronic vicon mx motion capture system (vicon, oxford, uk) as a gold - standard for validation . For the robotic rehabilitation system, 3d joint center data was recorded for the wrist, elbow, and shoulder joints of the right arm using the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sdk version 1.5 and custom c++ software . Additionally, a synchronization signal was sent from the robotic rehabilitation system to indicate to the vicon system when data were being collected . For the vicon system, the reflective markers were placed on the subjects according to the vicon upper body model marker set . Vicon nexus software (version 1.8.2) was used to record the position of the markers during the exercise tasks as well as the synchronization signal . The data was post - processing in vicon nexus to obtain the joint center locations of the right wrist, elbow, and gleno - humeral (shoulder) joint . Analysis of the data was completed in matlab r2012a (mathworks, natick, ma, usa). A homogenous transformation was used to align the coordinate systems of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} and vicon measurement systems . To find the best parameters for the homogenous transformation, a minimum cost maximal matching optimization was used (cost function: average distance between a small, randomized set of paired points from each system). The data was then resampled to a common frame rate of 120 hz, and the synchronized subset of the data extracted . The pearson's correlation (r) coefficient and the root - mean - square error (rmse) were calculated between the resampled and synchronized vicon and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} data for each trial . Twelve young, healthy subjects \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm age}\! :29.8~{\rm years}\pm{4.2},~{\rm height}\! :169.0~{\rm cm}\pm 9.3,~{\rm male}={5})$\end{document} with no history of upper body impairment volunteered to participate in this study . The subjects wore close - fitting short sleeve shirts to allow for the placement of the two emg electrodes on their biceps and triceps . Each subject was first asked to complete 10 full cycles of one of two different training exercise tasks, either a vertical double figure 8 pattern (fig . 4, part a) or a vertical half figure 8 pattern (fig . 4, part b). After the neural network was trained, a related exercise task, consisting of either a full horizontal figure 8 pattern (fig . 4, part a) or half of a horizontal double figure 8 pattern (fig . 4, part b), was given to the neural network as input data (combined with the healthy emg data for that particular task), and a customized exercise task was output from the neural network . The subject then completed 10 full cycles of this customized output exercise task (custom), as well as 10 full cycles of the unmodified original task used as input to the neural network (standard) (fig . 4). A full exercise set consisted of the three linked exercise tasks (training, custom, standard), and each subject completed 4 full sets (two of each type of set), for a total of 12 exercise tasks per subject . Fig . Analysis of the data was completed in matlab r2012a (mathworks, natick, ma, usa). For the custom and standard exercise tasks, the root - mean - square error (rmse) was calculated between the subject's hand and the sphere for both position and velocity . For the emg signals, the peak value and the area under the linear envelope were calculated . Finally, the data from the subjects were merged, and paired \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $t$\end{document}-tests (two - tailed, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\alpha=0.05 $\end{document}) were conducted between the standard exercise data and the corresponding custom exercise data in the set for the aforementioned variables . For the system identification, the final approximated function (6) was tested experimentally by applying an external impulse torque disturbance to the robot and then recording the measurements from the encoder in the elbow joint; this test measured the passive properties of the robot because the valves were sealed after the initial impulse . The experiment was conducted five separate times, and the approximations for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\zeta$\end{document}, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{n}$\end{document}, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $a$\end{document} were determined using the nlinfit function in matlab . To test the pressure control algorithm, continuous sine wave pressure reference input signals were sent to the robot prototype at 60 hz . Sine wave frequencies of 0.25 hz, 0.5 hz, 0.75 hz, and 1 hz were tested (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm gain}=0.10 $\end{document} for all tests). During the tests, after the trials, the rmse and pearson's correlation coefficient (r) were calculated between the reference pressure and the measured pressure . As a comparison to the passive system identification testing, an external impulse torque disturbance was applied to the robot when the control algorithm was active, and the measurements from the encoder in the elbow joint were subsequently recorded . This test measured the closed - loop properties of the robot because control algorithm was active and the valves were open after the initial impulse . The experiment was conducted 15 separate times, and the approximations for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\zeta$\end{document}, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{n}$\end{document}, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $a$\end{document} were again determined using the nlinfit function in matlab ., a subject moved his right arm from full extension to full flexion at different speeds . The angle of the subject's right elbow was continuously updated in real - time based on the 3d position of the subject's shoulder, elbow, and wrist joints . This angle was then converted into a voltage signal (between 0 and 5 volts) that corresponded to the relative angle of the elbow (between a minimum of 35\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} when the hand touched the shoulder, to a maximum of 175\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} when the elbow was fully extended). Any angle measured beyond these limits was considered either 0 or 5 volts, depending on which limit was exceeded . A national instruments usb-6218 (bnc) data acquisition device (national instruments corporation, austin, tx, usa) was connected to the rehabilitation system, and the elbow angle voltage was sent as an output analog signal from this device . This signal was then connected to the analog input of the arduino uno to provide a real - time position reference signal for the robot prototype based on the subject's elbow angle . This position signal was then converted into two corresponding pressure reference input signals for the pams, where 0 volts corresponded to the minimum \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({-}45^{\circ})$\end{document} and 5 volts corresponded to the maximum \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({+} {45}^{\circ})$\end{document} allowable angle of the robot from the neutral position (0\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document}). In this way, the subject could actuate the robot by changing the angle of his elbow . Five 30-second duration tests were conducted . During the tests, the real - time angle of the subject's elbow (measured by the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document}) and the real - time angle of the robotic elbow joint (measured by the encoder) were simultaneously recorded . After the tests, the angle data were resampled and independently normalized on a scale of 0 to 1 . Finally, the rmse and pearson's correlation coefficient (r) were calculated between the normalized angle of the subject's elbow and the normalized angle of the robotic elbow joint . The results from the validation testing between the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} data and the vicon data are presented for all trials of the 10 subjects for the right arm joints (wrist, elbow, and shoulder) by exercise task in table i. for the rom task, the largest rmse was observed for the shoulder joint . For the visual follow task, the largest rmse was observed for the elbow joint, then the wrist joint, and last the shoulder joint . Furthermore, the rmse for the elbow and shoulder joints was greater during the visual follow task when compared to the rom task, while the rmse for the shoulder joint was greater during the rom task . Table ivicon - kinect comparisons by joint and task vicon - kinect comparisons by joint and task a comparison of the standard and custom exercise tasks by exercise set is presented in table ii . The subjects had greater rmse for the position and the velocity variables during the custom tasks (as compared to the standard tasks) for both sets . Table iikinematic and emg variables by set kinematic and emg variables by set the results from the external impulse response tests of the robot prototype are presented in table iii for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\zeta$\end{document}, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{n}$\end{document}, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $a$\end{document}, derived from the final approximated function (6). The joint angle measured by the encoder was then compared to the predicted joint angle, which was calculated by using function (6) and the experimentally - determined function parameters . Table iiiimpulse response the average values for the five passive properties tests were \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\zeta=0.064 $\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{n}=15.01~{\rm rad}/{\rm sec}$\end{document}. Compared to the predicted joint angle, the average rmse was 0.23\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} with a high correlation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm r}=0.99)$\end{document}. The average values for the fifteen active properties tests were \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\zeta=0.13 $\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\omega_{n}=14.84~{\rm rad}/{\rm sec}$\end{document}. Compared to the predicted joint angle, the average rmse was 0.90\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} with a high correlation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm r}=0.97)$\end{document}. The results from the pressure control algorithm tests are presented in table iv, figs . 5 and 6 . 5.reference vs. measured pressure in the pams by frequency for sine wave input signals . Fig . Table ivref - meas comparisons by frequency and pam reference vs. measured pressure in the pams by frequency for sine wave input signals . Ref - meas comparisons by frequency and pam the continuous joint angles during the pressure control algorithm tests are shown in fig . For the 0.25 hz and 0.5 hz continuous sine wave pressure reference input signals, the measured pressure accurately followed the reference pressure for both pams \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $({\rm r}=0.99,~{\rm average~rmse}=0.093~{\rm bar})$\end{document}. However, for the 0.75 hz and 1 hz input signals, the measured pressure did not follow the reference pressure during pressurization when the reference pressure was greater than 2.5 bars, indicating saturation of the pams . The range for the joint angles was 38.8\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} at 0.25 hz, 39.0\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} at 0.5 hz, 51.9\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} at 0.75 hz, and 53.6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} at 1 hz . The wearable robot prototype was able to accurately mimic the angle of the subject's elbow in real - time . The rmse between the normalized position of the subject's elbow and the normalized position of the robot elbow ranged from 0.073 to 0.103 (average 0.088), and the correlation ranged from 0.94 to 0.97 (average 0.96, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm p}<0.001 $\end{document}). The overall objective of this research was to create an adaptive home - use robotic rehabilitation system for the upper body . This paper presented results from comparison testing between different exercise tasks and from preliminary testing of a pneumatically actuated prototype for the wearable robot, all of which demonstrate that this robotic rehabilitation system achieved the stated objective . The first specific goal for this research was to collect data from a subject in real - time using simple and portable sensors . During the subject testing, the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sensor measured the 3d position and 3d velocity of the subject's hand at 30 hz . Experimental testing was conducted to determine the accuracy of the joint center measurements from the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} as compared to the vicon gold - standard measurements during two upper - body exercise tasks (table i). The results presented in table i show that the largest rmse for any joint during either task was 35.8 mm, while the smallest was 25.5 mm . These results are similar to a previous study comparing the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm\thinspace}$\end{document} measurements to vicon measurements for upper body exercise tasks . Clark et al . Showed that between the two systems, there was approximately 42.9 mm difference during a lateral reaching task and 13.1 mm difference during a forward reaching task for hand measurements . These results suggest that the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} measurements are sufficiently accurate for clinical significance, and therefore the robotic rehabilitation system is sufficiently accurate for measurements of rehabilitation exercises . Since traditional physiotherapy regimens for the upper body are managed by physiotherapists, easily identified visual landmarks are used to quantify the success of the exercises . One study, focused on rehabilitation after a thrower's elbow injury, used visual quantification landmarks such as extend elbow to full extension, raise arm to shoulder level and stand with shoulder abducted 90\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}$\end{document} to describe the exercises . The current standard is to evaluate the exercises based on visual observation (which can result in substantial errors when compared to a quantitative system like vicon). As such, rmse of 35 mm to 40 mm in joint center measurements can be considered insignificant for clinical purposes because visual observation error in a clinical setting has been shown to be approximately 10% and this rmse is less than 10% of the movement range . Therefore, the joint center measurements from the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sensor are accurate enough to quantify rehabilitation exercises of the upper body, and are an improvement to the current standard of visual observation by a physiotherapist . The second specific goal of the system was to create a customized exercise task for each subject . A neural network was created using the subject's training exercise data and was then used to modify a standard exercise task into a custom exercise task . For both sets of exercises, the subjects had greater rmse for the position and the velocity variables during the custom tasks; in other words, during the custom task, their kinematic performance was worse in terms of accuracy to the instructed task . These results show that the subjects were less able to replicate the custom task, suggesting that the custom task was more difficult to complete . This may be a result of the unconstrained nature of the neural network used to create the custom task . No output constraints were placed on the neural network, and the researchers did not manually alter the output . As a result, consequently, the subjects were continually reacting to the unpredictable trajectory of the moving sphere during the custom task but were able to anticipate the trajectory of the standard task, thereby making the standard task easier to complete . Adding constraints to the neural network previous successful adaptive rehabilitation systems, used trained physiotherapists to adapt the rehabilitation tasks to the subjects based on the quantitative data . Therefore, the therapist acted as a constraint on the output of the systems, and could modify the output exercise task to achieve the desired goal . Future iterations of the system will include constraints on the neural network and/or a trained physiotherapist who can modify the output task in order to ensure that the custom task is an anticipatory task, not a reactionary task . Both the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sensor and the emg system are easily transportable and simple to use after minimal training . Thus, it is feasible to use the entire rehabilitation system in a home setting, as the only additional requirements are a computer and a display device like a television . The third specific goal for the system was to build a simple and non - rigid wearable robot that will provide subject - specific resistance training . The results for the wearable robot prototype demonstrate the feasibility of using pams for actuation . Table iii shows that the exoskeleton can be modeled as a second order linear system described by (6). The measured damping coefficient \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $\zeta=0.064 $\end{document} was low, suggesting that it might be difficult to control the robot . However, it is likely that a human elbow will have a higher damping coefficient than the test platform and therefore will be more controllable . Furthermore, the measured damping coefficient was higher during the closed - loop comparison testing of the robot \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $(\zeta=0.13)$\end{document}, showing that the simple pressure control algorithm increased the damping of the robot . Overall, these results indicate that a robust controller can be built that will control the robot . The first controller developed for the robot was based on pressure control of the pams . The results from the pressure control algorithm tests indicate that there was saturation of the pams at 0.75 hz and 1 hz during pressurization; the valves did not pressurize the pams fast enough to follow the reference signal . This saturation most likely occurred because the reference pressure input signals were both high amplitude and high frequency, and as such required a high change rate (7 bar / second) of the pressure inside the pams (corresponding to an angle change of 100\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} $^{\circ}/{\rm second}$\end{document}). As the pressure rose inside the valves, the pressure differential between the valves and the pressure source decreased, thereby decreasing the flow rate to the valves . Eventually, the decreased flow rate was insufficient to achieve the high change rate required by the reference pressure input signals, and saturation occurred . This trend was not observed during depressurization because it would only occur near 0 bar, and the pressure reference input signals did not reach this level . While the motion bandwidths of normal human arm movements are centered at approximately 2 hz, most of these movements are relatively low amplitude and would not require high pressure change rates of the pams . Nevertheless, future versions of the wearable robot will use a higher pressure source to increase the pressure differential (and therefore the flow rate) of the pams so that the robot can follow reference pressure input signals of at least 5 hz . Additionally, force sensors will be added to the robot so that advanced control algorithms can be implemented, such as impedance or admittance control, in order to more accurately control the pneumatically actuated robot . The control performance of the wearable robot did not account for additional loading from the subject . In a wearable robot, the subject would also be exerting force on the robot (both assistive and resistive force). This force would need to be measured and compensated by the controller in order to accurately control the position of the robot . Future prototypes of the robot arm should include sensors to measure the force exerted on the robot by the subject . The results from the tests of the combined system indicate that the pressure loop can successfully follow low amplitude pressure reference input signals at frequencies greater than 1 hz . During these tests, saturation was not observed when the subject actuated the robot with a low amplitude reference input signal greater than 1 hz . Varying the rate of change of the elbow angle also did not affect the accuracy of the robot, as it was able to follow the subject's slow and fast movements . Due to a slightly noisy input signal (caused by small inaccuracies in the measured elbow angle from the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document}) and insufficient damping in the control algorithm, the robot sometimes oscillated about the desired angle . Furthermore, this test demonstrates the feasibility of the entire robotic rehabilitation system because the subject was able to actuate the wearable robot using only the movement of his elbow . Future iterations of the entire system will implement real - time smoothing of the elbow angle reference signal in order to compensate for the small inaccuracies in the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} measurements, and the damping terms in the control algorithm will be increased . This study described a promising home - use robotic rehabilitation system comprised of low - cost components . Further research is necessary to create a commercial version of this system, specifically on the prototype of the wearable robot . However, the data collection component of the system could be used in the near future for large scale research studies . The immediate next steps are to create a gui for the system to increase usability and to optimize the model generation code for speed and accuracy . Afterwards, this part of the rehabilitation system could be used by a variety of end - users for large scale research studies on rehabilitation exercises . Given the low cost and easy portability of the microsoft \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document}} {} ${\rm kinect}^{\rm tm}$\end{document} sensor, these studies should include home - based exercise components . Concurrently, the next prototype of the wearable robot should be developed with the goal of having a fully functioning wearable prototype within two years . A limitation of this study is that the current system does not provide force feedback to the subjects . The wearable robot was designed to provide force feedback during the exercise tasks, but the current prototype was not tested on human subjects . Future iterations of the robot prototype should be designed for wearability and human safety so that it can be worn by the subjects in order to provide force feedback . The addition of force feedback to the system should significantly enhance the results as the subject will have additional biofeedback information to help them complete the rehabilitation exercises.
International diabetic federation estimated that in india there are 66.8 million patients in 2013, with nearly 50% undiagnosed . Diabetes was related to one million deaths and mortality was higher under age of 60 years . By 2030 there will more than 79 million diabetics, making it one of the major public health challenge to the country . Observed retinopathy in 28.9%, nephropathy in 32.5%, neuropathy in 30.1%, cardio - atherosclerotic diseases in 19.2% and peripheral vascular diseases in 18.1% diabetic patients . The investigators of a1 cheive study observed high prevalence of macrovascular and microvascular complications among the indian diabetic patients . Different studies conducted in countries like united states have observed the positive association of patient's knowledge regarding the disease and self care to the treatment compliance . Hence, this study was planned to assess knowledge about diabetes and its correlation with pharmacological and non - pharmacological compliance, among the diabetics attending a rural health center from sangli district, maharashtra (india). This was a cross - sectional study conducted in a rural health center attached to a medical college from sangli district of maharashtra, india . The study population was adult type-2 diabetes patients on oral hypoglycemic drugs for at least 6 months attending the rural health centre . The patients attending for routine check up or drug re - prescription and consenting to participate were included in the study . Minors, patients on insulin, hospitalized patients, patients who had undertook any sensitization program other than routine counseling by physician and those who cannot read in the local language were excluded from the study . Prevalence of knowledge regarding diabetes in the pilot study was 52.71%; considering level of significance as 5% and error 15%; study instrument was a pretested, prevalidated, self - administered questionnaire with good test - retest reliability (spearman correlation coefficient, r = 0.81) and internal consistency (cronbach's, r = 0.78). It was developed in marathi language with the help of subject experts and published literature and finalized after the pilot study . It consists of initial section with general information of patient like age, gender etc ., the occupations reported by patients were classified as sedentary work, moderate work and heavy work . The second section consisted of questions regarding knowledge of patients about diabetes and its complications and the answers were scored . Those scoring under 50%, 5075% and more than 75% were considered to be having poor, moderate and good knowledge, respectively . Pharmacological compliance was self - reported, with participants reporting to have missed more than two doses in last 15 days were considered as non - compliant . Due to lack of exact definition of compliance to the non - pharmacological management, a scale was developed with equal importance to diet modifications and physical exercises . The maximum possible score was 12 and the participants scoring above 9 (75%) were considered as compliant to non - pharmacological management . Mean, standard deviation, percentage, chi - square and binary logistic regression were applied . Data from the pilot study and incomplete questionnaires were not included in the final analysis . Out of 329 questionnaires collected, 307 were complete and hence used in the final analysis . Two hundred twenty - three (72.6%) participants were male while 84 (27.4%) were female [table 1]. The mean age of study participants was 55.6 years (range 3585 years and standard deviation 12.22 years). Two hundred ninety - two (95.1%) participants were married . While considering the educational status, 106 (34.5%) had studied up to secondary school and 201 (65.5%) had attended college . Among the participants: 208 (67.8%), 56 (18.2%) and 43 (14%) were sedentary, moderate and heavy workers, respectively . The mean morbidity with diabetes was 10.7 years (range: 1 to 44 years and; standard deviation: 10.02 years). Age group and gender distribution of the participants the pharmacological compliance was reported by 234 (76.2%) participants . The mean score for the non - pharmacological compliance was 8.35 (standard deviation: 2.8). Based on preset criteria of score, 156 (50.8%) participants were considered to compliant to the non - pharmacological management . The mean score for knowledge regarding diabetes was 14.82 (standard deviation: 3.5). Only 29 (9.4%) participants had good knowledge, whereas 219 (71.3%) had moderate and 59 (19.2%) participants had poor knowledge . Age was not associated with the knowledge (chi - square 5.47, p = 0.49), however, higher percentage of older age group (75 yrs) participants had poor knowledge . Gender was associated with knowledge (chi - square 10.78, p = 0.005); higher percentage of male participants (81.6%) had moderate to good knowledge as compared to females (78.6%). Marital status and occupation education was not associated with the knowledge (chi - square 2.23, p = 0.33), however higher percentage of participants who had attended college had moderate to good knowledge . Pharmacological compliance was associated with knowledge . Among the participants with good compliance, 88.5% had moderate to good knowledge as compared to only 56.2% participants with poor compliance . The moderate to good knowledge was present in 87.8% participants with good compliance as compared to only 73.5% with poor compliance [table 2]. Association between knowledge regarding diabetes with pharmacological compliance and non - pharmacological management compliance binary logistic regression was applied with knowledge regarding diabetes as dependent variable, while age, gender, education, occupation, pharmacological compliance and non - pharmacological management compliance as independent variables . The chance accuracy rate for the model is 83.6%, which was greater than calculated chance accuracy rate (67.3%). Pharmacological and non - pharmacological management compliance were the highly significant predictors, while education and age were also significant predictors for knowledge regarding diabetes [table 3]. Binary logistic regression model - association of age, gender, education occupation, pharmacological compliance and non - pharmacological management compliance with knowledge regarding to diabetes in the current study, we observed that the patients compliant with pharmacological and non - pharmacological management had good knowledge regarding diabetes . Al - qazaz hkh et al ., (2010), observed significant correlations between the knowledge about diabetes and adherence to the treatment in subjects from penang . When carried out a study at al - makhfia governmental diabetes primary healthcare clinic in nablus, palestine in 2012, observed that diabetic patients with high knowledge were less likely to be non - adherent . Chaudhary et al ., from multan (pakistan) in 2010, observed that male gender, higher education and older age (> 40 years) were associated with the knowledge regarding diabetes mellitus . However, age groups used by them were only two: <40 years and> 40 years, while in the current study four age groups were considered with lowest being 3550 years . Abdella and mohammad studied awareness of diabetic patients about their illness and associated complications in ethiopia (2012); majority of patients (67%) had good knowledge on actions to be taken on the occurrence of acute complications and reasons for developing acute complications . While we observed that less than 10% participants had good knowledge . However, there is difference in the questionnaire used in both the studies and most of the participants from abdella study had undergone sensitization unlike the current study subjects . (bangladesh) 2013, observed that 19% respondents had poor knowledge, while 68% had average and 13% had good knowledge . They also deduced that male gender and higher educated people are likely to have better knowledge regarding diabetes . Conducted a study on adults in tamaka village, kolar (india) in 2009 . Observed that male gender, higher education and younger age of respondents was significantly associated with level of knowledge, while type of occupation is not associated with it . Bansal et al . Observed compliance to medication in 82.5% patients from rural area of ludhiana district, india . Rao et al . Observed that 83.6% patients from southern karnataka were on regular medication . Santhanakrishnan et al . Observed compliance to oral hypoglycemic agents in 76% subjects, dietary modifications in 81.4% and physical activity in 37% subjects from puducherry, india . While in the current study, pharmacological compliance was reported by 76.2% participants and compliance to the non - pharmacological management was reported by 50.8% participants . Being a self - administered questionnaire reporting bias cannot be totally eliminated; there may be over - estimation of compliance . Confounders like economical state, availability of medications, counseling by health professionals, exposure to information through media, additional use of ayurvedic / homeopathic / other indigenous systems of medicines etc ., may be present and are not considered in the current study . Only self - reported compliance was considered without confirming . . However, many of the patients have poor knowledge regarding the disease, the situation is worse among the females . Lower level of literacy and old age hinder the presence of knowledge regarding the diabetes . Seminars, counseling sessions and workshop should be arranged periodically for diabetic patients to increase their awareness regarding diabetes disease in total per say.
