Phenotype changes of oral epithelial stem cells after in vitro culture

Abstract The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.

Differential effect of the route of inoculation of rabies virus on NeuN immunoreactivity in the cerebral cortex of mice / Efecto diferencial de la vía de inoculación del virus de la rabia sobre la inmunorreactividad de NeuN en la corteza cerebral de ratones

Rabies is a lethal disease caused by a neurotropic virus that produces inconspicuous morphological changes hardly observable with conventional histopathology. The fatal outcome caused by rabies could be attributed to specific biochemical changes that severely impact neuronal function. The neuronal nuclear protein (NeuN) has become a widely used neuronal marker for the research and the histopathological diagnosis of nervous system diseases. To evaluate the distribution of the protein NeuN in the motor cortex of normal and rabies-infected mice adult ICR mice were inoculated with rabies virus either intramuscularly or intracerebrally. Rabies-infected mice were sacrificed at the terminal stage of the disease. Control mice were also euthanized at the same age. The brains were removed and cut into coronal sections on a vibratome. Immunohistochemistry was used to study the expression of NeuN in the motor area of the cerebral cortex. Neuronal counts, cellular optical densitometry and neuronal diameter measurements were performed to analyze the immunoreactivity of the protein. All parameters revealed decreased immunoreactivity for NeuN in cortical neurons of mice intracerebrally infected with rabies. In contrast, the changes were not statistically significant in mice inoculated intramuscularly. Either the immunoreactivity of NeuN or its expression is affected by the presence of rabies virus in the cerebral cortex depending on the inoculation route. These results contribute to the knowledge of the dynamics of cellular infection on rabies pathogenesis.

La rabia es una enfermedad mortal causada por un virus neurotrópico que produce discretos cambios morfológicos difícilmente observables con la histopatología convencional. El desenlace fatal causado por la rabia puede atribuirse a cambios bioquímicos específicos que afectan gravemente la función neuronal. La proteína nuclear neuronal (NeuN) es un marcador ampliamente utilizado para la investigación y el diagnóstico histopatológico de enfermedades del sistema nervioso. Este trabajo se realizó con el propósito de evaluar la distribución de la proteína NeuN en la corteza motora de ratones normales y ratones infectados con virus de la rabia. Ratones ICR adultos fueron inoculados con virus de la rabia por vía intramuscular o por vía intracerebral. Los animales infectados con rabia fueron sacrificados en la etapa terminal de la enfermedad. Ratones de la misma edad no inoculados con el virus (controles) fueron sacrificados simultáneamente. Se extrajeron los cerebros y se obtuvieron cortes coronales en un vibrátomo. Mediante inmunohistoquímica se estudió la expresión de NeuN en el área motora de la corteza cerebral. Se realizaron conteos neuronales, densitometría óptica celular y mediciones del diámetro de los perfiles neuronales para analizar la inmunorreactividad de la proteína. En los ratones inoculados por vía intracerebral hubo disminución significativa de la inmunorreactividad de NeuN manifestada en los diferentes parámetros evaluados. En contraste, estos cambios no fueron estadísticamente significativos en los cerebros de ratones inoculados por la ruta intramuscular. La inmunorreactividad de NeuN o su expresión es afectada por la presencia del virus de la rabia en la corteza cerebral pero dependiendo de la vía de inoculación. Estos resultados contribuyen al conocimiento de las dinámicas de infección celular en la patogénesis de la rabia.

La combinación de atorvastatina y meloxicam inhibe la neuroinflamación y atenúa el daño celular en la isquemia cerebral experimental por embolia arterial / Atorvastatin-meloxicam association inhibits neuroinflammation and attenuates the cellular damage in cerebral ischemia by arterial embolism.

Introducción. El accidente cerebrovascular es la segunda causa de muerte y la primera de discapacidad en el mundo, y más de 85 % es de origen isquémico. Objetivo. Evaluar en un modelo de infarto cerebral por embolia arterial el efecto de la atorvastatina y el meloxicam, administrados por separado y de forma conjunta, sobre la respuesta neuronal, los astrocitos y la microglia. Materiales y métodos. Se sometieron ratas Wistar a embolia de la arteria carótida y a tratamiento con meloxicam y atorvastatina, administrados por separado y conjuntamente, a las 6, 24, 48 y 72 horas. Se evaluó la reacción de las proteínas COX-2, GFAP y OX-42 en las neuronas, los astrocitos y la microglia mediante inmunohistoquímica y estudios morfológicos y de densitometría. Los datos obtenidos se evaluaron por medio de un análisis de varianza y de pruebas no paramétricas de comparación múltiple. Resultados. La isquemia cerebral por embolia arterial incrementó significativamente (p<0,001) la reacción de los astrocitos y la microglia, en tanto que la atorvastatina y el meloxicam, administrados por separado y de forma conjunta, la redujeron. La isquemia produjo acortamiento de las proyecciones de los astrocitos, engrosamiento celular, ruptura de las expansiones protoplásmicas (clasmatodendrosis) y cambios morfológicos en la microglia propios de diversas etapas de actividad. En las zonas circundantes del foco se incrementó la reacción inmunológica de la COX-2 y se redujo en el foco isquémico, en tanto que el meloxicam y la atorvastatina redujeron significativamente (p<0,001) la reacción inmunológica en la zona circundante del foco, restableciendo la marcación de la ciclooxigenasa en el foco isquémico. Conclusión. La combinación de meloxicam y atorvastatina atenúa la respuesta de los astrocitos y la microglia en el proceso inflamatorio posterior a la isquemia cerebral por embolia arterial, reduciendo la degeneración neuronal y restableciendo el equilibrio morfológico y funcional del tejido nervioso.