Lactic acid was first found and described in sour milk by the swedish chemist karl wilhelm scheele (17421786) in 1780 . The swedish chemist jns jakob berzelius (17791848) found lactic acid in fluid extracted from meat in 1808 [2, 3], and the german chemist justus von liebig (18031873), who established the world's first school of chemistry at giessen, proved that lactic acid was always present in muscular tissue of dead organisms . In 1859, emil heinrich du bois - reymond (18181896) published several articles on the influence of lactic acid on muscle contraction [59]. Araki and zillessen found that if they interrupted oxygen supply to muscles in mammals and birds, lactic acid was formed and increased [1014]. This was the first demonstration of the relationship between tissue hypoxia and the formation of lactate . The occurrence of increased lactic acid in blood (hyperlactataemia) nowadays reflects severe illness, in which the increased blood lactate levels may result from both anaerobic and aerobic production or from a decreased clearance . It was the german physician chemist johann joseph scherer who first demonstrated the occurrence of lactic acid in human blood under pathological conditions after death in 1843 and 1851 [15, 17], and carl folwarczny in 1858 who first demonstrated lactic acid in blood of a living patient . In this article we wish to honour scherer's forgotten observations and describe the influence of his finding on further research on lactic acid at the end of the 19th century . Born on 18 march 1814 in aschaffenburg, germany, scherer studied medicine, chemistry, geology and mineralogy at the university of wrzburg between 1833 and 1836 . He obtained his phd in medicine and surgery in 1838 with a thesis entitled versuche ber die wirkung einiger gifte auf verscheidene thierclassen he practised medicine in wipfeld, but inspired by the chemist ernst von bibra (18061878) he completed his studies in chemistry at the university of munich between 18381840 . In 1840 he was employed at the laboratory of justus liebig at giessen, and became professor at the medical faculty in 1842, professor of organic chemistry in 1847, and later professor of general, anorganic and pharmaceutical chemistry . His work especially concerned quantitative research on blood and urine in pathological conditions . In 1843 he published his book chemische und mikroskopische untersuchungen zur pathologie angestellt an den kliniken des julius - hospitales zu wrzburg (chemical and microscopic investigations of pathology carried out at the julius clinic at wrzburg) (fig . 1), in which he described 72 case reports, giving details on clinical course, diagnosis, and results obtained during autopsy and analysis of body fluids . In one chapter in his 1843 book entitled' untersuchungen von krankhaften stoffen bei der i m winter 18421843 in wrzburg und der umgegend herrschenden puerperal - fieber - epidemie' (investigations of pathological substances obtained during the epidemic of puerperal fever which occurred in the winter of 18421843 in and around wrzburg) scherer described the cases of seven young women who all died peripartum . One of the women, the 23-year - old primipara eva rumpel, gave birth to a healthy child on 9 january 1843 . The same night she developed a painfully swollen abdomen and became ill, feverish, and sweaty, with rapid pulse and severe thirst . The next evening she deteriorated, became delirious, with anxious breathing, a tense abdomen, cold extremities and rapid pulse, finally losing consciousness . A.m., 36 h after the onset of the first symptoms, she died . During autopsy, severe purulent endometritis, vaginal pus, pulmonary oedema, and shock liver and shock spleen were found . The blood that was obtained directly from the heart was chemically analysed, in which lactic acid was found . Most likely this unfortunate woman had died from a fulminant septic shock caused by group a haemolytic streptococci (streptococcus pyogenes). Another patient, the 28-year - old, 7 months pregnant (second pregnancy) margaretha glck, was, after being icteric, nauseous, vomiting and complaining about epigastric pain for 8 days, admitted to the lying - in birth clinic on 6 february 1843 . Four days later she was transferred to the hospital with severe nosebleeds and generalised exanthema or purpura . In the evening she suffered from severe gastric bleeding and epistaxis, showing rapid pulse, cold extremities and dizziness . The next morning, she was transferred back to the birth clinic, where she gave birth to a premature child (30 weeks) and suffered from a severe post - partum fluxus . She was again transferred to the hospital with the following symptoms: cold clammy skin, tachycardia, severe lochia and persistent exanthema or purpura, but without signs of an acute abdomen . During the night of february 11 autopsy revealed a small intracerebral haematoma, normal lungs without pulmonary oedema, ascites and an anaemic, foul smelling uterus filled with purulent and decayed tissue and pus . Blood was also obtained directly from the heart during autopsy and lactic acid was found . In this case we could think of a haemorrhagic shock and cerebral haemorrhage due to clotting disorders possibly resulting from either acute fatty liver of pregnancy / hellp syndrome, idiopathic thrombocytopenic purpura, thrombotic microangiopathy (ttp / hus) or dic . In the conclusions of his 1843 book, scherer attached high importance to the fact that he found lactic acid in cases of puerperal fever, which he had not found before in healthy persons . He held the opinion that lactic acid was formed in blood during bodily deterioration in severe diseases like puerperal fever . Lactic acid was thus described for the first time in human blood and was demonstrated for the first time as a symptom of septic and haemorrhagic shock . In the same period a junior obstetrician in vienna, ignaz philipp semmelweis (18181865), discovered in 1847 that physicians carried infectious particles on their hands from the mortuary to the obstetrical clinic, causing puerperal fever and puerperal sepsis, and he introduced a successful method for its prevention . Louis pasteur (18221894) found in 1879 that infection with streptococci was the most important cause of puerperal fever . Scherer worked closely with the famous pathologist rudolf virchow (18211902) on several projects (fig . 2). In 1851 virchow performed an autopsy on a patient who had died from leukaemia and offered scherer blood from this patient for analysis . The results of this analysis were published the same year in the verhandlungen der physikalisch - medicinischen gesellschaft in wrzburg . Virchow and scherer had previously studied the spleens of patients who died from leukaemia, and were curious if they could find the same results in the blood . Scherer reached the conclusion that: the blood of this patient contains: ameisensure, essigsure und milchsure, die gleichfalls von mir schon frher als in der milzflssigkeit vorkommend bezeichnet wurden (formic acid, acetic acid, and lactic acid, as also found by me previously in fluids from the spleen). 2johann joseph scherer (left) and rudolf virchow (right) in 1849 johann joseph scherer (left) and rudolf virchow (right) in 1849 scherer's observations inspired others to conduct further research, primarily in patients with leukaemia [1922], but also in patients with other conditions and diseases and in animal experiments with dogs and rabbits . While scherer found lactic acid in blood obtained after death during autopsy, mosler and krner mention an observation made by carl folwarczny, published in the allgemeinen wiener medicinischen zeitung in 1858, where blood was withdrawn from a leukaemia patient during life, analysed according to scherer's method, and found positive for lactic acid . In addition, carl folwarczny described in 1863 in his' handbuch der physiologischen chemie' that lactic acid can be found in the blood of patients with leukaemia, septicaemia (pyaemia) and in conditions leading to septicaemia like puerperal fever, the latter probably after scherer's observations . In an extensive article, the berliner physician georg salomon, who had serious doubts that the occurrence of lactic acid in blood was mostly related to leukaemia, proved in 1878 that lactic acid was also present in the blood of patients who were suffering and died from other diseases . He studied blood obtained during autopsy from cadavers, but also blood from patients obtained by bloodletting or cupping, and in some cases he compared the blood before and after death . He was able to demonstrate lactic acid in the blood of patients suffering from leukaemia, (pernicious) anaemia, congestive heart failure, chronic obstructive pulmonary disease, pleuritis, pericarditis, pneumonia and several solid malignant tumours . Gaglio is often erroneously mentioned as the first author to find lactic acid in blood [2729]. He was able to demonstrate lactic acid in fresh arterial blood withdrawn from dogs and rabbits after bloodletting . Both gaglio and berlinerblau, however, neglected previous research, as indignantly described by salomon in 1888 [ich erlaube mir, den inhalt meiner arbeiten, die von gaglio nur ganz flchtig, von berlinerblau gar nicht berhrt sind, in krze zu reproduciren (i take the liberty of summarizing the contents of my work, which was mentioned only briefly by gaglio and not at all by berlinerblau)]. The japanese chemist trasaburo araki showed that the amount of lactic acid in exhausted muscle results from muscle activation . Irisawa, inspired by the results obtained by salomon and gaglio, obtained fresh blood of 11 dying patients with serious conditions . In six cases he found hyperlactataemia, in four cases normal values . He speculated on the aetiology of hyperlactataemia, the most plausible cause being the severe hypoxia during the dying process . In an experiment in which he made a dog anaemic for several days, he found a rise in lactic acid levels during the time leading up to death . In cambridge (uk), walter morley fletcher (18731933) and frederick gowland hopkins (18611947) worked together on the metabolic changes occurring in muscular contractions and rigor mortis under anaerobic conditions, and found that lactate was the product of carbohydrate metabolism . Their classic 1907 paper demonstrated rigorously that muscle contraction is accompanied by the anaerobic formation of lactic acid, which is removed aerobically, at a rate depending on the level of exposure to oxygen . Poul astrup and john severingshaus mentioned scherer's 1851 article as first demonstration of lactic acid in blood, but overlooked the 1843 cases and folwarczny's work . In conclusion, scherer's 1843 case reports should be cited as the first description of lactic acid in human blood and also as the first demonstration of lactic acid as a pathological finding in septic and haemorrhagic shock . Folwarczny, in 1858, was the first to demonstrate lactic acid in blood in a living patient.
Panax ginseng (ginseng) has been used traditionally in eastern asia over thousands of years . It has been used orally to treat various diseases including hypertension, diabetes mellitus, liver and kidney dysfunction, mental disorders, and postmenopausal disorders . In addition, topical applications have also been used to heal wounds and reduce skin inflammation . In the past few decades, it has been proved that ginseng extracts actually show a wide range of effects against human diseases . Their potential therapeutic effects have been mainly attributed to its immunomodulatory, neuroprotective, antioxidative, antitumor, and hepatoprotective activities . Ginseng contains a number of active ingredients including ginsenosides, polysaccharides, phytosterols, peptides, polyacetylenes, fatty acids, and polyacetylenic alcohols, which have different effects on carbohydrate and lipid metabolism, cognition, angiogenesis, and the neuroendocrine, immune, cardiovascular, and central nervous systems . Among the active constituents of ginseng, ginsenosides are known to be the major biologically active components of ginseng and the most widely studied . Several studies have shown that ginsenosides play important roles in the pharmacological effects of ginseng . . However, ginseng contains other constituents, including ginsenoyne, phenolic compounds, polyacetylenes, sesquiterpenes, methoxypyrazine, alkylpyrazine derivatives, sesquiterpene alcohols, panasinsanols, and -carboline . White ginseng is peeled, dried, ginseng root and red ginseng is produced by steaming fresh ginseng root at 98100c for 23 h, and then drying until the moisture content is <15% . Red and white ginseng have both been shown to have immunomodulatory, anti - inflammatory, antioxidant, and antiatopic activities . Moreover, red ginseng has been reported to have more potent pharmacological activities than white ginseng in some respects [2022]. The differences in biological activities between red and white ginseng are caused by the chemical changes of ginsenosides after the steaming process . Steaming partially converts the original ginsenosides to deglycoslated derivatives . As a result, the species and amounts of ginsenosides are quite different based on the processing method used . Chu et al showed that a total of 53 and 43 compounds were tentatively identified in white ginseng and red ginseng samples, respectively . The featured compounds are mainly malonyl ginsenosides in white ginseng, and decarboxyl products of mal - ginsenosides and the dehydrated compounds from polar ginsenosides were characteristic in red ginseng . It is interesting that ginsenosides show a wide variety of biological activities, although the absorption rates from orally administered intact ginsenosides are very low . In the human intestinal tract, thus, the pharmacological actions of these ginsenosides have been closely related to their biotransformation by human intestinal bacteria . In this context several studies showed that the transformation of ginsenosides into deglycosylated ginsenosides is needed to increase ginseng's effectiveness in vivo . Abnormal changes in skin color induce significant cosmetic problems with a negative effect on quality of life . There are two groups of pigmentary disorders: disorders of the quantitative and qualitative distribution of normal pigment and the abnormal presence of exogenous or endogenous pigments in the skin . Hyperpigmentation - related diseases include melasma, lentigines, nevus, ephelis, freckles, postinflammatory hyperpigmentation, and age spots . Postinflammatory hyperpigmentation appears in many skin conditions, including acne, eczema, and contact dermatitis . Skin color is determined by various factors including melanin content, oxygenation state of hemoglobin in capillary vessels, carotenoid content, water content, and organization of collagen fibers in the dermis . Among these factors, melanin is the major determinant of skin color . In this context, melanogenesis is a biochemical pathway responsible for melanin synthesis that is controlled by complex regulatory mechanisms . Melanogenesis occurs in melanocytes confined in separate cytoplasmic organelles called melanosomes, which contain key enzymes of melanogenesis . Differences in skin color are related to the size, number, shape, and distribution of melanosomes, whereas melanocyte density typically remains relatively constant . Although tyrosinase is the key regulatory enzyme of melanogenesis, tyrosinase - related protein (trp)-1, dopachrome tautomerase (dct / trp2), and melanosomal matrix proteins (pmel17, mart-1) carry out important roles in regulating melanogenesis . The genes of tyrosinase, trp-1, and dct contain common transcription starting sites, the microphthalmia - associated transcription factor (mitf) binding sites . The intracellular signal transduction pathways of protein kinase c, cyclic amp (camp), and nitrogen oxide are involved in the regulation of melanogenesis . Various endogenous and exogenous factors, such as estrogen and ultraviolet (uv) radiation, affect melanogenesis via signal transduction pathways . These endogenous / exogenous factors exert their actions directly on melanocytes or indirectly via surrounding skin cells . Melanocytes, keratinocytes, dermal fibroblasts, and other skin cells communicate with each other by factors that are secreted and cell cell contacts . It has been shown that the interactions between keratinocytes and melanocytes are critical in the regulation of melanogenesis . In addition, dermal factors have been found to be involved in the regulation of melanogenesis . At the same time, stimulated melanocytes secret a number of signal molecules targeting not only keratinocytes but also skin immune cells . Soluble factors released by melanocytes include proinflammatory cytokines and chemokines such as interleukin (il)-1/1, il-6, il-8 il-10, tumor necrosis factor (tnf)-, transforming growth factor (tgf)-, catecholamines, eicosanoids, serotonin, -melanocyte stimulating factor (-msh), and nitric oxide (no). A variety of hypopigmenting agents including hydroquinone, arbutin, tretinoin, kojic acid, azelaic acid, vitamin c, n - acetylglucosamine, niacinamide, linoleic acid, ellagic acid, methimazole, dioic acid, soy extract, licorice extract, rucinol, and glycolic acid have been used alone or in combination to treat abnormal hyperpigmentation . These agents can interfere with the pigmentation process at several different steps of skin pigmentation . However, the treatment of hyperpigmented conditions still remains challenging and the results are often discouraging . Thus there is a need for novel skin - whitening agents that are highly effective and tolerable . In this article, we review recent reports investigating the skin - whitening effect of ginseng and its components and the underlying mechanisms of action, and then discuss their potential as candidates for novel skin - whitening agents . P. ginseng is one of the most widely used medicinal plants in traditional oriental medicine . Over thousands of years, it has been used to improve the overall condition of skin, as well as to treat a wide variety of diseases . However, genuine scientific approaches to clarify the efficacy of ginseng in skin have only been made in recent years . Several reports have shown that ginseng extract, powder, or some other constituents could inhibit melanogenesis in vitro and in vivo . Table 1 summarizes the direct effects of ginseng and its components on skin color and key enzymes involved in melanogenesis . Song et al reported that red ginseng powder improved melasma in a human clinical trial . They orally administered korean red ginseng powder for 24 weeks to female patients with melasma . After 24 weeks, the melasma area and severity index score decreased and melasma quality of life scale showed improvement in 91% of patients . In addition, 74% of the patients showed some improvement on the patient- and investigator - rated global improvement scales . Most of reports investigating the antimelanogenic effect of ginseng were conducted in vitro used purified tyrosinase or melanocyte cell lines . In melan - a cells treated with ethanol extract of ginseng seeds, melanin content and in addition to the crude extract or powder, several studies tested the effects of specific constituents of ginseng . The phenol compounds inhibited tyrosinase activity while ginsenoside prevents uvb - induced intracellular increase of reactive oxygen species [4648]. In some reports, ginsenosides alone exerted antimelanogenic effects . Aglycone of ginsenoside rh4 inhibited melanin synthesis in b16 melanoma cells, possibly by involvement of protein kinase a pathway . It significantly reduced melanin content and tyrosinase activity in -msh and forskolin - stimulated b16 melanoma cells . It reduced the camp level and camp response - element binding protein level in b16 melanoma cells, and this might be responsible for the downregulation of mitf and tyrosinase . In addition, ginsenoside rb1 inhibited melanogenesis through the inhibition of tyrosinase activity in -msh - stimulated b16 cells in a dose - dependent manner . The crude methanol extract of the fresh leaves of p. ginseng showed inhibitory activity on mushroom tyrosinase, and p - coumaric acid was characterized as the principal tyrosinase inhibitor in the extract . P - coumaric acid inhibited melanogenesis in b16f10 melanoma cells stimulated by -msh, and was suggested to interfere with melanogenesis by its structural similarity with tyrosine . Interestingly, p - coumaric acid showed weaker inhibition against mushroom tyrosinase but more strongly inhibited human or murine tyrosinase in comparison with kojic acid and arbutin . Enzyme kinetics analysis indicate that p - coumaric acid is a mixed type (for tyrosine) or competitive inhibitor (for dopa) of human tyrosinase . In addition, p - coumaric acid potently inhibits melanogenesis in human epidermal melanocytes exposed to uvb . Cinnamic acid, one of the major components of cinnamomum cassia blume, is found in the root and seed of p. ginseng . Cinnamic acid significantly reduced melanin production, tyrosinase activity, and tyrosinase expression in the melan - a cells . In addition, cinnamic acid showed depigmenting activity on the uvb - tanned skin of brown guinea pigs . It is already known that the pharmacological actions of these ginsenosides have been closely related to their biotransformation by human intestinal bacteria . Although the contents of total ginsenosides in red ginseng and fermented red ginseng using lactobacillus brevis were not significantly different, the ginsenoside metabolite content was higher in fermented red ginseng compared to red ginseng . The tyrosinase inhibitory activity of fermented red ginseng extract was more potent compared with red ginseng extract in a test using mushroom tyrosinase . As reviewed above, crude extract or some components of ginseng and its components showed antimelanogenic activities by direct inhibition on key enzymes of melanogenesis, such as tyrosinase . Moreover, ginseng and its components could exert antimelanogenic activity via action on the several factors related in melanocyte physiology . Among a large number of soluble factors produced from melanocytes, keratinocytes, fibroblasts, and immune cells in skin, adrenocorticotropic hormone (acth), -msh, endothelin-1, prostaglandin e2, prostaglandin f2, no, and histamine are well - known stimulators of melanogenesis [37,5963]. Il-1/1 and granulocyte - macrophage colony - stimulating factor (gm - csf) stimulate melanogenesis, while il-6, tgf-1, and tnf- downregulate melanin production . Gm - csf has been reported to be involved in regulating the proliferation and differentiation of epidermal melanocytes . Treatment of melan - a cells with conditioned media from uv - irradiated sp-1 keratinocytes increased melanocyte proliferation, and the proliferative effect of the conditioned media was blocked by anti - gm - csf antibody treatment . When uv - irradiated sp-1 keratinocytes were treated with red ginseng extract or saponin of red ginseng, the increased melanocyte proliferation by the conditioned media was blocked . In that report, red ginseng extract or saponin of red ginseng treatment decreased the expression of gm - csf induced by uv - b irradiation in sp-1 keratinocytes . As mentioned above, inflammatory cytokines such as il-1 and tnf- take part in the regulation of melanogenesis . Ginseng extracts and ginsenosides have been reported to have anti - inflammatory activities in several different studies . Ginsenosides inhibit different inducer - activated signaling protein kinases and transcription factor nuclear factor (nf)-b, and then decrease the production of proinflammatory cytokines and mediators of inflammation . Korean red ginseng extracts decreased tnf- and il-8 production in lipopolysaccharide (lps)-stimulated hacat keratinocytes and show radical scavenging and antioxidant activity in human dermal fibroblasts . These findings suggest that ginseng extracts and ginsenosides might affect melanogenesis through their anti - inflammatory activities . The effect of ginseng on no production is still questionable . Sun ginseng, a new processed ginseng prepared by steaming at high temperature, reduced uv - b - induced cell damage and decreased no production by inhibition of inducible no synthase mrna synthesis in hacat keratinocytes and human dermal fibroblasts . Red ginseng marc oil inhibited inducible no synthase and cyclooxygenase-2 via nf-b and p38 pathways in lps - stimulated raw264.7 cells . In addition, ginsenjilinol, a protopanaxatriol - type saponin obtained from the roots of p. ginseng, shows inhibitory activity on no production in lps - stimulated raw264.7 cells . By contrast, there are some controversial reports that ginseng extract enhanced no production or no signaling . Hong et al reported that ginseng extract administration stimulated nongenomic endothelial no synthase activation and enhanced no production in spontaneously hypertensive rats . In another report, water extract of korean red ginseng exerted vasoprotective effects through augmentation of no production by inhibiting arginase . Therefore, the effect of ginseng on melanogenesis via no signaling remains to be clarified by further study . Considerable numbers of immune cells including langerhans cells, macrophages, mast cells, and t cells are working actively in skin tissue . Because the immunostimulatory activities of many ginsenosides are known, it is not surprising that ginsenosides could enhance the reactivity of skin immune cells . In a recent paper, a cream containing 0.1% ginsenoside f1 (a metabolite of ginsenoside rg1) showed a significant whitening effect on artificially tanned human skin . However, ginsenoside f1 did not directly inhibit mrna expression of tyrosinase or dct in normal human epidermal melanocytes . Instead, ginsenoside f1 enhanced production of il-13 from human epidermal t cells, and il-13 significantly reduced the mrna expression and protein amount of both tyrosinase and dct resulting in visible brightening of normal human epidermal melanocyte pellets . These results suggest that ginsenosides might be able to regulate melanogenesis via their effect on skin immune cells . Recently, several reports have shown that extract, powder, or some constituents of ginseng could inhibit melanogenesis in vivo or in vitro . The underlying mechanisms of the antimelanogenic effect of ginseng or its components included the direct inhibition of key enzymes of melanogenesis (tyrosinase and dct), inhibition of transcription factors (mitf, nf-b) or signaling pathways (protein kinase a pathway and protein kinase c pathway) involved in melanogenesis, decreasing the production of inducers of melanogenesis (camp, gm - csf), and enhancing production of antimelanogenic factor (il-13). Fig . Although issues surrounding the antimelanogenic activity of ginseng still remain controversial, especially in its effect on the production of proinflammatory cytokines and no, these recent findings suggest that ginseng and its constituents might be potential candidates for novel skin - whitening agents.
Chronic kidney disease (ckd) is associated with a number of comorbidities and prognosis is poor, because many patients experience progression to end - stage renal disease . Also, in the majority of patients disease progression may be altered due to more or less suitable immunosuppressive protocol treatments and therapeutic approaches . The mechanisms of injury underlying progression are blurred, but traditional opinion pointed to an association between decline in renal function with the degree of proteinuria and histological findings of glomerulosclerosis and interstitial fibrosis [2, 3]. Kidney injury molecule-1 (kim-1) is a new specific biomarker of proximal tubule injury that can be measured both in urine and kidney tissue samples . It is an apoptotic - cell phagocytosis and scavenger receptor that is most highly upregulated in proximal tubular epithelium in acute and chronic kidney injury . Also, much attention has been paid to its possible pathophysiological role in modulation of tubular damage and repair [57]. Besides a close relationship between tissue kim-1 expression and urinary kim-1 concentration, our previous six - month prospective pilot study showed that kim-1 expression in tissue correlated better with tin features and renal function in different chronic kidney diseases than with urinary kim-1 concentration . Therefore, that investigation was the basis of this long - term retrospective study, where only kim-1 tissue expression as a predictor of long - term kidney function was examined . The aim of this retrospective investigation was to evaluate (1) possible associations between tissue kim-1 expression and tubulointerstitial (tin) inflammation, atrophy, and fibrosis in different chronic kidney diseases and (2) possible associations between tissue kim-1 expression and some demographic and laboratory parameters as well as kidney function and proteinuria at the time of biopsy and 6, 12, 24, and 36 months later . The retrospective study included 60 patients (27 men) of mean age 34.42 12.15 years (range 1859 years), who were hospitalized in the clinical center of serbia for kidney biopsy from 2006 to 2009 . Indications for kidney biopsy were nephrotic syndrome, pathologic proteinuria without nephrotic syndrome, and abnormal urinary sediment (erythrocyturia and leukocyturia) in several samples . Pathohistological analysis revealed minimal change glomerulonephritis (mcgn) in two patients, non - iga gn in nine, iga gn in six, membranous and membranoproliferative gn in seven patients each, focal glomerulosclerosis in eleven, lupus nephritis in ten, and crescentic gn in eight patients . After diagnosis, the participants were treated according to established protocols for each type of gn with immunological and nonimmunological therapy (nonsalt diets, statins, angiotensin - converting - enzyme - ace inhibitors, and angiotensin receptor type 1 blockers atb). The institutional review committee approved our study protocol therebys following local biomedical research regulations (irb no . A complete blood count; serum urea and creatinine, albumin; lipid status, and urine sediment were determined in blood and urine samples collected 1 day before kidney biopsy as well proteinuria / day . Only samples with a sterile urine culture were processed . Urine sediments with more than 3 rbc / hpf or 5 wbc / hpf were defined as clinically significant erythrocyturia or leukocyturia . Estimated glomerular filtration rate (egfr) was calculated with a shortened version of the modification of diet in renal disease (mdrd; patients 18 years). Alteration in tin were assessed after routine hematoxylin - eosin staining by a qualified nephropathologist who was not familiar with the preset histopathologic or clinical diagnosis, nor the identity of patients . Inflammation activity was then scored numerically (0: no inflammation, 1: slightly marked inflammation, 2: moderate inflammation, and 3: strong inflammation) as well as atrophy and fibrosis advancement (0: no atrophy and no fibrosis, 1: slightly marked atrophy and fibrosis, 2: moderate atrophy and fibrosis, 3: very pronounced atrophy and fibrosis). After deparaffinization tissue samples were rehydrated and stained immunohistochemically for kim-1 using the tissue kim-1/tim-1 kit (r & d systems inc ., minneapolis, mn, usa) with contrasting hematoxylin and eosin staining . A tubule was considered to be kim-1 positive if it contained at least one kim-1 positive cell regardless of whether the kim-1 expression was cytoplasmic or apical . Kim-1 staining was scored semiquantitatively by estimating the percentage of cortical tubules expressing kim-1 per field (the complete biopsy area was scored, with a minimum of five fields; in controls 30 fields were scored). Unaffected parts of kidneys from patients with renal cell carcinoma were used as the negative control . The 6-, 12-, 24-, and 36-month followup for all patients included measurement of serum creatinine concentration, egfr, and proteinuria . Data are presented as mean 1 standard deviation (sd) and the range of values . The one - sample kolmogorov - smirnov test was used to check for normal distribution of the variables . Relationships between variables were estimated using pearson's rank and spearmen's correlation tests as appropriate . Independent predictors of renal function 6, 12, 24, and 36 months after the kidney biopsy were identified by stepwise multivariant regression analysis . Table 2 presents the demographic, clinical, and laboratory data for the patients at the time of kidney biopsy . Tissue kim-1 expression was significantly induced in all kidney biopsies except in one patient with mcgn . Tissue kim-1 had a typically apical localization, but it could also be found in the cytoplasm with no apparent affinity for the apical membrane of tubulocytes . Kim-1 positive tubules were located mainly in the tin regions affected by inflammation or fibrosis but were not observed in regions with end - stage fibrosis . Kim-1 univariate analysis showed significant positive associations between tissue kim-1 expression and age (r = 0.313; p = 0.015), tin inflammation (r = 0.4563; p = 0.004), tin fibrosis (r = 0.317; p = 0.021), and hemoglobin (r = 0.440; p = 0.001) as well as negative associations with albumin concentration (r = 0.376; p = 0.011) and egfr (r = 0.572; p <0.001) at the time of biopsy and 6 (r = 0.442; p = 0.002), 24 (r = 0.398; p = 0.012), and 36 (r = 0.412; p = 0.015) months later . Tissue kim-1 expression correlated significantly only with proteinuria / day 6 months after biopsy (r = 0.394; p = 0.026) but not with proteinuria at the time of biopsy and 12, 24, and 36 months later . Table 3 gives the correlation coefficients between egfr at the time of biopsy and 6, 12, 24, and 36 months later and the examined variables . Multivariant stepwise regression analysis showed that the best predictor of kidney function at the time of biopsy (egfr 0) was tin inflammation (p <0.001), as well as of egfr12 (p = 0.015), egfr 24 (p = 0.039), and egfr 36 (p = 0.012). However tissue kim-1 expression was the best predictor of egfr 6 (p = 0.016) along with tin inflammation (p = 0.016) (table 4). Figure 2 presents the linear correlation between tin inflammation (p <0.001), tissue kim-1 expression (p <0.001), and egfr 6 . In our previous 6-month prospective study, we found higher urine kim-1 concentrations in twenty chronic renal patients than in control subjects and a significant positive correlation between urine kim-1 level and kim-1 expression in tissue . Although, urine kim-1 content reflected tissue kim-1 expression, tissue kim-1 correlated better with tin inflammation and fibrosis as well as with kidney function at the time of biopsy and 3 and 6 months later . Therefore, we retrospectively examined sixty patients with different kidney diseases in order to evaluate the importance of tissue kim-1 expression in predicting kidney function in the ensuing 36 months after kidney biopsy . In the present study tissue in addition, kim-1 positive tubules were located in parts of the cortex that were imbued with inflammatory infiltrate and/or fibrotic changes but not in completely atrophic areas . It is well known that the expression of kim-1 in tubules is associated with inflammation and tin damage . Double - labeling studies have shown simultaneous expression of kim-1 and markers of prefibrotic changes, repair, and chemotaxis [10, 11]. The study of van timmeren et al . Also demonstrated a connection between kim-1 expression in tissue, interstitial fibrosis, and macrophage accumulation . . First showed that tubular epithelial cells expressing kim-1 on their surface could act as phagocytes for apoptotic and necrotic renal epithelial cells . Kim-1 is expressed predominantly in renal tubules, which, judging by the simultaneous expression of markers of dedifferentiation, are currently under the influence of agents that cause injury, but is absent from completely atrophic tubules . Controversy remains about the function of kim-1: is it actively regulating the inflammatory process? Or is its expression just a response to damage, attempted recovery, and/or repair? . The present study revealed a significant negative correlation between tissue kim-1 expression and all estimated gfr at the time of kidney biopsy and 6, 24, and 36 months later . However multivariate analysis pointed to tin inflammation, but neither tin fibrosis nor proteinuria, as the best predictor of kidney function at biopsy and 6, 12, 24, and 36 months afterwards . However, along with tin inflammation, tissue kim-1 expression was the best predictor of kidney function 6 months after biopsy . Interstitial infiltration of inflammatory cells can be seen in a variety of immune and nonimmune kidney diseases and is thought to play a significant role in tin damage and fibrosis . Upon exposure to high protein concentrations, renal tubule cells produce a host of chemokines, vasoactive mediators, and adhesion molecules, which may contribute to interstitial fibrosis . Protocol immunosuppressive therapy and treatment with ace inhibitors and at blockers for chronic renal patients with different chronic immune and nonimmune - mediated kidney disorders proven by biopsy have the strongest effects during the first 6 months of therapy . The severity of pretreatment tin damage predicts a dulled response to renoprotective intervention, with a worse long - term renal outcome . Therefore, we can speculate that patients with less interstitial inflammation at the time of kidney biopsy will have a more favorable response to treatment and better kidney function afterwards . Also, the finding that kim-1 was the strongest predictor of kidney function 6 months after biopsy along with tin inflammation may support the hypothesis about its potential role in the development of interstitial fibrosis and an unsatisfactory treatment response . Association between higher urinary kim-1 levels and poorer kidney function was found in some human studies in native [10, 14] and transplanted kidneys but data on human tissue kim-1 expression are scarce . Zhang et al . Found that kim-1 expression in transplant biopsies is a sensitive measure of cell injury . In addition, they showed that more intense kim-1 staining predicts a better graft outcome over the ensuing 18 months and speculated that its level of expression may be an indicator of graft function recovery . It is well known that proteinuria is an ominous biomarker of progressive kidney disease and our study revealed that only proteinuria 6 months after kidney biopsy correlated negatively with kidney function 24 and 36 months after the biopsies and positively with kim-1 expression in tissue . Earlier investigations [10, 14, 17] have yielded conflicting data regarding the relationship between urinary kim-1 level or tissue kim-1 expression and proteinuria . One explanation may be that proteinuria is not always accompanied by tin damage and a progressive decline in renal function [18, 19]. Data regarding the selectivity of proteinuria were not available for the purposes of this study . The present study exposed a strong association between kim-1 tissue staining and tin inflammation and fibrosis but not with tin atrophy . Although multivariate analysis pointed to tin inflammation, as the best predictor of kidney function at biopsy and 6, 12, 24, and 36 months later, tissue kim-1 expression is one of the best predictor of kidney function 6 months after biopsy, the time when treatment effects are the strongest . Therefore, we can speculate that kim-1 has a potential role in the development of interstitial fibrosis and poor treatment responses . The major limitation of this investigation is the relatively small number of patients examined but it could be the basis for a larger prospective follow - up study for evaluation of urinary kim-1 concentration and tissue kim-1 expression in patients with various chronic and acute renal diseases.
These range from various slow - growing benign tumors and low - grade malignancies to fast growing high - grade malignancies . Angiofibromas are one such group of tumors arising in this region, which present as swellings . Of these, nasal angiofibromas (nas) are the most common and nas occurring in the region other than the nose are called as extranasopharyngeal angiofibromas (ena). A 54-year - old male reported to our department with history of swelling over the right side of face since 5 years . The patient reported insidious onset of swelling with a gradual progress to the present size . He also reported mild, intermittent, and dull pain in the region of swelling . Clinical examination confirmed a solitary, oval, well - defined swelling measuring approximately 7 5 cm in the preauricular region [figure 1]. It extended superioinferiorly from the level of outer canthus of the eye to inferior border of mandible and anterioposteriorly extended from 2 cm in front of anterior border of ramus to the mastoid notch [figure 1]. On palpation, the swelling was firm, non - tender, free from skin, and mobile over the underlying structures . Ultrasonography revealed a large lobulated solid mass of 6 4 cm in the right parotid region with multiple vessels with low velocity flow and without any areas of calcification or cystic changes . Findings were suggestive of a low - grade vascular tumor . Computed tomography scan with contrast medium revealed a large well - circumscribed, solitary isodense lesion measuring 6.36 4.39 cm [figure 2]. It was observed to be extending anteriorly over the superficial lobe of the parotid gland, with anterior displacement of parotid gland . Fnac revealed numerous vascular channels with occasional singly scattered and single cluster of plump spindle cells . Ct- scan transverse section classical superficial parotidectomy was performed under general anesthesia using a modified appiani incision . Tumor mass was dissected anteriorly and excised along with the superficial lobe of parotid gland [figure 3]. Facial nerve was located using the anterograde method and all the branches of facial nerve were preserved . Follow up, facial nerve function was intact and no sign of reoccurrence was observed . Histopathological sections revealed connective tissue with numerous vascular spaces of variable sizes ranging from small capillary - like vessels to partly lined vessels . Around the endothelial cells, cells were plump or stellate - shaped with mild inflammatory cell response and composed predominantly of plasma cells and a few lymphoid follicles [figure 4]. Photomicrograph shows numerous endothelial lined blood capillaries with collagen fibres interspersed with fibroblasts and fibrocytes (h&e stain, 40). (b) photomicrograph shows endothelial lined blood vessels with connective tissue wall in the background of collagen fibres interspersed with fibroblasts and fibrocytes (h&e stain, 400) photomicrograph of the sections showing the blood vessels positive for cd 34 (ihc stain, 100) various soft tissue tumors, both benign and malignant, present as swelling in the parotid region . Clinical examination confirmed a smooth - surfaced, solitary, oval, firm, well - defined swelling measuring approximately 7 5 cm in the preauricular region . Based on the clinical findings of location, consistency and borders a differential diagnosis of pleomorphic adenoma, solitary fibrous tumor (sft), low - grade fibrosarcoma, and lipoma was made . As the most common tumors found in the region are parotid tumors, a provisional diagnosis of mixed parotid tumor was made . However, fnac and contrast - enhanced ct scan were in favor of a tumor with vascular component, shifting the differential diagnosis towards tumors with a vascular component presenting as slow - growing, circumscribed firm swellings . Histopathological examination of the excised lesion revealed features suggestive of angiofibroma and had to be differentiated from lesions with similar features . The differential diagnosis included low - grade vascular lesions, spindle cell neoplasms, comprising of a large range of benign and low - grade malignant soft tissue lesions, including cellular angiofibroma, sft, low - grade fibromyxoid sarcoma (lgfms), low - grade myxofibrosarcoma, myxoid liposarcoma, giant cell angiofibroma (gca), angiomyolipoma, and angiomyofibroblastoma . Cellular angiofibroma, usually occurs in the pelviperineal region, which would be an unusual location for the tumors reported herein; however, cases have been reported to have occurred in the buccal mucosa, raising a diagnostic possibility . Cellular angiofibroma shows more rounded, non - branching vessels, often of medium size with thicker walls . The tumors are generally more uniformly cellular, composed of lesional cells with short, stubby nuclei resembling those of spindle cell lipoma . Sfts occur in a variety of locations in the head and neck region; myxoid examples may be particularly difficult to identify and should be considered in the differential diagnosis . Microscopically, besides the so - called patternless architecture, there is pronounced regional variation in cellularity, prominent thick collagen bundles and characteristic branching ectatic staghorn vessels, which are not accompanied by the abundant smaller - sized vessels present in angiofibroma of soft tissue . In addition, the tumor cells in sft express strong and diffuse cd34 in most cases . This entity lacks the innumerable, evenly distributed, arborizing thin - walled vessels characteristic of angiofibroma . Lgfms is a distinctive fibroblastic malignant neoplasm characterized by a peculiar tendency to give rise to very late metastases . Histologically, it shows alternating collagenous and myxoid areas with a usually swirling or whorled growth pattern, a frequently lobular appearance, and deceptively bland spindle cell morphology . The lesions tend to be more hypocellular, with a fibrous component that has uniform collagen deposition rather than the fibrillary or coarsely banded collagen fibers in angiofibroma of soft tissue . Although lgfms may contain arcades of thin - walled vessels, vascularity is usually not prominent . Usually occurring in subcutaneous tissues of the extremities, it shows distinctive histologic features including a lobulated growth pattern with infiltrative margins and fusiform, round, or stellate tumor cells with frequently slightly eosinophilic cytoplasm and hyperchromatic atypical or pleomorphic nuclei . Obvious features of malignancy are usually present, characteristic elongated curvilinear vessels with perivascular hypercellularity, these features bear little or no resemblance to the rich vascular network of angiofibroma . Myxoid liposarcoma contains a prominent plexiform network of thin - walled capillaries (which has been referred to as a chicken wire or however, it also shows scattered univacuolar and multivacuolar lipoblasts throughout, as well as stromal mucin pools not seen in angiofibroma . Gca has been reported as a benign mesenchymal tumor with 2 cases originating in the buccal mucosa . Although benign, the lesion has potential for local reoccurrence, especially after incomplete resection . Histopathologically, it has similar presentation to that of sft, with presence of multinucleated giant cells not seen in angiofibroma . Angiomyolipoma differs from angiofibromas because of the presence of prominent muscular arteries while angiomyofibroblastoma differs because of the presence of fibrovascular component with a loose myxoid fibroelement . Enas have interstitial stromal tissue predominance with less vascular elements such as that of long - standing na . Other distinctive histologic features are angiofibroma of soft tissue, consisting of a vaguely lobular, variably cellular proliferation of bland, uniform spindle cells set in an abundant, variably myxoid or collagenous extracellular matrix with numerous small, and thin - walled, branching blood vessels . The complexity of the vascular pattern, often also including larger vessels of varying size and shape, is the most noticeable feature . Immunohistochemical analyses may show that stromal cells have strong cytoplasmic reactivity for vimentin and are generally immunonegative for smooth muscle actin and desmin . Histopathology coupled with immunohistochemistry usually confirms the diagnosis in favor of angiofibroma . In our case, the tissue specimen showed features consistent with the diagnosis of ena . In 1980, de vincentiis and pinelli reviewed a series of 704 cases of angiofibroma and found that 13 cases manifested outside the nasopharynx, thus suggesting that extra nasopharyngeal localization of this tumor is a possible although rare occurrence . A review article by windfuhr and remmert in 2004 summarized 65 patients with enas . The most common site reported for enas is the maxillary sinus followed by the ethmoid, nasal cavity, and nasal septum . Enas are also reported to originate from the ethmoid sinus, nasal cavity, nasal septum, larynx, sphenoid sinus, cheek, conjunctiva, oropharynx, retromolar area, middle turbinate, inferior turbinate, and tonsil . Other rare sites for enas occurrence have also been described - external nose, hard palate, external ear, lacrimal sac, carotid bifurcation, oesophagus, trachea, facial nerve, middle cranial fossa, and infratemporal fossa . The most common age of incidence was the second decade (46%) in comparison to 80% of patients with nas . Enas have a higher mean age, i.e, 22 years compared to nas, which have a mean age of 18 years . This lesion has been described as different from that of the classical nasopharyngeal variety in being more common in females, older individuals with an early presentation, and being capsulated with poor vascularity and infrequent recurrence . Unlike nas, enas present non - specific and numerous symptoms . Imaging modalities such as ct scan, mri, ct angiography, and ultrasonography are required for optimal preoperative evaluation of nas and enas . Imaging of nas with a contrast agent leads to diffuse and usually homogenous involvement in ct and mri scans with strong contrast enhancement . In contrast, enas enhances moderate amount of contrast or none due to its weak vascular involvement . Surgical excision is the prime modality of treatment for enas; radiotherapy maybe applied for non - resectable lesions . Occurrence of enas, although rare in the maxillofacial region, should be considered in differential diagnosis of swellings in this region.