Introduction: Stroke is the second leading cause of death and the first cause of disability in the world, with more than 85% of the cases having ischemic origin. Objective: To evaluate in an embolism model of stroke the effect of atorvastatin and meloxicam on neurons, astrocytes and microglia. This evaluation was done administering each medication individually and in association. Materials and methods: Wistar rats were subjected to carotid arterial embolism and treatment with meloxicam and atorvastatin at 6, 24, 48 and 72 hours. Using immunohistochemistry, we evaluated the immunoreactivity of COX-2 protein, GFAP and OX-42 in neurons, astrocytes and microglia by densitometric and morphological studies. Data were evaluated by variance analysis and non-parametric multiple comparison. Results: Cerebral ischemia by arterial embolism increased significantly the reactivity of microglia and astrocytes (p<0.001), whereas it was reduced by atorvastatin, meloxicam and their association. Ischemia produced astrocytic shortening, cellular thickening, protoplasmic rupture expansions (clasmatodendrosis) and microglial morphological changes characteristic of various activity stages. In perifocal areas, immunoreactivity of COX-2 was increased and in the ischemic focus it was reduced, while meloxicam and atorvastatin significantly reduced (p<0.001) perifocal immunoreactivity, restoring the marking of cyclooxygenase in the ischemic focus. Conclusion: These results suggest that the meloxicam-atorvastatin association attenuates astrocytic and microglial response in the inflammatory process after cerebral ischemia by arterial embolism, reducing neurodegeneration and restoring the morphological and functional balance of nervous tissue .

Parallel determination of NeuroD1, Chromogranin-A, KI67 and androgen receptor expression in surgically treated prostate cancers

PURPOSE: Neuroendocrine differentiation is a hallmark of prostate cancer. The aim of our study was the detection of the parallel expression of neuroendocrine related markers using a prostate tissue microarray (TMA). MATERIALS AND METHODS: Our study was aimed at detecting the parallel expression of NeuroD1, Chromogranin-A (ChrA), Androgen Receptor (AR) and Ki-67 by immunohistochemistry on prostate cancer tissue microarray. The data was analyzed using SAS version 8.2 (SAS Inc, Cary, NC). The relationships between NeuroD1, ChrA and AR expressions and patients' characteristics were investigated by multivariate logistic regression analysis. Progression and Overall Survival (OS) distributions were calculated using Kaplan-Meier method. RESULTS: Tissue reactivity for NeuroD1, ChrA and AR concerned 73 percent, 49 percent and 77 percent of the available cases, respectively. Regarding overall survival, there were 87 deaths and 295 patients alive/censored (6 years of median follow-up). Seventy-seven disease progressions occurred at the median follow-up 5.4y. A significant correlation between NeuroD1, ChrA and AR expression was observed (p < 0.001 and p < 0.03, respectively). Additionally, ChrA was strongly associated in multivariate analysis to Gleason score and Ki67 expression (p < 0.009 and p < 0.0052, respectively). Survival analysis showed no association between markers neither for overall nor for cancer-specific survival. CONCLUSIONS: The results highlight that NeuroD1, Chromogranin-A and Androgen Receptor are strongly associated, however their expression does not correlate with overall survival or disease progression.

A murine model of xenotransplantation of human glioblastoma with imunosupression by orogastric cyclosporin / Modelo murino de xenotransplante de glioblastoma humano com imunossupressão utilizando ciclosporina orogástrica

Several animal experimental models have been used in the study of malignant gliomas. The objective of the study was to test the efficacy of a simple, reproducible and low cost animal model, using human cells of glioblastoma multiforme (GBM) xenotransplantated in subcutaneous tissue of Wistar rats, immunosuppressed with cyclosporin given by orogastric administration, controlled by nonimunosuppressed rats. The animals were sacrificed at weekly intervals and we have observed gradual growth of tumor in the immunosuppressed group. The average tumor volume throughout the experiment was 4.38 cm³ in the immunosuppressed group, and 0.27 cm³ in the control one (p<0.001). Tumors showed histopathological hallmarks of GBM and retained its glial identity verified by GFAP and vimentin immunoreaction. Immunosuppression of rats with cyclosporin was efficient in allowing the development of human glioblastoma cells in subcutaneous tissues. The model has demonstrated the maintenance of most of the histopathological characteristics of human glioblastoma in an heterotopic site and might by considered in research of molecular and proliferative pathways of malignant gliomas.