Atherosclerotic renal artery hypertension is reported in almost 7% of adults older than 65 years1) and is associated with cardiovascular events, and may double risk of mortality2). In the 1990s, radiological angioplasty began to replace surgical revascularization . Galaria et al . Showed that percutaneous and open renal revascularization had equivalent long - term functional outcomes3). Over time, the less - invasive procedure became more accessible . Most cases of atherosclerotic renovascular disease (arvd) are located in the ostium, and are extensions of calcified aortic plaques4). Stent placement might also provide additional force to increase the rates of technical success5) and to reduce the rate of restenosis at 6 months after the initial procedure6). Intervention with stents has become a standard procedure; in patients with stenosis of the renal artery, placement of a stent is likely to be the initial form of treatment . However, there is limited evidence to support revascularization over medical therapy for patients with atherosclerotic renal artery stenosis7, 8). Retrospective studies previously reported that renal artery lesions might progress to severe stenosis and ultimately to renal artery occlusion9). Michael et al . Performed a prospective study that reported that the progression of renal artery disease was a frequent occurrence with an annual rate of progression of renal artery lesions reported to be 7%10). However, at the time of these studies, statins were being used in fewer patients . A retrospective study of the effects of statins on the progression of arvd has shown that the use of statins reduces the risk of progression and the development of arvd11). Another prospective population - based study reported that the progression to significant arvd was observed in only 4.0% during 8 years of follow - up (annualized rate, 0.5% per year), and no case of arvd progressed to occlusion12). Essential hypertension and clinically silent renal artery stenosis often coexist, and essential hypertension also coexists with renovascular hypertension13). Renal injury distal to an atherosclerotic renovascular obstruction is due to multiple intrinsic factors producing parenchymal tissue injury (fig . Non - traditional mediators of arvd such as inflammatory pathways, reactive oxygen species production, ischemia / reperfusion damage and modulation of matrix turnover have been proposed as causes of the renal failure related to arvd15). This complexity of the pathophysiology might explain why the severity of the stenosis is not correlated with renal dysfunction . Clinical data support dissociation of improved renal artery patency from clinical outcome in patients with atherosclerotic renal artery stenosis; this may illustrate the effects of irreversible injury on the post stenotic kidney . A prospective clinical study reported by wright et al . Showed absence of a correlation between renal artery anatomy and baseline renal function or functional outcome, and a good correlation between renal functional outcome and proteinuria; these findings suggest that renal parenchymal damage is a major determinant of renal dysfunction and outcome rather than the severity of the renal artery stenosis in arvd16). Reported that the mean arterial pressure did not decrease by 10 mmhg or more after revascularization and renal function declined in most patients with arvd that had high resistance - index values before revascularization17). Chrysochou et al . Showed a correlation between baseline proteinuria and decline in estimated glomerular filtration rate (gfr) with time after revascularization18). Therefore, post stenotic renal injury can lead to renal parenchymal injury reflected by high intrarenal resistance and/or the presence of proteinuria . Proteinuria and other factors involved in intrarenal resistance are predictors of a poor outcome after renal revascularization in arvd . When patients with chronic kidney disease undergo renal angioplasty with / without stent placement, the response with regard to renal function can be negative as well as positive . Positive responses include improvement of renal function, and stabilization or attenuation of declining renal function, as expected . Possible causes of these adverse outcomes include contrast induced nephropathy, atheroembolism, and restenosis or stent thrombosis19). The administration of contrast might increase the risk of acute renal dysfunction, especially in patients that have preexisting renal impairment . An ex vivo study reported that each manipulation of atheroma specimens, from simply advancing the guidewire through the atherosclerotic lesion to positioning and deploying the wallstent, releases thousands of fragments, and that these atherosclerotic fragments are of sufficient size to create vascular occlusion and initiate significant renal parenchymal damage20). The nature of this disorder suggests that a systemic approach is necessary to provide cardiovascular protection . Previously, statin therapy was discussed as a method of altering the natural history of arvd11). Angiotensin - converting enzyme inhibitors and angiotensin receptor blockers effectively reduce blood pressure in patients with renovascular disease . In addition, a population - based cohort study of over 3,500 patients with arvd in canada found that angiotensin inhibitors could cause acute renal toxicity in a small subset of vulnerable patients; however, they still improved the cardiovascular and renal outcomes in patients with arvd, but at the expense of acute renal toxicity21). Recently three randomized controlled trials have been performed: the stent placement and blood pressure and lipid - lowering for the prevention of progression of renal dysfunction caused by atherosclerotic ostial stenosis of the renal artery (star) trial, the angioplasty and stenting for renal artery lesions (astral) trial, and the cardiovascular outcomes in renal artherosclerotic lesions (coral) trial (table 1). Two large randomized trials of intervention vs. medical therapy showed negative results for the intervention . The star trial was a european multicenter trial that enrolled 140 patients with ostial renal artery stenosis greater than 50%, blood pressure controlled to less than 140/90 mmhg, and creatinine clearance 15 to 80 ml / min7). All patients received angiotensin blocking agents and a statin, regardless of their lipid levels . After a 2-year interventional period, no difference was observed in the decline of renal function, the degree of blood pressure control, and the rates of cardiovascular morbidity and death . Investigators in the astral trial enrolled 806 patients with at least one stenotic renal artery considered suitable for balloon angioplasty, stenting, or both in their international, multicenter trial8). The mean estimated gfr was 40 ml / min, and most of the patients were on statin and angiotensin blocking therapy . At a mean follow - up of 33.6 months, no difference was noted between the treatment groups in the decline of renal function or blood pressure control, and the renal function worsened slightly in both groups . Most of the enrolled patients in the two clinical trials had relatively asymptomatic atherosclerotic renal artery stenosis . Therefore, the practice of indiscriminately performing revascularization, without strong evidence, is no longer acceptable . The coral trial is an ongoing multicenter randomized controlled trial in the united states22). It is still enrolling patients that have drug - resistant hypertension or a gfr <60 ml / min . It is using a standardized medical protocol to control blood pressure, and embolic protection devices during procedures are encouraged . Thus far, intervention has not been recommended if renal function has remained stable over the past 6 to 12 months and if hypertension can be controlled medically . According to a clinical classification of atherosclerotic renal artery stenosis with guidelines for vascular intervention of surveillance published in 2008 by the atherosclerotic peripheral vascular symposium23), additional recommendations favoring medical therapy were as follows: very advanced age and/or limited life expectancy, extensive co - morbidities that make revascularization too risky, high risk for or previous experience with atheroembolic disease, and other concomitant renal parenchymal diseases that cause progressive renal dysfunction (e.g., interstitial nephritis, diabetic nephropathy)23). They also recommended factors favoring medical therapy and intervention as follows: progressive decline in gfr during treatment of systemic hypertension, failure to achieve adequate blood pressure control with optimal medical therapy (medical failure), rapid or recurrent decline in the gfr in association with a reduction in systemic pressure, decline in the gfr during therapy with angiotensin - converting enzyme inhibitors or angiotensin receptor blockers, and recurrent congestive heart failure in a patient in whom the adequacy of left ventricular function does not provide an explanation23). The best evidence supporting intervention is for bilateral stenosis with " flash " pulmonary edema, but the evidence is from retrospective studies24 - 26). The aim of treatment for arvd should include the prevention of cardiovascular and/or renal events . Treatment of patients with arvd should include consideration of the following factors: age, co - morbidity, blood pressure, renal function, and kidney size . The results of two recent studies suggest that patients with arvd and stable renal function can be controlled medically . Intervention should be considered only in patients with arvd and rapidly progressive cardiac or renal dysfunction . The results of the coral and the post hoc analysis of astral might provide additional evidence for revascularization.
Tuberculosis (tb) caused by mycobacterium tuberculosis (m. tb) continues to be a significant global health problem, affecting millions of people worldwide [1, 2]. It is a prevalent infectious disease in china, with 250,000 deaths from tb annually and 6 million active tb patients at present . The global incidence of tb is raising due to coinfection with the human immunodeficiency virus (hiv) and the emergence of multidrug - resistant (mdr) m. tb strains [5, 6]. According to the report of world health organization (who), m. tb will cause 1 billion new cases and about 35 million deaths worldwide by 2020 . Therefore, effective treatment and control strategies are urgently needed to counteract the global threat of tb . The current vaccine against tb, m. bovis bacilli - calmette - gurin (bcg), is a live attenuated vaccine which has been widely used throughout the world for many decades . Bcg protects children efficiently against miliary and meningeal tb, but protective efficiency against pulmonary tb in adults has been found to vary highly from 0% to 80% . Much effort has been devoted to improving bcg efficacy by genetic engineering technology because of its strong immunostimulatory properties and proven safety for human use [9, 10]. Recombinant bcg (rbcg) expressing different immunodominant antigens of m. tb, such as secreted antigens (ag85b, ag85c, esat-6, etc .) Or latency associated antigens (-crystallin, rv2659c, rv3407 and rv1733c, etc . ), have been tested as candidate vaccines against tb and are demonstrated to have an enhanced ability to induce th1 immune response and protection against m. tb challenge in animal models [11, 12]. Also, it is definitely no doubt that doses of antigens could subtly influence the magnitude of host immune response as well as protection efficacy, no matter antigen is administered in the form of rbcg, protein, dna, or rna . We have previously reported the construction of a m. tb fura gene operator / promoter (pfura)-based differential expression system, from which it is feasible to express target antigens of interest in a modular fashion . M. tb chimeric antigen ag856a2, which is coded by a recombinant ag85a gene with 2 copies of esat-6 gene inserted at the acc i site of ag85a (see figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2014/196124), shows improved immunogenicity in mice when it is inoculated intramuscularly as a dna vaccine . For the current study, we selected two rbcg strains overexpressing the same chimeric antigen ag856a2 at the maximum difference: rbcg186 and rbcg486 overexpressing the fusion protein under control of the wild - type or the optimized double - mutated fura promoters, respectively . We tested their efficacy as vaccines in c57bl/6 mice, comparing immune response and protection against m. tb challenge . The results showed that mice vaccinated with rbcg186 or rbcg486 generally induced higher antigen - specific effector and memory immune responses, as well as protective efficacies compared to mice vaccinated with the parent bcg strain . However, the two rbcg strains between themselves, which expressed the chimeric antigen ag856a2 at different levels, induced different antigen - specific ifn- production and comparable number of m. tb - specific cd4 t cells expressing il-2 . And the protective efficacies imposed by the two rbcg strains displayed no significant differences although higher protection was observed in rbcg486 vaccinated mice than that in rbcg186 vaccinated mice . Female specific pathogen - free (spf) c57bl/6 mice aged 68 wks were purchased from shanghai slac laboratory animal co., ltd . (shanghai, china) and kept under spf conditions with food and water ad libitum until challenge . Infected mice were maintained in a biosafety level 3 (bsl-3) biocontainment animal facility . All animal experiment protocols were approved by chinese science academy committee on care and use of laboratory animals and were performed according to the guidelines of the laboratory animal ethical board of shanghai public health clinical center . Bcg and its derivative recombinant strains were grown in liquid middlebrook 7h9 broth (bd difco, usa) supplemented with 10% oleic acid - albumin - dextrose - catalase enrichment (oadc, bd difco, usa), 0.2% glycerol, and 0.05% tween 80 . Cultures in the exponential phase were frozen and stored at 80c . When required, kanamycin was added at a final concentration of 50 or 20 g / ml for e. coli or mycobacteria, respectively . Two rbcg strains overexpressing m. tb chimeric immunodominant antigen ag856a2 at different levels were constructed as previously described . Briefly, the ag856a2 coding gene, which is a recombinant ag85a gene with 2 copies of esat-6 gene inserted in its acc i site, was amplified from the plasmid template of dna vaccine hg856a and then cloned into mycobacterial differential expression vectors pmfa11 and pmfa41 under control of the prototypical and double - mutated (mutations: initial codon change from gtg to aug and 6 bp substitution at upstream at - rich region) fura promoter, respectively . The resulting constructs were electroporated into bcg - danish competent cells and selected on middlebrook 7h11 agar with kanamycin . The rbcg transformants were grown to midexponential phase in complete middlebrook 7h9 broth and then verified the recombinant protein expression by routine western - blotting assay . Mice were vaccinated subcutaneously (s.c .) With 2 10 colony - forming units (cfu) of bcg or rbcgs in 100 l saline . Eight weeks after vaccination, groups of 6 mice were either sacrificed for assessment of antigen - specific t cell responses in splenocytes or exposed to an aerosol of virulent m. tb h37rv strain to deposit an inhaled dose of 100200 cfu per lung by an inhalation exposure system (glas - col, usa). Ifn- elispot assay kit (bd biosciences, usa) was used as described by the manufacturer . Plates were coated with anti - ifn- mab overnight at 4c and then blocked with rpmi 1640 medium containing 10% fetal bovine serum (fbs) for 1 h at room temperature . Splenocytes (2.5 10 cells / well) from immunized mice were isolated, plated, and cultured with 10 g / ml ppd (statens serum institute, denmark) or 2 g / ml recombinant ag85a, 6 g / ml recombinant esat-6 to provide stimulation at 37c, 5% co2 for 20 h. after washing the plates with pbs - t20 (1 pbs, ph 7.4, 0.05% tween 20), biotinylated anti - ifn- was added for 2 h at room temperature . Streptavidin - hrp was added for 45 min, and the color was developed with 3-amino-9-ethylcarbazole (aec) substrate (bd biosciences). An immunospot analyzer (cellular technology, usa) was used to count the spots . Splenocytes (2 10 cells / well) isolated at 8 weeks after immunization were plated in 96-well plates and stimulated with 10 g / ml ppd for 14 h in the presence of 1 g / ml anti - cd28 (bd biosciences) and subsequently incubated for an additional 5 h at 37c following the addition of 0.5 l / ml monensin / golgistop (bd biosciences). Following overnight incubation at 4c, the cells were washed in facs buffer (pbs containing 0.1% sodium azide and 1% fbs) and subsequently stained for 30 min at 4c for surface markers with mabs as indicated using anti - cd3-pacific blue, anti - cd8-fitc, and anti - cd44-v500 (all from bd biosciences). Cells were then washed in facs buffer, fixed, permeabilized using the cytofix / cytoperm kit (bd biosciences) according to the manufacturer's instructions and stained intracellularly for 30 min at 4c using anti - ifn--apc - cy7, anti - tnf--percp - cy5.5, and anti - il-2-apc mabs (all from bd biosciences). Cells were subsequently washed, resuspended in facs buffer, and then analyzed by multiparameter flow cytometry using a bd facsaria flow cytometer (bd biosciences). For each sample, at least 300,000 events were collected and responses were analyzed using flowjo software (tree star, usa). Five weeks after infection, mice were sacrificed and the mycobacterial burden was determined by plating homogenates of lung, excluding right postcaval lobe, and entire spleen onto middlebrook 7h11 agar plates supplemented with 10% oadc enrichment and a 4-antibiotic mixture (40 u / ml polymycin b, 4 g / ml amphotericin, 50 g / ml carbenicillin, 2 g / ml trimethoprim) that prevents growth of contaminating microorganisms . Plates were incubated at 37c for 3 weeks in semisealed plastic bags and then cfu were counted and expressed as log10 cfu per organ . Then, the embedded lung lobes were sectioned in thickness of 5 m, stained with haematoxylin and eosin (h & e) and photographed using a olympus ckx41 microscope (olympus, japan) fitted with an olympus dp20 camera connected to a computer . The image pro plus program (media cybernetics, usa) was utilized to objectively assess the level of inflammation present in each image . The mean percent of area inflamed was quantified averaging from three to five lung sections of each of the different groups of mice . The antibodies were rabbit polyclonal anti - mouse tnf- (abcam, uk), rabbit polyclonal ifn- antibodies (invitrogen, usa), and rabbit polyclonal anti - mouse inos antibody (cayman chemical, usa). All sections were examined by light microscopy, and the expression of tnf-, ifn-, or inos was semiquantified by intensity of positive signal using image pro plus software . Immune responses, protective efficacies, and histopathological staining were tested by one - way anova followed by tukey's multiple comparison tests of the means . We have previously developed a novel mycobacterial differential expression system (pmfa series) based on the m. tb fura gene operator / promoter (pfura) or its derivatives . Ag856a2 was cloned into two of these plasmids, pmfa11 and pmfa41, which drives low and high gene expression under the control of the wild - type and modified fura promoters, respectively . By transformation of bcg, we obtained two strains, rbcg186 and rbcg486, which drove correspondingly low and high expression of chimeric immunodominant antigen ag856a2 (figure 1, upper panel). Quantification of the band intensities of western - blot indicated that rbcg486 roughly expressed> 3-fold of ag856a2 than rbcg186 did (figure 1, lower panel). Eight weeks after vaccination, elispot assay of splenocytes showed that more cells in the rbcg486-vaccinated mice expressed ag85a - specific ifn- compared to those of rbcg186- and bcg - vaccinated mice (figure 2, left panel). Also, significantly elevated numbers of splenocytes expressed esat-6-specific ifn- in both rbcg186- and rbcg486-vaccinated mice compared to that of bcg - vaccinated mice (figure 2, middle panel). Additionally, esat-6-specific ifn- was induced at much higher level in rbcg486-vaccinated mice compared to rbcg186 group (figure 2, middle panel). A similar pattern of ppd - specific ifn- responses as ag85a - specific response was also observed but the difference was not statistically significant, regarding to the comparisons of rbcg486-vaccinated mice and other immunized groups (figure 2, right panel). We used flow cytometry to measure the capacity of m. tb - specific cd4 t cells from spleens of vaccinated mice producing cytokines ifn-, tnf-, and il-2 at single cell level after stimulation in vitro with ppd . The cytokine - producing cd3cd4 cells were classified into seven subpopulations based on their production of ifn-, tnf-, and il-2 in any combination (figure 3(a)). Significantly increased frequencies of ppd - specific il-2 cd4 t cells were identified in rbcg - vaccinated mice, whereas increased frequencies of ifn- cells were identified in bcg - vaccinated mice even though statistically insignificant (figure 3(a)). The pie chart of this data clarified the dominance of il-2 cd4 t cells in rbcg - vaccinated mice, while ifn- cd4 t cells dominated the responses of bcg - vaccinated mice (figure 3(b)). Rbcg and bcg - vaccination did not differ in their ability to induce m. tb - specific cd4 t cells producing other combinations of cytokines (p> 0.05). In accordance, we also observed higher integrated mean fluorescence intensities (imfi =% frequency mfi) of il-2 in il-2-producing cd4 t cells, even though it is statistically insignificant (figure 3(c)). In general, rbcg induced higher antigen - specific cytokine responses as compared to bcg (figures 2 and 3), and rbcg486 induced higher antigen - specific ifn- response (figure 2) and comparable frequency of m. tb - specific cd4 t cells expressing il-2 (figure 3). Then, we further compared the protective efficacies of rbcg486, rbcg186, and bcg against m. tb - challenge . As shown in figure 4(a), 5 weeks after challenge all vaccinated mice had a significantly reduced bacillary load in lungs, when compared to the saline - treated mice . Vaccination with bcg and rbcg186 resulted in a comparable reduction in bacillary load (figure 4). However, even though rbcg486 vaccination induced a significantly greater protection when compared to the bcg - vaccinated mice, it showed no difference of protection when compared to the rbcg186-vaccinated mice (figure 4(a)). The bacillary loads in spleens shared the similar pattern as those in lungs, with rbcg486-vaccinated mice having far fewer bacilli when compared to the saline - treated or bcg - vaccinated mice and having comparable bacilli compared to the rbcg186-vaccinated mice (figure 4(b)). Five weeks after challenge, m. tb infection caused severe pathology changes in saline - treated mice, with about 24.3% of the tissue showing extensive multifocal granulomatous infiltration, characterized by numerous foamy macrophages surrounded by inflammatory cells (figure 5). However, all the vaccinated groups of mice had significantly reduced pulmonary granulomatous consolidation compared to the unvaccinated mice (i.e., 13.42% consolidation in bcg - vaccinated group, 7.24% in rbcg186-vaccinated group, and 4.87% in rbcg486-vaccinated group). The rbcg - vaccinated mice showed the mildest pathology, and all of the mice in these two groups had mainly well - preserved alveolar spaces with only a few scattered areas of diffused infiltration (figure 5). Immunohistochemical staining of the lung tissues showed the presence of tnf-, ifn-, and inos in all groups of infected mice and staining was strongest in the granulomatous lesions compared to that in the nongranulomatous areas . Five weeks after infection, a very high level of tnf- was observed in the lungs of saline - treated mice (figure 6(a)); tnf- staining was extensive in necrotic areas within the advanced coalescent granulomas . Vaccination with bcg resulted in the reduced amounts of tnf- expression, even though statistically insignificant . In contrast, rbcg - vaccinated mice, especially rbcg486-vaccinated mice, showed only a little weak staining for tnf- and this was restricted primarily to the granuloma core (figure 6(a)). Similar patterns of ifn- and inos staining were also observed except that there was relatively much weaker staining in the lungs of (r)bcg - vaccinated mice compared to the saline - treated mice (figures 6(b) and 6(c)). Similar pattern of tnf-, ifn-, and inos staining was also observed in the infected spleens of vaccinated mice, with the highest staining in saline - treated mice, moderate staining in bcg - vaccinated mice, and the lowest staining in rbcg - vaccinated mice (figure s2). During the past decades, great efforts have been focused on modifications of the current bcg vaccine to develop new anti - tb vaccine candidates . Some modified rbcg strains, such as rbcg30 and rbcgurec::hly, have been demonstrated to yield improved protection against m. tb infection in experimental animal model compared to the existing bcg vaccine and have entered into clinical trial . Nevertheless, it is promising to keep on optimization of bcg protective immune if two points are being issued . One is the fact that the best immunodominant antigen for tb should be precisely defined . Another is that the expression levels of such antigens should be optimal enough to elicit effective immune responses . Here, we constructed two rbcg strains overexpressing immunodominant chimeric antigen ag856a2 at varying levels depending upon the strengths of the different fura promoters and then compared the cellular immune response and protection in mice induced by these two rbcg strains . One way to improve bcg efficacy is to overexpress mycobacterial immunodominant antigens to induce optimal host immune responses in the life cycle of bcg within host [12, 19]. This kind of strategy reflects that the doses of antigens are one of pivotal factors influencing the protective efficacies of vaccines . Demonstrated that protective efficiency of tb subunit vaccines is highly dependent on the antigen dose . They vaccinated mice with different doses of fusion protein ag85b - tb10.4 which were emulsified in adjuvant ic31, and the higher immune response and protective efficacy were only observed when the antigen was administered in proper doses, and decreasing or increasing of the antigen dose would dramatically dwarf the protection efficacies of the antigens . In our study, the cognate antigen ag856a2 in rbcg186 and rbcg486 was expressed under the control of promoters pfura and pfurama (figure s1). These two promoters, by their nature, were verified to have varied promoter activities, with pfura the lower one and pfurama the higher one, and were consequently used to develop the rbcg strains overexpressing chimeric antigen ag856a2 at different levels, with lower expression in rbcg186 and higher expression in rbcg486 (figure 1). And different ag856a2 antigen loading in rbcgs resulted in differential host immune responses, with the higher antigen - specific effector immune response in the rbcg486-vaccinated mice as validated through in vitro ifn- elispot assay (figure 2). However, we did not observe the significant differences in the qualities of m. tb - specific cd4 t cells coexpressing ifn-, tnf-, and il-2 (figure 3), nor the protection efficacies and lung inflammations, between the two groups of rbcgs - vaccinated mice (figures 4 and 5). Interestingly, subtly higher percent of polyfunctional cd4 t cells (ifn-il-2tnf-) was observed in bcg - vaccinated mice compared to other groups of mice (figure 3(a)); however, the protective efficacy elicited by bcg vaccination is not that effective as rbcgs (figure 4). This contradictory result could be explained with the fact that the lower imfi values of ifn-, il-2, and tnf- in bcg - vaccinated mice were observed (see the case of il-2 in figure 3(c) as representative). Mfi provides one measure of the quality of the immune response since the cells that are more actively producing cytokine stain more brightly, thus the lower imfi values of cytokines reflected poor quality although mildly higher frequency of polyfunctional cd4 t cells was seen in bcg - vaccinated mice, and this further emphasizes that not only the magnitude but also the quality of vaccine - induced t cells responses are critical to guide development of effective immunization strategies . In addition, higher il-2 secretion, both in the levels of percentage and imfi, were seen in the rbcg486-vaccinated mice than that of bcg group; this data support our recent findings that il-2 production in the spleens of vaccinated mice after vaccination can predict vaccine efficacy (kang h, et al . Immunology, 2014; in press). The same explanation might also be used to account for the fact that although the saline - treated mice showed high numbers of cells producing tnf-, the imfi is relative low (data not shown). The quality of t cell response has significant effect on the establishment of protective memory . As with the phenotypic heterogeneous nature of t cells thus, in addition to monitoring exclusively the ifn- response after vaccination, researchers have been focusing on the coexpression of more cytokines at single cell level through flow cytometry technique [24, 25]. The rbcg186 or rbcg486, at least at the time we tested, induced much higher frequencies of il-2 cd4 t cells responding to ppd stimulation in splenocytes compared to the saline - treated or bcg - vaccinated mice after vaccination, which was further confirmed by higher il-2 production when cytokine concentration was measured as imfi value (figure 3). Although il-2 has little direct effector function, it has the ability to expand effector functions of other t cells . In the linear model of differentiation for cd4 th1 cells, il-2 cd4 t cells belong to memory cells and have the potential to differentiate into ifn--producing cells after recalling by the relevant antigens . Thus, rbcg186 and rbcg486, because of the incorporation of chimeric antigen ag856a2, enhance the memory capacity of host to m. tb pathogen . However, we did not detect any differences of cd4 t cells between rbcg186-vaccinated and rbcg486-vaccinated mice . This may attribute to the short vaccination time window we chose, or the real differences lies in other functions of t cells which is beyond the scope of the t cell functions currently tested and may need to be further exploited in the future . Those relevant th1 cytokines (e.g., tnf- and ifn-), in a larger extent, function through activation of macrophages . Tnf- and ifn- synergistically inhibit the growth of m. tb in macrophages through stimulating the production of reactive nitrogen intermediates (rnis) [27, 28]. As for rnis, inos is the vital enzyme involved for the production of rnis [29, 30]. Tnf-, ifn-, and inos give proper containment of m. tb in the early stage . At later stage of infection when inhibition or killing of m. tb is well established, their levels of expression will go down to a reasonable value; otherwise immune - pathological response would happen . Rbcgs, especially rbcg486, induced enhanced protection against m. tb infection in this study (figure 4). Consistent with the protective efficacy, the inflammation responses in the infected lungs alleviated greatly in rbcgs - vaccinated mice after infection (figure 5). When measuring the expression levels of inflammatory molecules, the rbcgs - vaccinated mice also displayed reduced levels of expression which were in accordance with the remissive granulomatous inflammation (figures 5 and 6).
A 6-year - old boy presented with a history of penetrating injury to the upper left eyelid sustained while diving head - first into a pond . On presentation, he could only perceive hand movements very close to his face . Since a computed tomography (ct) scan done at this time did not reveal the presence of any foreign body, the patient was treated for orbital cellulitis . One month later, he returned reporting discharge from the eyelid wound, which had formed a fistula . A repeat ct scan showed an abscess with an empty sinus tract but no distinguishable foreign body [fig . 1]. The patient subsequently developed septic arthritis of the left hip joint, which was treated separately . Subsequent re - evaluation of the patient's left eye revealed a queried perception of light, relative afferent pupillary defect, optic disc pallor, and inferotemporal displacement of the globe . A superonasal quadrant granulation mass was observed from the non - healing wound, but elevation and depression of the globe in adduction were minimally restricted . Re - evaluation of the ct scan showed a single hypodense superomedial lesion corresponding to the previously noted sinus tract . A superomedial orbitotomy resulted in the removal of 4 wooden foreign bodies, each approximately 1.5 inches in length . By post - operative day 5, the patient's edema and proptosis had resolved . At 1-month follow - up, the eyelid wound was completely healed, and left eye visual acuity had improved to 20/120 . Axial (a) and coronal (b) ct of a 6-year - old boy with a history of penetrating injury shows an empty abscess tract without obvious foreign body . Subsequent orbitotomy recovers four wooden fragments (d) a 12-year - old boy presented to an outside hospital with fever, vomiting, and a prominent right eye with a painful, swollen lower lid . He had a history of a penetrating injury sustained while diving head - first into a river 12 days previously . After 1 week of treatment with systemic antibiotics, the patient presented to our institution with continued swelling at the injury site . He had an indurated swelling of the lower lid with a discharging sinus, mechanical ptosis, diplopia, eccentric upward displacement of the globe, and 3-mm of proptosis . Repeat ct scan showed a hypodense body surrounded by secondary granulomatous inflammation [fig . 2]. Post - operatively, the patient showed symptomatic improvement with a reduction in proptosis and a visual acuity of 20/30 in the affected eye [fig . 3]. Coronal (a) and axial (b) ct scans taken one week after initial ct showed no evidence of retained foreign body . Ct now shows a hypodense body surrounded by secondary granulomatous inflammation pre - operative (a) and post - operative (b) photographs of 12-year - old boy with a history of penetrating injury by a 2 inch wooden foreign body (c) sustained while pond - diving a 6-year - old boy presented with a history of penetrating injury to the upper left eyelid sustained while diving head - first into a pond . On presentation, he could only perceive hand movements very close to his face . Since a computed tomography (ct) scan done at this time did not reveal the presence of any foreign body, the patient was treated for orbital cellulitis . One month later, he returned reporting discharge from the eyelid wound, which had formed a fistula . A repeat ct scan showed an abscess with an empty sinus tract but no distinguishable foreign body [fig . 1]. The patient subsequently developed septic arthritis of the left hip joint, which was treated separately . Subsequent re - evaluation of the patient's left eye revealed a queried perception of light, relative afferent pupillary defect, optic disc pallor, and inferotemporal displacement of the globe . A superonasal quadrant granulation mass was observed from the non - healing wound, but elevation and depression of the globe in adduction were minimally restricted . Re - evaluation of the ct scan showed a single hypodense superomedial lesion corresponding to the previously noted sinus tract . A superomedial orbitotomy resulted in the removal of 4 wooden foreign bodies, each approximately 1.5 inches in length . By post - operative day 5, the patient's edema and proptosis had resolved . At 1-month follow - up, the eyelid wound was completely healed, and left eye visual acuity had improved to 20/120 . Axial (a) and coronal (b) ct of a 6-year - old boy with a history of penetrating injury shows an empty abscess tract without obvious foreign body . A 12-year - old boy presented to an outside hospital with fever, vomiting, and a prominent right eye with a painful, swollen lower lid . He had a history of a penetrating injury sustained while diving head - first into a river 12 days previously . After 1 week of treatment with systemic antibiotics, the patient presented to our institution with continued swelling at the injury site . He had an indurated swelling of the lower lid with a discharging sinus, mechanical ptosis, diplopia, eccentric upward displacement of the globe, and 3-mm of proptosis . Repeat ct scan showed a hypodense body surrounded by secondary granulomatous inflammation [fig . 2]. Post - operatively, the patient showed symptomatic improvement with a reduction in proptosis and a visual acuity of 20/30 in the affected eye [fig . 3]. Coronal (a) and axial (b) ct scans taken one week after initial ct showed no evidence of retained foreign body . Ct now shows a hypodense body surrounded by secondary granulomatous inflammation pre - operative (a) and post - operative (b) photographs of 12-year - old boy with a history of penetrating injury by a 2 inch wooden foreign body (c) sustained while pond - diving the injuries sustained in the cases we present are troubling since swimming and diving in rivers, ponds, and other natural bodies of water is a common pastime of children in rural areas worldwide . Penetration injuries by wooden foreign bodies embedded in shallow riverbeds have not been previously recognized as a risk of outdoor swimming and diving . However, in our experience these types of injuries represent a true threat to vision . This danger is compounded by the fact that retained organic foreign bodies are not often immediately apparent on imaging studies, making prompt removal difficult . Previous studies report that wood, depending on its level of hydration, can be isodense to either air or orbital fat on ct scan . In our two cases, ct scans taken within a few days of injury did not show a distinct foreign body, but only edema and signs of inflammation . Only after the foreign body became infected did a discharging sinus cavity form and become evident on ct scans . While nasr et al . Found magnetic resonance imaging (mri) to be slightly better at identifying organic foreign bodies, other studies recommend using ct scans for the initial identification of wooden foreign bodies, so the best imaging method remains controversial . As in our case, serial imaging in patients with a high - risk mechanism of injury may be a useful non - invasive method of identifying a retained wooden orbital foreign body . Unfortunately, orbital exploration does not always identify the presence of a wooden orbital foreign body . As in our two cases, these fragments are often not lodged in a tissue, but embedded within orbital fat . They are thus mobile within the orbital cavity until becoming enclosed within a discharging sinus . Thus, even after a negative orbital exploration, it is important to keep a high index of suspicion for a retained wooden orbital foreign body . In summary, ophthalmologists attending to cases of penetrating orbital injuries sustained during river and pond diving should maintain a high index of suspicion for a retained wooden orbital foreign body and realize that neither orbital exploration nor imaging may be sufficient for diagnosis . Consideration should be given to serial imaging in the event of a negative initial scan . A thorough history as to the mechanism of injury, a meticulous exam, and frequent follow - up visits are also of utmost importance.
Postcataract surgery endophthalmitis is a relatively uncommon but devastating complication of modern cataract surgery, and there is not complete consensus regarding the ideal practice patterns for prophylaxis . The european society of cataract and refractive surgeons (escrs) prospective multi - center interventional trial (and many other case control, cross - sectional, and longitudinal studies) supported the hypothesis that the use of icc at the conclusion of cataract surgery reduces the incidence of postoperative endophthalmitis . It is estimated that two to four cases of endophthalmitis per 1000 surgeries can be avoided if surgeons adopt the use of icc, and the absence of an icc prophylactic regimen at a dose of 1 mg/0.1 ml was associated with an almost 5-fold increase in the risk of postoperative endophthalmitis . The injection is expedient, painless, and achieves high antibiotic concentrations in the immediate postoperative period . In addition, intracameral injection may be particularly effective in patients who sustain posterior capsule rupture and would otherwise incur an increased the risk of endophthalmitis . When considering the implementation of any intervention, cost - effectiveness is another important variable . Estimated the cost - effectiveness ratio for icc to be $1403 per case of postoperative endophthalmitis prevented . Many commonly used topical antibiotics are not cost - effective compared with icc, even under optimistic assumptions about their efficacy . Another economic analysis comparing different prophylaxis regimens concluded that icc provided the best cost - effectiveness ratio . Therefore, the decision to adopt this therapy is supported by studies demonstrating efficacy and cost - effectiveness . The final variable that must be included when one weakness inherent in any meticulously performed clinical trial is that the results may not be generalizable to a more diverse patient population and varying scenarios of care . Most clinical trials make great effort to reduce the risk of enrollment in the study, and therefore the safety profile reported may be a best - case scenario . The purpose of this review is to provide an evidence - based review and synthesis of the literature regarding the risks of icc when administered for the prevention of postcataract surgery endophthalmitis, thereby informing decisions regarding best practices for cataract surgery . The categories of risk to be reviewed are: risk of anaphylaxis, especially in penicillin or cephalosporin allergic patientsrisk of toxicity at routine clinical dosesrisk of toxicity at increased doses due to compounding errorsidiosyncratic reactions, including toxic anterior segment syndrome (tass). Risk of anaphylaxis, especially in penicillin or cephalosporin allergic patients risk of toxicity at routine clinical doses risk of toxicity at increased doses due to compounding errors idiosyncratic reactions, including toxic anterior segment syndrome (tass). We performed a systematic review of the existing scientific literature using pubmed and google scholar . The key words used were cefuroxime, endophthalmitis and cataract surgeries . All articles were read in full by both authors with the exception of two that were available only as abstracts . The highest level of evidence for each aspect of the intervention was assigned utilizing the oxford center for evidence - based medicine guidelines . Cross - reactivity between penicillins and most second- and all third- and fourth - generation cephalosporins is negligible . It is generally considered safe to administer a cephalosporin with a side chain that is structurally dissimilar to that of penicillin . In patients with a documented ige - mediated reaction to penicillin, use of cephalosporins with a similar side chain however, cephalosporins with different side chains (such as cefuroxime) may be given . It should be noted that the escrs study excluded patients with penicillin or cephalosporin allergy, as did several other large studies . Icc injection during cataract surgery was well tolerated in a prospective study of forty penicillin - allergic patients with a negative preoperative cefuroxime skin test . The use of icc in patients with penicillin allergy was explored between 2004 and 2012 in a case control registry study; the control group was the cohort of patients undergoing cataract surgery under a hospital policy of excluding patients with self - reported penicillin allergy . After a critical review of the literature and pilot study in 817 patients with reported penicillin allergy, this policy was altered, and all patients without a specific history of cephalosporin anaphylactic reaction were administered icc . Out of 13,592 subsequent cataract surgeries, there were no reported cases of anaphylaxis or allergic reaction . This study is limited by the registry design, which may underreport or miscategorize adverse events . A similar longitudinal observational study by shorstein et al . Reported a decreasing endophthalmitis rate after instituting a standard icc protocol; there were no reports of anaphylaxis or allergic reaction among 12,609 surgeries, but some patients received other intracameral antibiotics, and it is unclear if penicillin - allergic patients routinely received cefuroxime or other antibiotics such as moxifloxacin or vancomycin . Control study comparing the endophthalmitis rates before and after instituting a standard icc regimen; patients with cefuroxime allergy were excluded, but penicillin - allergic patients received icc . There were no cases of anaphylaxis among 2289 patients receiving icc, but the prevalence of penicillin allergy was not reported . There are two case reports of an anaphylactic reaction in penicillin or cephalosporin allergic patients who received icc . The number of patients from these centers that had received icc were not reported, preventing an estimation of the incidence of this complication . Adverse effects that have rarely been reported with routine clinical doses include serous macular detachment, cystoid macular edema (cme), increased central foveal thickness, decreased best - corrected distance visual acuity, anterior chamber inflammation, and vitritis . However, a prospective study found that icc at the standard dose of 1 mg/0.1 ml did not have a statistically significant effect on postoperative macular thickness compared with nonadministration of intracameral antibiotic; although, this study was under - powered to detect rare events . The escrs study and other longitudinal cohort studies were not designed to assess safety or adverse events as primary endpoints, but a large number of patients enrolled without a reported increase in these adverse events suggests that they are not associated, are masked by confounding factors, or are exceedingly rare . A lingering concern for some ophthalmologists is the risk of ocular toxicity attributable to inadvertent exposure to elevated concentrations (typically due to compounding errors). High doses of cefuroxime are associated with anterior and posterior segment inflammation with fibrin formation, corneal edema, elevated intraocular pressure, serous macular detachment, cme, hemorrhagic retinal infarction, and reduced rod photoreceptor cell function by electroretinography . Table 1 summarizes the reported complications related to inadvertent administration of increased concentrations or volumes of cefuroxime . A limitation in assigning risk based on studies such as this is the lack of incidence data; it is unknown how many patients have received increased doses of icc, and therefore, the rate of these adverse events is unknown . The incidence rate of compounding errors for icc is also unknown and is presumably highly dependent on local factors such as the mechanisms of medication preparation and quality control measures . Adverse events attributed to cefuroxime compounding errors tass after cataract surgery has been reported in association with the intracameral use of cefuroxime . Reported an ongoing cluster of tass cases at a single center, with a resolution of the outbreak after discontinuing icc in favor of intracameral moxifloxacin . As expected, there was no rechallenge in affected patients to determine conclusively if cefuroxime or other factor contributed to tass . There was also no reported analysis of cefuroxime concentration to ascertain the potential role of dilution errors or other factors related to preparation . No other studies were found that studied the relationship of cefuroxime to tass, or the effects of specific interventions to reduce the incidence of tass related to cefuroxime . Cross - reactivity between penicillins and most second- and all third- and fourth - generation cephalosporins is negligible . It is generally considered safe to administer a cephalosporin with a side chain that is structurally dissimilar to that of penicillin . In patients with a documented ige - mediated reaction to penicillin, use of cephalosporins with a similar side chain however, cephalosporins with different side chains (such as cefuroxime) may be given . It should be noted that the escrs study excluded patients with penicillin or cephalosporin allergy, as did several other large studies . Icc injection during cataract surgery was well tolerated in a prospective study of forty penicillin - allergic patients with a negative preoperative cefuroxime skin test . The use of icc in patients with penicillin allergy was explored between 2004 and 2012 in a case control registry study; the control group was the cohort of patients undergoing cataract surgery under a hospital policy of excluding patients with self - reported penicillin allergy . After a critical review of the literature and pilot study in 817 patients with reported penicillin allergy, this policy was altered, and all patients without a specific history of cephalosporin anaphylactic reaction were administered icc . Out of 13,592 subsequent cataract surgeries, there were no reported cases of anaphylaxis or allergic reaction . This study is limited by the registry design, which may underreport or miscategorize adverse events . A similar longitudinal observational study by shorstein et al . Reported a decreasing endophthalmitis rate after instituting a standard icc protocol; there were no reports of anaphylaxis or allergic reaction among 12,609 surgeries, but some patients received other intracameral antibiotics, and it is unclear if penicillin - allergic patients routinely received cefuroxime or other antibiotics such as moxifloxacin or vancomycin . Control study comparing the endophthalmitis rates before and after instituting a standard icc regimen; patients with cefuroxime allergy were excluded, but penicillin - allergic patients received icc . There were no cases of anaphylaxis among 2289 patients receiving icc, but the prevalence of penicillin allergy was not reported . There are two case reports of an anaphylactic reaction in penicillin or cephalosporin allergic patients who received icc . The number of patients from these centers that had received icc were not reported, preventing an estimation of the incidence of this complication . Adverse effects that have rarely been reported with routine clinical doses include serous macular detachment, cystoid macular edema (cme), increased central foveal thickness, decreased best - corrected distance visual acuity, anterior chamber inflammation, and vitritis . However, a prospective study found that icc at the standard dose of 1 mg/0.1 ml did not have a statistically significant effect on postoperative macular thickness compared with nonadministration of intracameral antibiotic; although, this study was under - powered to detect rare events . The escrs study and other longitudinal cohort studies were not designed to assess safety or adverse events as primary endpoints, but a large number of patients enrolled without a reported increase in these adverse events suggests that they are not associated, are masked by confounding factors, or are exceedingly rare . A lingering concern for some ophthalmologists is the risk of ocular toxicity attributable to inadvertent exposure to elevated concentrations (typically due to compounding errors). High doses of cefuroxime are associated with anterior and posterior segment inflammation with fibrin formation, corneal edema, elevated intraocular pressure, serous macular detachment, cme, hemorrhagic retinal infarction, and reduced rod photoreceptor cell function by electroretinography . Table 1 summarizes the reported complications related to inadvertent administration of increased concentrations or volumes of cefuroxime . A limitation in assigning risk based on studies such as this is the lack of incidence data; it is unknown how many patients have received increased doses of icc, and therefore, the rate of these adverse events is unknown . The incidence rate of compounding errors for icc is also unknown and is presumably highly dependent on local factors such as the mechanisms of medication preparation and quality control measures . Tass after cataract surgery has been reported in association with the intracameral use of cefuroxime . Reported an ongoing cluster of tass cases at a single center, with a resolution of the outbreak after discontinuing icc in favor of intracameral moxifloxacin . As expected, there was no rechallenge in affected patients to determine conclusively if cefuroxime or other factor contributed to tass . There was also no reported analysis of cefuroxime concentration to ascertain the potential role of dilution errors or other factors related to preparation . No other studies were found that studied the relationship of cefuroxime to tass, or the effects of specific interventions to reduce the incidence of tass related to cefuroxime . The efficacy of icc as a prophylaxis for postcataract surgery endophthalmitis has been well established, but concerns regarding the risks of this intervention remain . The consensus of the literature from systemic administration of antibiotics is that the risk of cross - reactivity between second - generation cephalosporins (such as cefuroxime) and penicillin is very low . Our review of the literature supports a low rate of anaphylactic reactions, even among penicillin - allergic patients . A postal survey conducted among consultant ophthalmic surgeons working in the national health service ophthalmic departments in england revealed that of 262 consultants, 103 (37%) used cefuroxime in patients allergic to penicillin . We propose that an acceptable practice pattern would be to consider icc in all cataract surgery patients, including those with a history of penicillin allergy; administration to those patients with cephalosporin allergy may be considered, but skin testing may be indicated to identify those patients that are at increased risk of anaphylaxis and should not receive icc . Alternative intracameral antibiotics, such as vancomycin and moxifloxacin, may reduce the risk of anaphylaxis in cefuroxime - allergic patients, but the efficacy of those interventions has been less well established . The latest survey of members of the american society of cataract and refractive surgeons revealed that 30% of the united states ophthalmologists were utilizing intracameral antibiotics, compared to 70% of european respondents . However, many us cataract surgeons believe that intra - cameral antibiotics are unnecessary, based on concerns about the methodology of the escrs and other studies, or that the absolute benefits do not outweigh the risks of dilution errors and toxicity . Not surprisingly, a clinical study demonstrated that the mathematical accuracy of a dilution protocol does not ensure dosage accuracy in a real - world clinical scenario . The authors suggest that a commercial preparation would likely reduce the risk of dilution errors, but commercial preparations of intracameral antibiotic agents may not be financially viable in all health care environments . Implementation of icc should include measures to audit and provide quality assurance of the cefuroxime dilution protocol . Half of the risk - benefit equation of icc has been solved to the satisfaction of most clinicians; the efficacy is clear . The remaining barriers to more widespread adoption include concerns about risks such as anaphylaxis, dilution errors, and toxicity (especially with noncommercial preparation) and finally, the additional costs of this therapy . Since endophthalmitis is a rare event, even a slight increase in the risk of prophylactic therapy may negate the potential benefits . Our review and synthesis of the literature regarding the risks of cefuroxime therapy support the following recommendations: cefuroxime may be used safely in patients with penicillin allergy (level 2a evidence)efforts to reduce the risk of dilution errors may include the use of a commercially prepared product, or strict quality assurance measures (level 4 evidence)routine measures to reduce tass should also apply to the use of cefuroxime (level 5 evidence); even though, there is no proven association . Cefuroxime may be used safely in patients with penicillin allergy (level 2a evidence) efforts to reduce the risk of dilution errors may include the use of a commercially prepared product, or strict quality assurance measures (level 4 evidence) routine measures to reduce tass should also apply to the use of cefuroxime (level 5 evidence); even though, there is no proven association . Future studies may further define the cost - effectiveness of icc, and continued efforts to reduce the risk attributable to dilution errors are indicated . Finally, comparative studies of the efficacy and safety of cefuroxime compared to other intracameral antibiotics are needed to help define the optimal endophthalmitis prophylaxis regimen for our patients undergoing cataract surgery.
Thyroid cancer is the most common endocrine neoplasia and accounts for 1% of all human cancers (1). Medullary thyroid carcinoma (mtc) is a rare neoplasm of the calcitonin - secreting thyroid cells and accounts for 510% of all thyroid cancers . Mtc can occurs as sporadically (75%) or as a part of the autosomal dominantly inherited forms (25%), (2, 3). The inherited type of mtc can be divided in three clinically distinct forms: multiple endocrine neoplasia type2a (men2a), multiple endocrine neoplasia type2b (men2b), and familial mtc (fmtc) (4). Men2a is defined by mtc, bilateral pheochromocytoma, and multiple tumors of the parathyroid glands (parathyroid adenomas, primary hyperparathyroidism or hpt), within a single patient or family . Men2b is characterized by the early development of an aggressive form of mtc in all affected individuals (typically during the first year of life), pheochromocytoma, the absence of hyperparathyroidism, and visible physical stigmata such as raised bumps on the lips and tongue, ganglioneuromas of the intestine, marfanoid body habitus with skeletal deformations . Fmtc is characterized by a strong predisposition to mtc in families with a very low incidence of other endocrinopathies related to men2 (1, 5). The molecular pathology of inherited mtcs is constitutive of ret (rearranged during transfection) proto - oncogene . The ret gene is located on chromosome 10q11.2, consisting of 21 exons and encodes a tyrosine kinase receptor . This complex plays a critical role in cell proliferation and differentiation of tissues derived from neural crest cells, such as c - cells of thyroid gland (5, 6). Ret is a tyrosine kinase protein and includes three domains; a large extracellular domain containing a cysteine - rich region and a series of cadherin homology domains, a trans - membrane domain, and an intracellular tyrosine kinase domain, required for ret phosphorylation and downstream signaling (5, 7). This protein is activated by binding a soluble ligand of the glial cell - line - derived neurotrophic factor (gdnf) family and requires a co - receptor of the gdnf family receptors (gfr) (8). The majority of men2a families are associated with one of the six point mutations in conserved cysteine residues in exon10 (codons 609, 611, 618, and 620) or exon11 (codons 630, 634) in the extracellular domain of the ret (2, 5, 810, 12). In fmtc cases, ret mutations are mainly detected in the same six codons as for men2a and also in exon13 (codon 768), or in exon15 (codon 891), in the intracellular region of ret (5, 8, 12). Mutation of codon 918 in ret exon16 in the tyrosine kinase domain, accounts for the vast majority of patients with men2b (5, 8). For the first time, ret polymorphisms g691s and s904s were described in 1994 (13, 14). Recent studies have found g691s in exon11, s904s single neucleotide polymorphisms in exon15 of the ret pathway to be associated with the risk of developing sporadic mtc (smtc) (7). It seems, two of these ret snps (g691s and s904s) may modify the age at onset of mtc tumor in family members (5, 15) but fabienne lesueur et al . Have rejected this hypothesis (7). In the present study, we report the frequency of g691s / s904s haplotype in iranian mtc patients and their relatives . Over the last ten years, one hundred ninety mtc patients, were referred to cellular and molecular research center of shahid beheshti university of medical sciences in order to ret genetic screening . This study has been approved by the ethics committee of cellular and molecular research center, research institute for endocrine sciences, shahid beheshti university of medical sciences . A questionnaire was used to collect information about age, sex, and history of neoplasia . If any mutation was found in patients, their first - degree relatives were invited for screening . 121 first - degree relatives from 31 index cases comprised 57 males and 64 females, with meansd age 29.0916.34 years . Genomic dna was extracted from peripheral blood leucocytes using standard salting out / proteinase k method and stored at -20c . For identification of ret variant 691 (rs1799939, codon 691 of exon 11, ggt> agt, gly> ser) and 904 (rs1800863, codon 904 of exon 15, tcc> tcg, ser> ser,), dna samples were amplified using the polymerase chain reaction (pcr) and the specific oligonucleotides primers (table1). The exon11 running profile was constituted initial denaturation at 96 c for 1min and, followed by 30 cycles with 96 c for 45s, annealing at 60 c for 1min dependent on the primer sequences, and extension at 72c for 45s, with a final extension at 72 c for 10 min . Sequences of oligonucleotide primers used for pcr amplification of exons 11, 15 of the ret pcr reaction for exon 15 was constituted initial denaturation at 94c for 3min and, followed by 35 cycles of 94c for 30s, annealing starting at 56c for 1min, 72c for 1min, and a final extension at 72c for 10min . The amplimers were confirmed for the presence or absence of mutations by direct dna sequencing method (abi 3100 genetic analyzer and big dye terminator v3.1 cycle sequencing kit, applied biosystems, california, usa). To detect the ret mutations and snps, sequences were analyzed by chromas 2.33 software and were also compared with the reference ret gene sequence using web based tool ncbi blast . Over the last ten years, one hundred ninety mtc patients, were referred to cellular and molecular research center of shahid beheshti university of medical sciences in order to ret genetic screening . This study has been approved by the ethics committee of cellular and molecular research center, research institute for endocrine sciences, shahid beheshti university of medical sciences . A questionnaire was used to collect information about age, sex, and history of neoplasia . If any mutation was found in patients, their first - degree relatives were invited for screening . 121 first - degree relatives from 31 index cases comprised 57 males and 64 females, with meansd age 29.0916.34 years . Genomic dna was extracted from peripheral blood leucocytes using standard salting out / proteinase k method and stored at -20c . For identification of ret variant 691 (rs1799939, codon 691 of exon 11, ggt> agt, gly> ser) and 904 (rs1800863, codon 904 of exon 15, tcc> tcg, ser> ser,), dna samples were amplified using the polymerase chain reaction (pcr) and the specific oligonucleotides primers (table1). The exon11 running profile was constituted initial denaturation at 96 c for 1min and, followed by 30 cycles with 96 c for 45s, annealing at 60 c for 1min dependent on the primer sequences, and extension at 72c for 45s, with a final extension at 72 c for 10 min . Sequences of oligonucleotide primers used for pcr amplification of exons 11, 15 of the ret pcr reaction for exon 15 was constituted initial denaturation at 94c for 3min and, followed by 35 cycles of 94c for 30s, annealing starting at 56c for 1min, 72c for 1min, and a final extension at 72c for 10min . The amplimers were confirmed for the presence or absence of mutations by direct dna sequencing method (abi 3100 genetic analyzer and big dye terminator v3.1 cycle sequencing kit, applied biosystems, california, usa). To detect the ret mutations and snps, sequences were analyzed by chromas 2.33 software and were also compared with the reference ret gene sequence using web based tool ncbi blast . Among 311 participants, 18 dna samples did not have enough quality, so were excluded from the study . We analyzed germline dna mutations of ret gene in 181 iranian mtc patients and 112 relatives, with a total of 293 members . The patients included, 33 fmtc, 6 men2a, 2 men2b, 1 pheochromocytoma and 145 apparently smtc cases (table 2). The frequency of females in our population was a little higher than males, but this ratio was not significant (160 vs. 133, p> 0.05). Specifically, we found 2 germline polymorphisms of ret gene at codon691 (ggt> agt, exon 11, rs1799939) that causes glycine to serine amino acid substitution, and codon 904 (tcc> tcg, exon 15, rs1800863) that does not lead to an ami - no acid alteration . Furthermore, the allele frequencies of each of these snps were similar in all patients and relatives (21.5% and 10.75%, respectively). The frequency of patients with medullary thyroid carcinoma as the g691s and s904s variants were in complete linkage disequilibrium, so the results were grouped together and referred to as g691s / s904s haplotype (fig.1). The analysis of g691s / s904s ret gene haplotype showed that 104 of 181 (57.45%) mtc patients and 55 of 112 (49.1%) relatives had this haplotype . In particular, 82 mtcs and 47 relatives were heterozygous and 22 mtcs and 8 relatives were homozygous for the g691s / s904s haplotype (table 3). There was not significant correlation between age of diagnosis and the presence of g691s / s904s haplotype . The frequency of g691s and s904s variants in the population the frequency distribution of g691s and s904s variants among mtc patients and their relatives in this study, we found two variants of ret gene, g691s (ggt> agt, exon 11, rs1799939) and s904s (tcc> tcg, exon 15, rs1800863) that are cosegregated together as a haplotype, suggesting that these polymorphisms are in linkage disequilibrium with each other . The assessment of the occurrence of two polymorphic changes g691s, and s904s in our population is similar to the study performed by lucieli ceolin et al . Unfortunately, in our study we did not have control group to compared with healthy population, but some studies have demonstrated that the ret variant g691s is more frequent in mtc patients than in the general population (16, 17). It has also been postulated that such variants are relatively common in the population which may confer a much higher attributable risk in the general population than mutations in high penetrance cancer susceptibility genes (5, 11). Some studies support our data that both g691s and s904s snps are associated with sporadic mtc and men2a (5, 7, 18). Two studies have shown that this haplotype may modify the age at onset of mtc tumor in family members (5, 15), however, this hypothesis is controversial (7). The ret - g691s polymorphism has been suggested as a genetic modifier in men2a (15). Overall, these data strengthen the potential role of the ret - g691s polymorphism in mtc . One of the probable mechanisms is that the g691s polymorphism seems to enhance ret on - cogenicity of other distinct mutations affecting ret (1820), on the other hands, bases exchange in the dna molecule could create a new alternative splicing site, leading to the synthesis of a truncated protein, erroneous ligand binding, micro - rna binding, change of structure and mrna stability as well as a number of copies, and also the change in the secondary structure of proteins (16). The ret gene mutations are a critical factor in patient management, e.g. To decide on total or partial thyroidectomy or the age at which the child should be subject to surgery (6). Ret gene mutations are very important in mtc patient management, especially in decision making for total or partial thyroidectomy or the age at which the child should be subject to surgery . In the case of inherited mutations, those who test negative for germline mutations will be reassured; those who test positive for germline mutations will be screened for calcitonin levels and will be offered a prophylactic thyroidectomy depending on the complete clinical and biochemical picture . This data showed the frequency of g691s / s904s haplotype among iranian mtc patients and their family, for the first time . Even though g691s / s904s haplotype are not considered as oncogenic mutations at this time, its functional role should be investigated . Further researches on this issue can contribute toward clarifying the prevalence of this haplotype and its probable modifying effect on the phenotypic characteristic of mtc . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.
After initial establishment and derivation of human embryonic stem cells (hesc; thomson et al ., 1998; reubinoff et al ., 2000) first the infection free status of the donors has to be addressed, in europe couples are tested before any fertility treatment is offered, but the cells themselves have to be tested, too (hovatta, 2011). Second, optimized good manufacturing practice (gmp) compliant systems must be implemented for derivation, scaling - up, banking of cells, and their corresponding quality assurance controls (unger et al ., 2008; ausubel et al ., 2011). The culture systems currently encounter the problem of suboptimal quality of xeno - free culture constituents . Thus strategies are needed to overcome this difficulty (sidhu et al ., 2008). Steps have been taken; initially, hesc were grown on irradiated mouse feeders, later human fore - skin fibroblast were used (hovatta et al ., 2003), now we can use gmp compliant coating substrates specially designed for hesc growth (rodin et al ., steps were also taken for the generation defined xeno - free gmp compliant medium for derivation and for expansion (ludwig et al ., 2006; the potential of somatic cell reprogramming via expression of specific transcription factors and thus the generation of hesc induced pluripotent stem cells (hipsc; takahashi and yamanaka, 2006; takahashi et al ., 2007) has the advantage that they could be generated from the recipient patients own cells . There is no deep understanding of the effects that the reprogramming events have; for instance on extracellular signaling (okita et al ., 2011), and the way that this could lead to immune reaction . Hence fast reactivity is already present in healthy individuals for controlling any rapidly amplifying cells (dhodapkar et al ., 2010). Un - silenced expression of the reprogramming factor oct-4 might then cause undesired immunoreactivity on the transplanted cells . Immunoreactivity toward graft - derived hipsc of the same genetic background was also shown in animal models (zhao et al ., 2011). For successful reprogramming of somatic cells, many epigenetic changes must occur in an adequate manner . Dna methylation changes during reprogramming must occur in important developmental and oncogenic regions, which increases the oncogenic risk of the reprogrammed cells (doi et al . There is an additional risk for abnormalities and high tumorigenic potential, especially if c - myc is used as one of the transcription factors (okita et al ., 2007). Also, genetic and epigenetic stability and large - scale genomic rearrangements after reprogramming and subsequent culture (kim et al ., 2010; gore et al ., 2011; hussein et al ., 2011; lister et al ., 2011) it is also important to address the safety of long - term culture, as shown recently; the occurrence of chromosomal rearrangements in long - term culture of 125 hesc and 11 hipsc (amps et al ., 2011). There is a consensus that undifferentiated pluripotent stem cells (psc) will not be used directly in any clinical transplantations procedure, but instead their psc derived differentiated cells recently, results using hesc derived dopaminergic neurons have shown correct phenotype differentiation and grafting potential given by no tumor formation, maintenance of the grafted cells, and functional recovery in parkinsonian animal models in mice, rats, and monkeys (kriks et al ., 2011). The protocols designed for this cell replacement assay were optimal regarding the phenotype, quantity of the cells, functionality, and immunological properties . Integration of transplanted cells was achieved when single cells were transplanted, the use of proper biodegradable scaffolds must also be considered . In addition to this initial report regarding the neural lineage, differentiation protocols for other cell types are needed . Even if transplantation in animal models is successful, it is important to generate safety strategies before clinical trials to appropriately remove undifferentiated psc from their psc derived therapeutic cells . Strategies such as inserting suicide gene (drobyski et al ., 2003; uchibori et al ., 2009) might have controversial outcomes under clinical trials given their safety (yi et al . Alternatively, strategies such as removal of undifferentiated cells using antibodies might be safer (tang et al ., 2011). As discussed earlier, an optimal engraftment and cell replacement strategy should account for a minimal immune reaction in the recipient . This immune reaction occurs because the immune system of the recipient recognizes the grafted cells as foreign material or mismatched cellular components and thus generates a cascade of events that ultimately results in destruction and rejection of the grafted cells . This destruction can also compromise the recipient s immune status (petersen et al ., 1975). Immunoreactivity toward the graft is mainly caused by t cell response toward unmatched major histocompatibility complex (mhc); in humans called human leukocyte antigen (hla). If the profile is unmatched, it will result in rejection (lechler et al ., 2005). This rejection can occur via direct allorecognition of the donor antigen presenting cells (apc) or via indirect recognition of apoptotic cells ingested by the recipients apc, in both cases apcs presenting unmatched mhcs (walsh et al ., 2004). Several groups have studied mhc profiles of hesc and their differentiated cells (swijnenburg et al ., 2008; pearl et al ., 2011). Findings are that undifferentiated cells express mhc i antigens, though at low levels compared with somatic cells; but they do not express mhc ii molecules (drukker et al ., 2002, 2006; li et al ., 2004). During in vitro differentiation toward germ lineages, embryoid body (eb) formation, or teratoma formation mhc i expression increases dramatically (drukker and benvenisty, 2004). Also culture methods of hesc can change antigen expression levels (rajala et al ., 2010). Careful selection of culture conditions, both for the undifferentiated hesc and for their differentiated derivatives is needed . Human embryonic stem cells adopts the expression of non - human cell surface markers if exposed to such substances during culture (martin et al ., 2005; hisamatsu - sakamoto et al ., 2008) hence, optimal culture conditions must be xeno - free from the initial derivation and onward . These culture conditions must be carefully analyzed and scientific consensus must be achieved in order to raise current methodologies . Challenges with the immunoreactivity of the transplantable cells could be addressed by rigorous immunosuppressive treatments . Unfortunately, this is not desired, since there is a clear correlation between the length and intensity of exposure to immunosuppressive therapy and post - transplant risk of malignancy and tumor aggressiveness (gutierrez - dalmau and campistol, 2007). An interesting solution is costimulatory blockage of t cell response (grinnemo et al ., 2006, 2008; swijnenburg et al ., 2008; pearl et al ., 2011 . This immunosuppression strategy will generate tolerance to the grafted cells and thus increase graft survival; initial pharmaceutical agents have been developed and pending clinical applications to the fda are to give in the near future more information . In this mini - review we highlighted the most important areas to be considered under a cell replacement therapy . The possibility of using hipsc derived therapeutic cells in cell replacement therapies requires still long - term studies in non - human animal models addressing the questions of immunogenicity, epigenetic and genetic stability of these cells, and the optimized differentiation of the cells . The importance of profiling immunogenic markers as part of the stem - ness characterization and profiling of cells allocated in stem cell banks must be consider . Such information has to be well protected so that it will not be lost in any given situation . Adequate culture conditions, supporting correct immunogenicity of the cells under a transplantation assay is also required . Next, the management of immunosuppression schemes must aim to a minimal time influencing the immunological status of the recipient . From all the information obtained, these profiles can then be used in combination with methodologies focused at monitoring the status of the transplanted cells . In a given scenario that undesired cells persist in the transplant, adequate counteracting actions have immediately to be taken . Such possibilities have to be tested and the removal of undesired effects confirmed before starting cell transplantations . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
To report two cases of femtosecond laser - assisted small incision deep lamellar endothelial keratoplasty (dlek) for patients with corneal endothelial decompensation by fuchs dystrophy and glaucoma irvine, ca) with 15 khz of repetition rate, was used for a 9.5 mm diameter by 400 m thickness donor corneal lamellar dissection . In case 1, the graft was clear and compact without interface haze, orbscan showed smooth and regular corneal surface, specular microscopy was unremarkable without sign of corneal endothelial damage, and optical coherence tomography showed uniform graft well attached to recipient stroma with minimal interface reflection at 2 months postoperation . In case 2, the graft was clear and compact with minimal interface haze at 1 month postoperation . Femtosecond laser - assisted small incision dlek was safe and technically feasible in our cases; however, further evaluation is required to determine long - term effects . The donor corneoscleral button was mounted in a dedicated artificial anterior chamber (bausch & lomb, st . This was to assure a donor corneal thickness between 500 and 565 m, in order to approximate a graft thickness of 100 to 165 m . The femtosecond laser (intralase; intralase corp ., irvine, ca) was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness . Femtosecond laser parameter settings were as follows: raster energy 3.6 j, spot separation 10 m, line separation 10 m, side cut energy 3.8 j, spot size 2.4 m, and repetition rate 15 khz . After laser application, stromal and side cut adhesions were fully released and the anterior corneal flap was fully lifted . The donor corneoscleral button was then placed in medium and transported to the operating room, where the anterior flap was excised with scissors and an 8.0 mm trephine (katena products, inc ., denville, nj) was used to punch out the posterior donor corneoscleral button . The recipient corneal lamellar dissection and graft transplantation was performed as previously described.5,6 briefly, following an 8.0 mm epithelial marking and 5.0 mm scleral incision, a deep lamellar pocket was created to approximately 75 - 85% of corneal thickness . The posterior recipient disc was then excised with cindy scissors (bausch & lomb) using the 8.0 mm epithelial mark as a template . The excised posterior recipient disc was removed from the lamellar pocket and spread over recipient corneal epithelium to verify size . An 8.0 mm pre - prepared posterior donor disc was then folded, endothelial side inside, with a layer of viscoelastic (healon; pharmacia, peapack, nj) coating the endothelium and inserted into the anterior chamber through the small incision using forceps . The folded donor disc was opened and attached to the recipient stromal bed by injecting air underneath the graft . The scleral wound was closed with 2 or 3 interrupted 10 - 0 nylon sutures and air was replaced with balanced salt solution to normalize intraocular pressure (iop). Femtosecond laser - assisted small incision dlek was performed in the right eye of a 73-year - old male with fuchs dystrophy (fig . 1). Preoperative uncorrected visual acuity (ucva) and best spectacle corrected visual acuity (bscva) was 20/100 and 20/80 respectively and manifest refraction was + 0.75 -1.7530 . Donor corneoscleral button lamellar dissection was performed using intralase femtosecond laser (intralase corp . ), set at a depth of 400 m and flap diameter of 9.5 mm . Ucva and bscva was 20/150 and 20/100 respectively and manifest refraction was + 2.75 -1.0050 . Corneal topography showed smooth and regular corneal surface and specular microscopy was unremarkable without sign of corneal endothelial damage (fig . Optical coherence tomography (oct) findings showed compact and uniform graft, well attached to the recipient stromal bed with minimal interface reflection (fig . 3). Femtosecond laser - assisted small incision dlek was performed in the left eye of a 94-year - old female with corneal edema, glaucoma, and dry type age - related macular degeneration (armd). Her preoperative ucva and bscva was 20/400 and 20/200 respectively and her manifest refraction was -1.00 -1.7595 . Iop was well controlled with topical medication (cosopt; merck & co., whitehouse station, nj). Postoperation, the graft was clear and compact, iop was 19 mmhg, and ucva was 20/400 . At one month, the graft was clear and compact with minimal interface haze; iop measured 31 mmhg . Anti - glaucoma medication (alphagan; allergan, irvine, ca) was added and topical steroid tapered . We were not able to perform further examinations due to follow - up loss at two months postoperation . All methods adhered to the declaration of helsinki of the world medical association for research involving human subjects . The donor corneoscleral button was mounted in a dedicated artificial anterior chamber (bausch & lomb, st . This was to assure a donor corneal thickness between 500 and 565 m, in order to approximate a graft thickness of 100 to 165 m . The femtosecond laser (intralase; intralase corp ., irvine, ca) was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness . Femtosecond laser parameter settings were as follows: raster energy 3.6 j, spot separation 10 m, line separation 10 m, side cut energy 3.8 j, spot size 2.4 m, and repetition rate 15 khz . After laser application, stromal and side cut adhesions were fully released and the anterior corneal flap was fully lifted . The donor corneoscleral button was then placed in medium and transported to the operating room, where the anterior flap was excised with scissors and an 8.0 mm trephine (katena products, inc ., denville, nj) was used to punch out the posterior donor corneoscleral button . The recipient corneal lamellar dissection and graft transplantation was performed as previously described.5,6 briefly, following an 8.0 mm epithelial marking and 5.0 mm scleral incision, a deep lamellar pocket was created to approximately 75 - 85% of corneal thickness . The posterior recipient disc was then excised with cindy scissors (bausch & lomb) using the 8.0 mm epithelial mark as a template . The excised posterior recipient disc was removed from the lamellar pocket and spread over recipient corneal epithelium to verify size . An 8.0 mm pre - prepared posterior donor disc was then folded, endothelial side inside, with a layer of viscoelastic (healon; pharmacia, peapack, nj) coating the endothelium and inserted into the anterior chamber through the small incision using forceps . The folded donor disc was opened and attached to the recipient stromal bed by injecting air underneath the graft . The scleral wound was closed with 2 or 3 interrupted 10 - 0 nylon sutures and air was replaced with balanced salt solution to normalize intraocular pressure (iop). Femtosecond laser - assisted small incision dlek was performed in the right eye of a 73-year - old male with fuchs dystrophy (fig . Preoperative uncorrected visual acuity (ucva) and best spectacle corrected visual acuity (bscva) was 20/100 and 20/80 respectively and manifest refraction was + 0.75 -1.7530 . Donor corneoscleral button lamellar dissection was performed using intralase femtosecond laser (intralase corp . ), set at a depth of 400 m and flap diameter of 9.5 mm . Ucva and bscva was 20/150 and 20/100 respectively and manifest refraction was + 2.75 -1.0050 . Corneal topography showed smooth and regular corneal surface and specular microscopy was unremarkable without sign of corneal endothelial damage (fig . 2b and c). Optical coherence tomography (oct) findings showed compact and uniform graft, well attached to the recipient stromal bed with minimal interface reflection (fig . Femtosecond laser - assisted small incision dlek was performed in the left eye of a 94-year - old female with corneal edema, glaucoma, and dry type age - related macular degeneration (armd). Her preoperative ucva and bscva was 20/400 and 20/200 respectively and her manifest refraction was -1.00 -1.7595 . Iop was well controlled with topical medication (cosopt; merck & co., whitehouse station, nj). Postoperation, the graft was clear and compact, iop was 19 mmhg, and ucva was 20/400 . At one month, the graft was clear and compact with minimal interface haze; iop measured 31 mmhg . Anti - glaucoma medication (alphagan; allergan, irvine, ca) was added and topical steroid tapered . We were not able to perform further examinations due to follow - up loss at two months postoperation . All methods adhered to the declaration of helsinki of the world medical association for research involving human subjects . Posterior keratoplasty refers to removal of diseased posterior corneal layers (endothelium, descemet's membrane, and posterior stroma) and replacement by partial - thickness donor tissue . This procedure can be an attractive alternative to penetrating keratoplasty (pkp) in treating corneal endothelial diseases such as fuchs dystrophy and aphakic and pseudophakic bullous keratopathy.13 - 15 the endothelial replacement technique via scleral incision was first described by melles and later modified by terry and ousley to' deep lamellar endothelial keratoplasty' (dlek).3,4 recent modification includes introducing the folded donor disc through the a small incision.6 one problem in performing dlek is the technical difficulty associated with manual lamellar dissection . It is not only time consuming, but also has the potential risk of corneal perforation.8 moreover, even with uncomplicated dissection, the plane may not be smooth and uniform, which results in interface scarring and reduction in potential best vision.6,7 in a recent paper, azar and coauthors described microkeratome - assisted posterior keratoplasty.16 given that laser - assisted in situ keratomileusis (lasik) shows almost no interface haze on the cornea,17,18 this technique's potential advantage is less interface scarring than manual dissection . However, microkeratome - assisted lamellar dissection does have the risk of corneal perforation in deeper dissection . Recently, the femtosecond laser was introduced in refractive surgery fields for lasik flap creation . According to recent papers, it demonstrated improved flap uniformity and better flap thickness predictability than mechanical microkeratomes.9 - 12,19 similar to the microkeratome, femtosecond laser technology has evolved . After initial procedures with a 2 khz laser in 1996, 15 khz (2003), 30 khz (2005), and soon after, 60 khz (2006) engines were introduced . Pulses are scanned and placed in a vertical pattern for trephination (side) cuts or in a spiral or raster (zigzag) pattern to achieve lamellar cuts . The smoothness of optical surfaces is determined by programmable parameters: energy per pulse, separation of adjacent laser spots (spot separation), and raster pattern row spacing (line separation). The closer the laser spots, the less energy required.20 the intralase system scans tissue at a repetition rate which depends on laser engine capacity . Higher repetition rates allow use of lower pulse energy and closer spot / line separation settings, which results in a smoother lamellar interface, an easier anterior corneal flap lift, and a faster procedure . The newer 60 khz engine permits a line / spot separation down to 66 m and energy less than 1 j per pulse, which may show superior efficiency for harvesting the posterior corneal disk.21 one concern during preoperative femtosecond laser - assisted donor eye posterior lamellar discs (plds) preparation is the possibility of endothelial cell loss (ecl). Ecl has been reported at 4% after 150 to 200 m thick endothelial side plds preparations, and did not appear to be caused by laser pulse energy.22 another in vitro study reported 4.3% versus 7.7% ecl after 30 khz laser lamellar cutting compared to 15 khz laser for horizontal lamellar cuts at corneal depth of 400 m and 9.5 mm diameter.23 favorable outcomes are consistent with histological results showing adjacent thermal damage to be on the order of 1 m.24 clinical results from case 1 showed relatively intact postoperative endothelium status, which was comparable to previous reports . Moreover, the femtosecond laser is expected to be applied in full - thickness pkp . Cuts are customizable to achieve different graft geometric configurations, potentially allowing for sutureless, self - adhesive, or shaped keratoplasty . Two or more side / lamellar cut segments can be combined to create patterns for shaped keratoplasty, including top - hat (larger diameter cut posteriorly), mushroom (larger diameter cut anteriorly), zigzag, and christmas tree patterns.25 peripheral wound edge shaping can provide stronger healing by increasing surface area, reduce corneal astigmatism by decreasing suture number, reduce donor - host topographic distortion disparity, and quicken visual recovery . Following we describe small incision dlek by femtosecond laser for donor corneal lamellar dissection . Before performing femtosecond laser - assisted donor lamellar dissection, we measured donor corneal thickness by ultrasound pachymetry . If corneal thickness was thicker than 565 m, glycerin was applied to dehydrate the cornea, and avoid a thinner graft than intended, as well as to ascertain graft thickness of 100 to 165 m for transplantation . The laser was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness . Afterwards, two possible methods to obtain the posterior donor disc were used . In the first method, this bi - hinged donor corneoscleral button is then placed in medium, transported to the operating room, and punched out with an 8.0 mm trephine . Separation of the anterior and posterior donor corneal disc follows . In the second method, stromal and side cut adhesions this donor corneoscleral button is placed in medium and transported; following, the anterior flap is excised using scissors and the posterior part is punched out with an 8.0 mm trephine . In our cases although the disadvantage is using scissors for anterior cap removal, there are other advantages . By removing the anterior cap, it is easier to visualize the posterior donor button edge, which may reduce a decentered donor cut . Furthermore, anterior and posterior donor disc separation is much easier, since the anterior flap is fully lifted and removed before trephination . Case 1 clinical results showed a clear and compact graft without interface haze at two months postoperation . These findings were further confirmed in oct, which showed a clear and uniform graft with minimal interface reflection and good graft attachment . Despite good clinical findings, postop visual acuity was not as expected . However, the patient was satisfied and his vision fluctuation (previously worse in the morning) was eliminated . Considering findings and short - term follow - up, we may expect further improvement with time . However, postop visual acuity did not improve because of low visual potential from dry armd and glaucoma . Unfortunately, this patient had an iop increase at one month postoperation and was unable to follow - up . Visual acuity after either pkp or dlek is mainly a function of retinal macular potential and cornea optical quality . In studies of pkp in young patients with keratoconus, postoperative visual results are uniformly better compared to results in older fuchs' dystrophy patients, despite similar graft problems of high or irregular astigmatism.26 this suggests that older maculas, may play a role in visual loss after any form of corneal transplantation, including dlek . In contrast to pkp where the cornea contributes to refractive visual loss, often resulting from high or irregular astigmatism, in dlek, topography is usually normal and corneal visual loss is most likely attributed to the donor - recipient stromal interface . The interface in dlek may account, on average, for 1 line of visual loss compared to the individual macular potential vision.27 other potential corneal factors include increased corneal thickness and posterior corneal curvature . How this irregularity influences visual acuity requires further investigation . Due to lack of long term follow - up and small case number, we cannot fully determine potential advantages of' femtosecond laser - assisted small incision dlek' . However, these cases demonstrate graft uniformity, technical feasibility, and potential application of femtosecond laser in lamellar keratoplasty . Further evaluation with longer follow - up and more cases would reveal potential advantages . Unfortunately, the use of femtosecond laser in recipient corneas is currently limited by the institutional review board . Also the femtosecond laser is not approved for cuts deeper than 400 m; therefore recipient cornea posterior lip creation requires scissors . Hopefully in the near future, we can use the femtosecond laser and perform both donor and recipient corneal lamellar dissection.
The prevalence of diabetes has explosively increased worldwide, leading to an increase in the number of patients who suffer from diabetic vascular complications such as diabetic nephropathy (dn). Dn is not only the leading cause of end - stage kidney disease but also a significant risk factor for cardiovascular disease . Although the treatment for dn is important to improve patients' prognosis, the current treatment remains suboptimal and therefore novel approaches for dn are urgently needed . Dipeptidyl peptidase-4 inhibitors (dpp4i) have been recently introduced in clinic as a new oral hypoglycemic agent . Dpp4 is a serine exopeptidase and processes the substrates that have either n - terminal proline or alanine including glucagon - like peptide-1 (glp-1) and glucose - dependent insulinotropic polypeptide (gip) known as incretin hormones . These incretin hormones are secreted from the small intestine after foods intake and induce the release of insulin from the pancreatic beta cells in the islets of langerhans . In addition to the reduction in the blood glucose level, it has been reported that dpp4i possesses properties and can protect the cardiovascular system, kidney, liver, and bone from injuries . Since it has been reported that receptors for incretins were not expressed within glomerulus, protective effects of dpp4i for kidney might depend on another substrate for dpp4 . For instance, stromal cell - derived factor-1 (sdf-1), one of the substrates for dpp4, can be stabilized by dpp4i . Because sdf-1 is the most important protein for the recruitment and homing of bone marrow - derived regenerative stem cells, increased levels of sdf-1 through dpp4 inhibition has reportedly increased the intragraft number of progenitor cells that had contributed to the recovery from ischemia - reperfusion lung injury throughout the mammalian system . It has also been reported that dpp4i decreased the levels of urinary albumin excretion in diabetic patients [9, 10]; however, the mechanism behind how dpp4i ameliorates kidney injuries is not yet clear . Pituitary adenylate cyclase - activating polypeptide (pacap) is one of the substrates of dpp4 . Pacap belongs to the glucagon superfamily of peptides and was originally purified from sheep hypothalamus in 1989 . Pacap is able to potentiate cyclic adenosine monophosphate (camp) production in pituitary cells and has a diverse array of biological functions, particularly neuroprotective and general cytoprotective roles, such as anti - apoptosis and anti - inflammation . Although the highest concentrations are observed in the nervous system, a wide variety of tissues, such as heart, pancreas, liver, and kidney, produce pacap; secreted pacap also has protective effects on the different types of tissues and cells through the three different receptors: pac1, vpac1, and vpac2 . Pacap was found to exist in two forms: 38-amino - acid, a major form, and 27-amino - acid, a short one, truncated at c - terminal and to a much lesser extent in blood stream, respectively . Recently, it has been reported that dpp4 degraded pacap (127) and (138) to form pacap (327) and (338). It has been reported that pacap has protective effects in the kidney against various insults, including ischemia / reperfusion injury, drug - induced nephrotoxicity, and myeloma light chain - induced nephropathy . Pacap treatment in streptozotocin - induced diabetic animals decreased cytokine expression and prevented kidney injuries . These results indicated that pacap has protective roles in the kidney; however, it is unknown which cell types pacap affects and how it decreases cytokine expression . Therefore, in this study, we investigated the effects of pacap on kidney cells and the mechanisms of how pacap decreases the expression of inflammatory cytokines . Lipopolysaccharide (lps) and u-73122 (phospholipase c (plc) inhibitor) were obtained from sigma aldrich and h89 (protein kinase a (pka) inhibitor) was obtained from cell signaling . Cultured mouse podocytes, transformed by ectopic expression of cyclin - dependent kinase 4, were kindly provided by dr . Podocytes were maintained in roswell park memorial institute (rpmi) 1640 medium containing 10% fetal bovine serum (fbs, sigma aldrich, usa), penicillin (100 u / ml), and streptomycin (100 u / ml, sigma aldrich, usa). All procedures were performed in accordance with the guidelines of the research center for animal life science of chiba university of medical science . Six- to nine - week - old male c57bl/6j mice were purchased from clea japan (japan) and housed in cages and maintained on a 12 h light/12 h dark cycle . The glomeruli were isolated by dynabeads (invitrogen, norway) perfusion technique as previously described . The kidney tissues were dissected from the mouse, fixed in oct compound, and stored at 80c until use . Several 6 m thick frozen sections were prepared and fixed with ice - cold methanol, air - dried for 30 min at room temperature, and then blocked with blocking buffer containing 2% bovine serum albumin (bsa) and 0.05% tween-20 in pbs . After washing with tween in pbs (pbst; 0.1% tween-20 in pbs), the slides were coincubated with vpac1 antibody (1: 50 dilution, santa cruz: sc-30019), podocalyxin (1: 200 dilution, r&d systems: mab1556), and toll - like receptor 4 (1: 50 dilution, santa cruz: sc-10741). The slides were imaged by the axio observer d1 (zeiss) or with a confocal laser scanning microscope (leica lsm5 pascal). The podocytes were cultured in lab - tek ii chamber slides (nalge nunc international), deprived of serum for 24 h, and stimulated with 100 ng / ml lps for 1 h. then the podocytes were fixed in ice - cold methanol for 10 min, rinsed with pbs, and incubated in a blocking buffer containing 0.5% bsa and 0.25% tween-20 in pbs for 1 h at room temperature . The slides were then incubated with an anti - nuclear factor - kappa b (nf-b) antibody (1: 50, santa cruz: sc-372) overnight at 4c, washed several times with pbs, and then incubated with 1: 1000 dilution of fluorescent - conjugated secondary antibody (alexa fluor 488 goat anti - rabbit igg, invitrogen) for 1 h at room temperature . The slides were then washed with pbst, nuclear - stained with hoechst 33342, and mounted with a fluorescence mounting medium . For the evaluation of immunostaining for nf-b, the cells that had accumulated nf-b in their nuclei were counted at 10 randomly selected areas . The total rna was extracted using the purelink rna mini kit (ambion: 12183 - 018a) according to the manufacturer's protocols . Two micrograms of total rna were reverse - transcribed with the superscript iii reverse transcriptase kit (invitrogen: 18080). The complementary dna product was then subjected to pcr using the system with different pairs of oligonucleotide primers that were shown in supplemental table 1, available online at http://dx.doi.org/10.1155/2015/727152 . The cycling conditions were as follows: a denaturation step at 94c for 30 s followed by 30 cycles, annealing at 55c for both pac1 and -actin and 58c for vpac1 for 30 s, and elongation at 72c for 30 s with the final extension at 72c for 7 min . The pcr products were separated by gel electrophoresis on a 2% agarose gel with ethidium bromide; the signals were quantified using a chemidoc mp imagelab pcsystem (bio - rad). Quantitative pcr was performed in the 7500 fast real - time pcr system (applied biosystems) using the fast sybr green master mix (applied biosystems: 4385612). Pcr conditions were set for incubation at 95c for 20 s followed by 40 cycles of 3 s at 95c and 30 s at 60c . Podocytes were lysed in a sds sample buffer containing 0.5 m tris - hcl, 10% sds, glycerol, bromophenol blue, and 3% 2-mercaptoethanol . They were boiled at 95c for 10 min, and then the protein was fractionated on 10%15% polyacrylamide gels (e - pagel, atto corporation, japan). The protein was transferred to pvdf membranes (immobilon - p transfer membrane); the membranes were blocked for 1 h at room temperature to block the nonspecific binding of the protein and incubated for 18 h at 4c with primary antibodies . The primary antibodies that were used were as follows: anti - vpac1 antibody (1: 200 dilution, santa cruz: sc-30019), anti - phospho - p44/42 mapk (extracellular signal - regulated kinase (erk1/2)) antibody (1: 1000 dilution, cell - signaling: number 9106), and anti - p44/42 mapk (erk1/2) antibody (1: 1000 dilution, cell - signaling: number 9102). The blots were then washed and incubated with second antibodies, peroxidase - conjugated anti - rabbit immunoglobulins (1: 2500 dilution, ge healthcare), or goat anti - mouse igg - hrp (1: 2500 dilution, santa cruz: sc-2055) for 1 h at room temperature . After washing for several times, the antibody binding sites were visualized using an ecl western blotting detection system (ge healthcare: rpn2106). The blots were quantified using a chemidoc mp imagelab pcsystem (bio - rad). Podocytes were plated in 96-well dishes at 50% confluency and transfected with 10 ng per well camp response element- (cre-) lux construct that contained consecutive camp response element by lipofectamine ltx and plus reagents (invitrogen). At six hours after transfection, cells were deprived of serum for 48 h. and then, the cells were stimulated with pacap for 1 h. the luciferase activities in the cell lysate were measured in a 1420 arvo sx multilabel counter (wallac, inc ., gaithersburg, md) using the dual luciferase reporter assay system (promega, madison, wi). To correct for potential variation in transfection efficiency, prl - tk vector (promega) was cotransfected in all experiments . Statistical analyses were done using sas 9.3 and/or microsoft office excel by student's unpaired t - test . Pacap works as a ligand and binds with specific receptors in order to transduce intracellular signals . Therefore, we first examined in which cell types pacap transduces intracellular signal within the glomeruli . Reverse transcriptase pcr revealed that mrna for vpac1 but not pac1 was detected in glomeruli as shown in figure 1(a). Immunohistochemistry revealed that vpac1 was primarily expressed in podocalyxin - positive cells (figures 1(b) and 1(c)). Complete absence of signal was observed when primary antibodies were omitted (data not shown). Rt - pcr (figure 1(a)) and western blotting (figure 1(d)) also confirmed that the vpac1 mrna and protein were expressed not only in isolated glomeruli but also in cultured podocytes . Because vpac1, a pacap receptor, was expressed on podocytes, we next examined whether pacap acted on podocytes . It has been reported that pacap binds with vpac1, which is coupled with gs protein and induces rapid camp production, which ultimately activates the pka pathway . Ten nm pacap significantly increased the cellular contents of camp in a time dependent manner (supplemental figure 1). Increased levels of camp in the presence of pacap was associated with increased levels of camp responsive element promoter activities (figure 2(a)) and phosphorylated camp response element binding protein (creb) (figure 2(b)). These results indicated that pacap primarily sent signals to glomerular podocytes, especially activating the camp / pka pathway in the podocytes . It has been reported that pacap also played an anti - inflammatory role in peripheral and central tissues . Toll - like receptors (tlrs) are the principal mediators of innate immunity and are reportedly activated by bacterial endotoxins . The tlr4 proteins were localized to podocytes and endothelial cells by immunohistochemistry as shown in figure 3 . As shown in figure 4, lps, a ligand of tlr4, significantly increased the expression of il-6 and mcp-1 . In the presence of pacap, the increased expressions of il-6 and mcp-1 were significantly attenuated . The expression of tlr2, tlr4, and myeloid differentiation primary response gene 88 (myd88), a tlr adaptor protein, but not that of il-1 receptor associated kinase-1 (irak1), a down steam signaling molecule of myd88, was decreased in the presence of pacap . It has been reported that pacap activates not only adenylate cyclase (ac), which eventually activates the pka signaling pathway, but also plc, which leads to an increase in intracellular calcium signaling . Then, we examined the effect of h89, an inhibitor of the pka signaling pathway, and of u-73122, a known plc inhibitor, on the increased levels of mcp-1 . Mcp-1 expression was reversed by h-89 but not by u-73122 as shown in figure 5 . These results indicated that pacap suppressed the expression of mcp-1 through the camp / pka - dependent signaling pathway . It has been reported that lps can activate tlr4 and subsequently the myd88 transfers signals by activating nf-b and mitogen - activated protein kinase, which eventually results in the expression of proinflammatory cytokines . Therefore, we examined the effects of pacap on the nf-b transnuclear localization and phosphorylation of erk . Figures 6 and 7 showed that, in the presence of lps, nf-b transnuclear localization and phosphorylation of erk were significantly increased and pacap significantly ameliorated both . In the present study, we reported that vpac1, a pacap receptor, was exclusively expressed in glomerular podocytes . Pacap, a substrate for dpp4, activated the camp / pka signaling pathway in cultured podocytes and inhibited the expression of inflammatory cytokines, which were induced by lps / tlr4 signaling . Pacap was first identified 25 years ago and has become one of the most studied neuropeptides [11, 12]. Pacap is expressed not only throughout the nervous system but also in peripheral tissues, such as the gastrointestinal tract, the endocrine tissues, and the urinary tract . In addition to widespread expression of pacap, it reportedly has a variety of biological functions that are primarily neuroprotective and general cytoprotective roles through the specific receptors, such as pac1, vpac1, and vpac2 . For instance, pacap has protected the kidney from ischemia - induced kidney injuries, myeloma injuries, cisplatin - induced renal failure, cyclosporine a - induced nephrotoxicity, and dn . These renoprotective actions appeared to depend on the anti - inflammatory actions on circulating cells, glomerular cells, and tubular cells . The kidney is injured by a wide variety of insults leading to chronic kidney diseases (ckd). Among ckd reported that pacap - treated streptozotocin - induced diabetic mice had fewer histological changes, such as pas - positive areas within the glomeruli, tubular damage, and arteriolar hyalinosis compared with the controls . Pacap treatment also decreased the expression of a number of cytokines that were upregulated in diabetic conditions . However, the specific cell types that are affected by pacap directly have not been reported . Jean cr reported that vpac1, but not pac1 and vpac2, was expressed in human glomeruli . In agreement with this previous report, we found that vpac1 was expressed within the glomerulus and localized its expression to podocytes . Recent studies of podocyte - expressed genes have considerably enhanced our knowledge of the molecular mechanisms of glomerular filtration . A number of podocyte - expressed genes, such as nephrotic syndrome type-1 (nphs-1), nephrotic syndrome type-2 (nphs-2); actin - related proteins such as -actinin 4, inverted formin 2, and cd2-associated protein (cd2ap); and cytoplasmic signaling molecules, such as phospholipase c, epsilon-1 (plce1) and transient receptor potential cation channel, subfamily c, member 6 (trpc6), were all involved in maintaining the cytoskeletal dynamics . Therefore, among the glomerular cells, the podocytes are becoming the most highlighted cell type in the field of glomerular research of most glomerular diseases . Thus, it is intriguing that pacap may give signals to podocytes . Because it has been reported that pacap has anti - inflammatory effects in a wide variety of disease models, we focused on the anti - inflammatory effects of pacap on cultured podocytes . Among the inflammatory signals, tlr, a sensor in the innate immune system, has been highlighted in the development of dn . It has been reported that tlr2 and tlr4 have been expressed in podocytes and activated in diabetic conditions . Activated tlrs have been reported to induce proinflammatory cytokines in podocytes and a disorganized podocyte cytoskeletal structure which eventually led to proteinuria . Therefore, our finding about the inhibition of podocytes' tlr - related signaling in the presence pacap is significant in terms of treating dn . Indeed, it has been recently reported that the treatment of pacap effectively counteracted diabetes induced podocyte injury in vivo . Tlr is a conserved family of pattern recognition receptors, which is triggered by microbial pathogens, fatty acids, uric acids, oxidative stress, and high glucose . When tlr is activated, it recruits different adaptor molecules, such as myd88 and irak-1 . Activated myd88-dependent or myd88-independent pathway engages the activation of erk and nf-b signaling, which results in inflammatory cytokines . Because pacap attenuated both activation of erk and nf-b in our case it has reported that administration of pacap attenuated the expression of tlrs and its adaptor protein in kidney . In agreement with these previous reports, lps - induced inflammation related genes, such as mcp-1 and il-6, and tlr signals related genes, such as tlr2, tlr4, and myd88 but not irak-1 were suppressed by pacap in this study . It has been reported that lps - induced expression of tlr2 has been suppressed by ciprofloxacin through the production of prostaglandin (pg) e2 but not through pka in monocyte / macrophages . Because pge2 was reportedly induced in the presence of pacap, ns398, an inhibitor of pge2, was tested and was not able to reverse the effects of pacap, which attenuated the tlr4 expression (data not shown). Thus, the precise mechanisms of how pacap inhibits the expression of tlrs and myd88 need to be further analyzed . Dpp4i has been introduced in clinic and has become one of the most promising options to treat diabetic patients owing to their effectiveness in glucose lowering and low risk of hypoglycemia and weight gain . Dpp4i stabilizes its substrates and incretins (glp-1 and gip), which are the main substrates for lowering blood glucose . Beyond the hypoglycemic action, it has been reported that dpp4i has numerous potential benefits in diabetic vascular complications, including dn . The possible renoprotective effects of dpp4i include the reduction of oxidative stress and anti - inflammation and the improvement of endothelial dysfunctions through incretin - dependent and -independent pathways . Since it has been reported that glp-1 receptor was not detected in glomerulus and we were also able to detect neither glp-1 receptor nor gip receptor both in glomerulus and cultured podocyte (supplemental figure 2: expression of glp-1 receptor and gip receptor were evaluated by rt - pcr), incretin independent pathway might have roles in protecting against dn . Pacap is n - terminally truncated by dpp4, and it has been reported that dpp4-degraded pacap loses its insulinotropic effects . Because the n - terminal of pacap is a high - affinity site for vpac1 binding, dpp4-degraded pacap may reduce the signals through vpac1 . We also confirmed that the treatment of linagliptin, dpp4i, inhibited the degradation of pacap which secreted from cultured cells (supplemental figure 3: linagliptin protect pacap from degradation). In this study, we demonstrated that pacap had anti - inflammatory effects on podocytes, leading to an assumption that dpp4i protects kidney from injuries through the stabilization of pacap . Nevertheless, we have not confirmed that pacap has protective roles in vivo . Because the half - life of pacap injected into mice and humans is between 2 and 10 min due to enzymatic degradation, truncated pacap is required to be produced in order to get pacap more stable in vivo without losing receptor activating effects . In conclusion, we have shown that pacap has anti - inflammatory effects on glomerular podocytes . Because treatment options for dn are still limited, pacap may be a good candidate for prevention / attenuation of dn . However, more study is definitely needed to prove this possibility.
Opsoclonus - myoclonus - ataxia syndrome (omas), also called kinsbourne syndrome or dancing eye syndrome, is a serious, rare, and often chronic neurological disorder . Omas consists of three main symptoms: opsoclonus (conjugate, multidirectional, chaotic eye movements), myoclonus (nonepileptic limb jerking that can also involve the head and face) and truncal ataxia, which cause gait imbalance . Omas is generally a paraneoplastic or parainfectious entity, but in children, it is most commonly associated with occult neuroblastoma (nb) in about 50% of cases and between 2% and 3% of children with nb have omas . Although most patients with nb and omas have good survival rates, 70%80% of these children will have long - term neurologic sequelae . In pediatric age, omas may be associated with neuroblastic tumors (nb, ganglioneuroblastoma, or ganglioneuroma). The diagnosis of omas may be difficult in some patients and should be considered even when only some of the features are present . International consensus has described three of the following four diagnostic criteria should be present to describe the typical syndrome: (1) opsoclonus, (2) myoclonus / ataxia, (3) behavioral change and/or sleep disturbance, and (4) nb . We present a retrospective study of five children presented to the pediatric oncology clinic (poc) of all india institute of medical sciences (aiims) with a diagnosis of opsoclonus - myoclonus syndrome (oms). The objective of this study was to describe the clinical profile and outcome of this disorder . The medical records of all children presented to poc, department of pediatrics, aiims, with a diagnosis of opsoclonus - myoclonus were retrieved and reviewed . The diagnosis of opsoclonus - myoclonus was based on a constellation of any three of the four clinical features: opsoclonus, myoclonus, ataxia, and encephalopathy / irritability / behavioral change / sleep disturbance . Outcome was assessed on follow - up by direct assessment and by telephonic communication (in one patient). A total of six patients with a diagnosis of opsoclonus - myoclonus were admitted over 4-year period [table 1]. Opsoclonus, myoclonus, ataxia, and encephalopathy / behavioral abnormalities (irritability / sleep disturbance) were present in all children at presentation . The duration of symptoms at the time of presentation was in the range of 610 months . Clinical profile and outcome in six children with opsoclonus - myoclonus syndrome the reasons for the delay in diagnosis included misdiagnosis by peripheral physicians and delayed referral to our center . All children had paraneoplastic opsoclonus - myoclonus (two left paravertebral ganglioneuroblastoma, two left paravertebral nb, one left paravertebral plus left psoas muscle ganglioneuroblastoma, and one right paravertebral ganglioneuroblastoma) [figures 13]. The diagnosis of tumor was made on contrast - enhanced computed tomography scan (ct) and magnetic resonance imaging (mri) of the chest, abdomen, and pelvis (3 ct scan and 3 mri). Tests for urinary excretion of vanillylmandelic acid (vma) were negative in all cases . Iodine-131 metaiodobenzylguanidine scintigraphy scan (i-131 mibg) was performed in all cases, but it detected mibg concentrating tumor only in two cases . (case 2) (original) axial section showing well - defined enhancing soft - tissue mass lesion (3.5 cm 2 cm 1.4 cm) seen in the left upper psoas muscle extending into left neural foramen in between l2 and l3 vertebra up to dura mater (a and b) (case 3) axial section and coronal section showing right paravertebral mass of size 1.5 cm 0.6 cm 2.4 cm at the level of d6 to d8 vertebral body (a and b) (case 5) axial and coronal section showing well - defined left paravertebral mass lesion 2 cm 1.8 cm 2.9 cm at the level of d6 to d8 vertebral body an initial diagnosis of omas was made in all children . Three children received injection adrenocorticotropic hormone (acth); two children received injection acth plus injection methylprednisolone followed by oral steroids over 4 weeks . One child (case 1) also received intravenous immunoglobulin (ivig) and oral clonazepam and risperidone in view of abnormal behavior (excessive irritability, biting, and head banging). One child (case 5) had spontaneous improvement and complete recovery without specific immunomodulator therapy . Of the six cases with paraneoplastic oms, four cases were treated with surgical resection of tumor and chemotherapy . In one child (case 5), treatment was deferred by parents but on telephonic communication that the child was symptoms free and healthy at 9 months after presentation to us . In another case (case 6), she received only injection acth, surgery, and chemotherapy were denied by parents . After follow - up of 24 months, the child was asymptomatic, but ct of the abdomen revealed mass of same size that was documented 2 years back . One child (case 3) died of febrile encephalopathy (not related to disease). Other names for oms include omas, paraneoplastic opsoclonus - myoclonus ataxia, kinsbourne syndrome, myoclonic encephalopathy of infants, dancing eyes - dancing feet syndrome, and dancing eyes syndrome . In our study, all six children were between 2 and 3 years of age, similar to the trend seen in other reports, in which oma was uncommonly diagnosed before 1 year of age . One possible explanation for the low incidence in infancy may be due to ability to develop specific antibodies is less in younger infants . We observed, alike to previously published studies, a similar median age at clinical presentation, acute / subacute onset of presentation, association of nb, and response to immunomodulator therapy . We have found poor sensitivity of urine vma in our case series with similar findings reported by brunklaus et al . We also observed poorer sensitivities for mibg scintigraphy scan in contrast with the reported high sensitivity (up to 95%) for detection of nb as compared to an abdominal ct scan / mri . In adults, oms is seen in relation to malignancies of the breast and lung (small cell carcinoma), in association with antibodies which are directed against an rna binding antigen from the anti - hu antibody . This antibody is not found in children in oms of nb . In children, nb which presents with oms is more mature, shows a favorable histology, and absence of n - myc oncogene amplification than tumors which occur without symptoms of oms . Oms, the most frequent paraneoplastic syndrome in pediatric age group, remains a challenge for treatment . Various immunomodulatory therapies have been used including steroids, ivig, cyclophosphamide, and more recently, rituximab . Patients with nb and oma have been reported to have excellent survival . According to children's cancer group data, the 3-year survival rate for children with nonmetastatic nb and oma was 100% (reported from 675 patients who were diagnosed between 1980 and 1994) in compared to 77% in non - oma . Described a case series of 11 patients (largest case series from india) with a diagnosis of opsoclonus - myoclonus (of the 11, 4 had paraneoplastic etiology) and concluded that children with paraneoplastic opsoclonus had more relapses and had a poor outcome as compared to an idiopathic group ., in our series, all six children had paraneoplastic oms, and all had good outcome . This tumor is known to have spontaneous regression . Our one child (case 5) had spontaneous symptomatic resolution, and another child (case 6) is doing well without surgery and chemotherapy . We have seen a good therapeutic response with immunomodulators, including acth, ivig, and corticosteroids . Because of the small number of patients, it is difficult to compare these therapies . No one had relapse in our series contradicting to those reported by tate et al . (up to 52%) could be due to the small number of patients in our study . In most children with nb, the characteristic feature is the response of this syndrome to corticosteroids and acth and the resolution of the neurological signs when the nb is treated . Children often develop lifelong neurologic sequelae that impair motor, cognitive, language, and behavioral developments . Observed that in older children, late effects are less likely seen because basic motor and cognition have already been formed . Rudnick et al . Reported that children with more advanced stage disease had better outcomes with regard to neurologic sequelae . One possible explanation for this association may be that patients with advanced stage disease require more intensive therapy that usually includes chemotherapy . Russo et al . Suggested that chemotherapy may improve neurologic outcome in children with oma and nb, possibly due to its immunosuppressive effects . In our study, five children with oms responded with acth and steroids; one child recovered spontaneously . The outcome was good in our all children, whereas in the study by tate et al ., the outcome was independent of etiology . Other studies have also observed better outcome of idiopathic opsoclonus - myoclonus . We did not observe progressive developmental and behavioral problems in our patients, which could be due to small number of patients and short follow - up . Oms is a rare disorder, but it affects children more frequently than adults and exhibits an excellent rate of survival . Screening for an occult nb is necessary in all children with this syndrome . Oms (paraneoplastic) had a good outcome without significant neurological deficits in our experience.
To the surprise and deep disappointment of all involved in the treatment of lung cancer, several large trials did not demonstrate any benefit of tyrosine kinase inhibitors (tkis) as an addition to chemotherapy [13]. Basic and clinical research then focused on mutations of the gene for epidermal growth factor receptor (egfr) as a predictive factor for response to monotherapy with tkis and to development of new compounds with broader and/or irreversible inhibition . The biological basis for the negative experience with combined treatment gefitinib and erlotinib met all three standard criteria for inclusion in a combination with chemotherapy: activity as monotherapy, different mechanism of action, and different toxicity . Why, then, did the combination not work? As explained in a recent editorial, we believe that the cells of tumors sensitive to tkis are pushed into the g-0 phase of the cell cycle and therefore become resistant to cytotoxic drugs . If antagonism between the two classes of drugs is really the biological basis for the aforementioned negative experience, then an optimal combination of tkis and chemotherapy should be in an intermittent, rather than a continuous schedule . This brief report presents a single - institution experience on intermittent chemotherapy and tki in a small series of patients with advanced adenocarcinoma of the lung . Our hypothesis was that intermittent treatment would lead to superior time to progression, when compared to experience with chemotherapy alone . If confirmed, such a result would be a solid basis for a randomised clinical trial . Patients eligible for the trial were chemonave with microscopically confirmed adenocarcinoma of the lung, had stage iii b (wet) or iv according to uicc - tnm classification (6th edition), had smoking history of less than 10 packs in years, had an ecog performance status 0 or 1, and had adequate parameters of hematological, liver, and renal function to receive cisplatin - based chemotherapy . In the absence of neurological symptoms, patients with brain metastases were eligible and were treated with brain irradiation only in case of intracranial progression . All patients had their diagnosis confirmed by biopsy or cytology . At the time when the trial was initiated, testing for egfr mutations was not available . Within three weeks prior to treatment, the precise extent of the disease was determined by chest x - ray and ct scanning of the chest, upper abdomen, and brain . Since 2008, pet - ct scanning has been available and included in the initial diagnostics and in followup . The treatment started with four cycles of intermittent chemotherapy and erlotinib according to the following schedule: day 1: gemcitabine 1250 mg / m in 30-minute infusion, day 2: cisplatin 75 mg / m, with appropriate hydration and antiemetics, day 4: gemcitabine 1250 mg / m in 30-minute infusion, days 515: erlotinib 150 mg daily p.o . The number of cycles depended on tolerance to cisplatin - based chemotherapy and was determined individually . Immediately after the last cycle, patients continued with erlotinib 150 mg / m daily continuously until progression or unacceptable toxicity . Definition of complete response (cr), partial response (pr), stable disease (sd), and progression followed the recist criteria . The first evaluation of response was done during the third cycle of intermittent therapy, with confirmation of response during the fifth cycle . Control radiological examinations were repeated every 2 months for chest x - ray, every 4 months for ct, and at 6 and 12 months for pet - ct (only patients who had this examination during their initial diagnostics). In october 2010, all biopsy samples were reviewed, and specimens with more than 10% of tumour tissue were analyzed . Genomic dna was extracted from formalin - fixed, paraffin - embedded tissue sections using qiaamp dna ffpe tissue kit (qiagen, hilden, germany) according to the manufacturer's instructions . Quantification of extracted dna was done on qubit fluorometer (invitrogen, carlsbad, usa). To detect egfr gene - activating mutations, we used therascreen egfr29 mutation kit (dxs diagnostics, qiagen, manchester, uk). All realtime pcr reactions were performed in a 25 l final volume on abi 7500 instrument (applied biosystems, carlsbad, usa). After standard chemotherapy for metastatic nonsmall cell carcinoma, the expected ttp is 5 months . The size of this single - arm nonrandomised trial of intermittent therapy was based on the assumption of 9 months as the median time to progression (ttp). To obtain such a result with a confidence interval of 612 months, we planned to recruit 40 patients . The investigators strictly followed recommendations of the helsinki declaration (1964, with later amendments) and of the european council convention on protection of human rights in bio - medicine, as accepted in oviedo in 1997 . The protocol was approved by the institutional review board (institute of oncology, ljubljana) and by the national committee for medical ethics, ministry of health, republic of slovenia . One patient was later found to have metastatic carcinoma of the pancreas rather than primary lung cancer and was excluded from all further analyses . With 12 patients each, twelve patients were never - smokers, and most were in good general condition (ps 0 - 1 for 21 patients). With the exception of a single patient with wet bone metastases were the most common site of metastatic disease, followed by pleura / pericardium, contralateral lung metastases, and liver . Two or more sites of metastatic disease were documented in 4 and 12 patients, respectively (table 1). Three patients had only cytological diagnosis, and an additional 3 had biopsy samples too small to allow for analysis of egfr mutations in tumor cells . Of the 18 adequate samples, 8 were positive for egfr gene - activating mutations . The actual number of cycles of intermittent therapy was from 1 to 6 cycles (median: 4 cycles). Due to early progression, 7 patients were still on maintenance treatment with erlotinib, and an additional patient stopped treatment with erlotinib after 12 months in pet - ct confirmed complete remission (figures 1 and 2). For the remaining patients, median total duration of treatment was 10 months . During the initial phase, side effects of maintenance with erlotinib were skin toxicity (grade 3: 1 pt; grade 2: 11 pts) and diarrhea (grade 2 in 1 pt). All patients are evaluable for response, and no patient has been lost to followup . For the whole group of 24 patients, complete remission (cr) was seen in 5 pts; partial remission (pr) in 9 pts (response rate 58%), minimal response or stable disease (sd) in 8 pts, and progression in 2 pts . A clear and statistically significant (p <.05) correlation was seen between the presence of activating egfr mutations and response . Among the 8 patients who were positive for egfr gene - activating mutations, 4 complete and no cr and only 2 pr were seen among the 10 patients negative for mutations (table 2). For the whole group, median time to progression (ttp) was 13.4 months, and median overall survival (os) was 23 months . Median ttp and os for this group was 21.5 months and 24.2 months, respectively . For patients without egfr mutations, ttp was 5 months, and os was 7 months (table 2 and figures 3 and 4). This clinical trial was launched at a time when routine testing for egfr gene - activating mutations was not yet available . Selection of patients for a combination of chemotherapy and erlotinib was made on the basis of classical histopathology (adenocarcinoma) and smoking status . Since testing for egfr gene mutations is now available, it is clear that patients with activating mutations are those who really benefit from tkis . In addition, standard first - line treatment for patients with activating egfr mutations is now monotherapy with a tki [6, 7]. Since continuing a trial with the same selection criteria and without considering the status of egfr gene activating mutations was not justified, the research group made a decision to close the trial and analyse the experience . In order to get a longer interval for intermittent erlotinib, gemcitabine was given on days 1 and 4 of the cycle . When compared to the standard day 1 and day 8 schedule, this minor modification in timing of cytotoxic drugs did not have any adverse effect on the tolerance to treatment . Clearly, other platin - based schedules which apply chemotherapy on a 3-weekly basis (such as pemetrexed - cisplatin or paclitaxel - carboplatin) can offer an even longer interval for tkis and might be considered for future trials of intermittent treatment . Two other groups recently reported promising experience with intermittent chemotherapy and tkis . In a trial from the usa, two schedules of intermittent treatment were tested . In combination with pemetrexed (500 mg / m on day 1), erlotinib was given either as a pulse application in a high dose (range: 800 to 1400 mg) given on days 2, 9 and 16, or in lower doses (150250 mg daily) on days 2 to 16 . While tolerance to this treatment was good, the small number and heterogeneity of patients recruited into this trial do not allow for any clear conclusion regarding the effectiveness of intermittent treatment . Of more importance this study from asia compared gemcitabine and either cisplatin or carboplatin to a schedule with addition of intermittent application of erlotinib (150 mg on days 14 to 28 of the cycle) and reported significantly superior ttp with the intermittent schedule . Their experience is most valuable but may not be of direct relevance for the rest of the world, due to the well - known differences in sensitivity of lung cancer to tkis between asian and caucasian patients . Despite its small size, our trial can offer valuable experience for further research on optimisation of treatment with combinations of chemotherapy and tkis . Looking at the whole series of patients, we can conclude that intermittent chemotherapy and erlotinib is a treatment of very low toxicity . It is also clear that the efficacy of treatment is closely related to the presence or absence of egfr gene - activating mutations . The most important finding is the excellent response rate with a substantial proportion of complete responses and prolonged ttp and os for patients positive for egfr gene - activating mutations . For many years, the maximal expectation of a patient with metastatic nonsmall cell lung cancer was a partial remission of relatively short duration in the range of 5 to 9 months . With intermittent treatment, while the number of patients in our trial is small and any definitive conclusion would be premature, we nevertheless believe that further research of intermittent therapy for patients positive for egfr gene - activating mutations is warranted . A randomised trial comparing first - line tki as monotherapy to the intermittent schedule should clarify the real value of this new approach.
Using metagenomic deep sequencing, we analyzed fecal samples from 180 infants and children ages 7 days96 months (mean 18.7 months) in tunisia who had unexplained diarrhea that tested negative for rotavirus, norovirus, astrovirus, sapovirus, adenovirus types 40 and 41, and aichi virus by reverse transcription pcr (7). The fecal supernatants were filtered through a 0.45-m filter (millipore, darmstadt, germany) to remove bacterium - sized particles, and the filtrates were digested with a mixture of dnases (turbo dnase from ambion, carlsbad, ca, usa; baseline - zero from epicenter, madison, wi, usa; and benzonase from novagen, san diego, ca, usa) and rnase (fermentas, pittsburgh, pa, usa) to digest unprotected nucleic acids . Enriched viral nucleic acids (rna and dna) were then extracted and amplified by using scriptseq v2 rna - seq library preparation kit (epicenter) and analyzed in pools of 10 specimens in 2 illumina miseq run of 250-bp end reads, yielding 20,693,619 unique sequences . We compared the illumina sequences with the genbank nonredundant protein databases using blastx (http://blast.ncbi.nlm.nih.gov/blast.cgi). Using a blastx e - score cutoff of 10, we identified, in decreasing frequency, sequences related to the mammalian viruses: sapovirus (120,177 reads), anelloviridae (14,841 reads), parechovirus (10,557 reads), norovirus (4,551 reads), enterovirus (3,857 reads), circoviridae (2,127 reads), group a rotavirus (839 reads), adeno - associated virus (812 reads), picobirnavirus (274 reads), bufavirus (168 reads), wu polyomavirus (136 reads), bocavirus (62 reads), adenovirus (58 reads), papillomavirus (22 reads), cosavirus (20 reads), group c rotavirus (17 reads), human astrovirus 1 (14 reads), salivirus (4 reads), and aichi virus (2 reads). One pool showed a single read encoding a parvovirus - like protein segment with high levels of genetic similarity (blastx e - score of 5 10) to the nonstructural protein (ns) 1 of rat parvovirus (genbank accession no . Was then identified by using pcr and underwent further deep sequencing as above, generating 11 more parovirus sequences . No other eukaryotic viral sequences were identified from 260,000 unique sequence reads from this patient . The near complete parvovirus genome was then acquired by filling genome gaps by pcr and amplifying 5 and 3 extremities using race (rapid amplification of cdna ends, life technologies). We named this virus tusavirus 1 for tunisian stool - associated parvovirus . A nearly complete 4,424-bp genome (tusavirus 1, genbank accession no . Kj495710) was successfully acquired with partial 5 untranslated region (243 bp), complete ns1 open reading frame (625 aa), complete viral protein (vp) 1 open reading frame (715 aa), and a partial 3 untranslated region (68 bp). Tusavirus has a potential upstream start codon mss in a weaker kozak consensus sequence than maq (figure, panel a), which we selected as the start codon . The walker loop gpattgks [gxxxxgk(t / s)], which is an atp- or gtp - binding motif, was found in the ns1 . Potential splicing signals to express vp1 were identified on the basis of alignments to other protoparvoviruses and classic rna splicing motifs (figure, panel a). The phospholipase a2 (pla2) motif was identified in vp1 n - termini with expected calcium - binding site and catalytic residues . The methionine codon of vp2 was located upstream of glycine - rich sequence (gggaraggvg). An unusual serine - rich sequence (sssdsgpsss) was also seen near vp1 n - termini . The alignment of the pla2 regions of representatives of 5 protoparvovirus species show the calcium - binding region and catalytic residues in tusavirus . Pairwise sliding window of percentage nucleotide similarity of tusavirus aligns with the genetically closest kilham rat parvovirus . B) phylogenetic trees generated with nonstructural protein (ns) 1 and vp1 of tusavirus and of the 5 international committee on taxonomy of viruses designated species in the protoparvovirus genus . Fpv, feline parvovirus; mev, mink enteritis virus; cpv, canine parvovirus; rapv, raccoon parvovirus; rpv1, rat parvovirus 1; mvmp, minute virus of mice, prototype; hapv, hamster parvovirus; ppv - kr, porcine parvovirus kresse; simian bupv, simian bufavirus; bupv1, bufavirus 1; bupv2, bufavirus 2; amdv, aleutian mink disease virus; gfav, gray fox amdovirus; b19v - lali, human parvovirus b19-lali . Bootstrap values (based on 100 replicates) for each node are given if> 70% . Protein sequence alignments were made by using clustalx version 2.0.3 (http://www.clustal.org) with the default settings; a phylogenetic tree with 100 bootstrap resamples of the alignment datasets was generated by using the neighbor - joining method based on the jones - taylor - thornton matrix - based model in mega5 (http://www.megasoftware.net). Phylogenetic analysis showed that tusavirus 1 was distinct from known members of the protoparvovirus genus (figure, panel b). Vp1 and vp2 shared identities of 39% and 37%, respectively, to those of kilham rat parvovirus . According to the international committee on taxonomy of viruses, the members of the same parvovirus genus should share> 30% and members of the same species> 85% aa identity in ns1 . The protoparvovirus genus currently comprises species infecting carnivores, rodents, pigs, and humans (1). Tusavirus 1 is proposed as prototype for primate protoparvovirus 2 species that would join bufaviruses as human viruses in this genus . We used a nested pcr targeting ns1 to determine the prevalence of this virus in the 180 tunisia diarrhea samples . Primers tusa - f1 (5-gaagaagctggaaactgtggtca-3) and tusa - r1 (5-ctcgtctttctcccaggcatct-3) were used for the first round of pcr, and primers tusa - f2 (5-attgctccaacaccagtcatca-3) and tusa - r2 (5-tctggtctggtccaatcttcttc-3) for the second round of pcr . The pcr conditions were: 95c for 5 min, 35 cycles 95c for 30 s, 52c or 51c (for the first or second round, respectively) for 30 s, and 72c for 1 min, a final extension at 72c for 10 min . No samples except the one initially detected by deep sequencing were pcr positive, yielding a low prevalence of 0.56% (1/180) in this tunisian population . We detected fecal shedding of a previously uncharacterized parvovirus in a child with unexplained diarrhea . The 18-month - old girl showed twice daily liquid and greenish feces over 3 days but no fever (37.4c), vomiting, or dehydration . To identify other viral infections in this patient, no other mammalian virus was detected, suggesting a possible role for tusavirus 1 in this patient s gastrointestinal illness, although the lack of testing for pathogenic bacteria and parasites does not enable us to exclude these alternative explanations . Control pcr studies of unexplained diarrhea and serologic tests, are needed to define the prevalence and disease association of this new parvovirus species in different age groups and populations.

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