Vários modelos animais têm sido avaliados no estudo dos gliomas e até o momento nenhum pôde ser considerado ideal. O objetivo deste trabalho é verificar a eficácia de um modelo animal simples, reprodutível e de baixo custo. Utilizamos células humanas de glioblastoma multiforme (GBM) xenotransplantadas em ratos Wistar, submetidos a imunossupressão com ciclosporina administrada por via orogástrica. Células tumorais foram implantadas no tecido subcutâneo dos ratos imunossuprimidos com ciclosporina, sendo o controle feito em ratos não imunossuprimidos. Os animais foram sacrificados em intervalos semanais e foi observado crescimento progressivo do tumor no grupo imunossuprimido. A média do volume tumoral em todo o experimento foi de 4,38 cm³ no grupo imunossuprimido e 0,27 cm³ no grupo controle (p<0,001). Os tumores apresentavam características histopatológica do GBM e mantinham sua identidade glial, verificadas por imunoreação para GFAP e vimentina. A imunossupressão dos ratos com ciclosporina foi eficiente em permitir o desenvolvimento do glioblastoma no tecido subcutâneo. Uma vez que o presente modelo mantém a maioria das características histopatológicas do glioblastoma humano, ele pode ser considerado em estudos que avaliem as vias moleculares e proliferativas dos gliomas malignos.

Gliosarcoma with prominent smooth muscle component (gliomyosarcoma): A report of 10 cases.

Background and Aim: Gliosarcoma (GS) is an uncommon malignant tumor of the brain, consisting of malignant glial, usually a glioblastoma (GB), as well as sarcomatous component; the latter is usually in the form of fibrosarcoma. We report a series of 10 GSs with prominent smooth muscle component, which is a rare occurrence. Settings and Design: Out of a series of 225 cases of GB admitted in our hospital, 10 were diagnosed as GS with prominent smooth muscle component, gliomyosarcoma (GMS). Materials and Methods: This is an observational study based on the experience with 225 cases of GB, encountered between 1995 and 2008, in our hospital. The tumors showing prominent spindle cell component were stained with reticulin and 20 with strongly positive reticulin stain were diagnosed as GS. They were further studied by immunohistochemical staining for glial fibrillary acidic protein (GFAP), smooth muscle actin (SMA), desmin and factor VIII antigen. Results: Out of 225 cases of GB, 20 were diagnosed as GS. Ten of these showed prominent smooth muscle component and were diagnosed as GMS. They revealed varying degrees of SMA and factor VIII Ag positivity. In the sarcomatous component, SMA and factor VIII positive cells were seen close to the vessel walls as well as away from them. Conclusion: GMS containing prominent smooth muscle component may not be as rare as has been reported in the literature. Both GS and GMS appear to arise from the vessel wall at least in some cases, suggesting their possible vascular origin.

Nestin down-regulation of cortical radial glia is delayed in rats submitted to recurrent status epilepticus during early postnatal life / Atraso no desaparecimento da nestina na glia radial cortical de ratos submetidos a recorrentes status epilepticus durante o desenvolvimento pós-natal precoce

OBJECTIVE: Nestin is temporarily expressed in several tissues during development and it is replaced by other protein types during cell differentiation process. This unique property allows distinguishing between undifferentiated and differentiated cells. This study was delineated to analyze the temporal pattern of nestin expression in cortical radial glial cells of rats during normal development and of rats submitted to recurrent status epilepticus (SE) in early postnatal life (P). METHOD: Experimental rats were submitted to pilocarpine-induced SE on P7-9. The cortical temporal profile of nestin was studied by immunohistochemistry at multiple time points (P9, P10, P12, P16, P30 and P90). RESULTS: We observed delayed nestin down-regulation in experimental rats of P9, P10, P12 and P16 groups. In addition, few radial glial cells were still present only in P21 experimental rats. CONCLUSION: Our results suggested that SE during early postnatal life alters normal maturation during a critical period of brain development.

OBJETIVO: A nestina, temporariamente expressa em diversos tecidos durante o desenvolvimento, é substituída no processo de diferenciação celular, o que permite a distinção entre células diferenciadas e indiferenciadas. O objetivo deste estudo foi verificar o padrão temporal da expressão da nestina nas células da glia radial cortical de ratos durante o desenvolvimento normal e nos ratos submetidos a sucessivos status epilepticus (SE) no periodo pós-natal precoce (P). MÉTODO: Os animais foram submetidos ao SE induzido pela pilocarpina em P7-9. O perfil temporal da nestina foi estudado por imuno-histoquímica em P9, P10, P12, P16, P30 e P90. RESULTADOS: Nos ratos experimentais, observamos atraso no desaparecimento da nestina nos grupos P9, P10, P12 e P16. Ainda, encontramos algumas glias radiais corticais apenas em P21 experimental. CONCLUSÃO: Nossos resultados sugerem que o SE durante o desenvolvimento pós-natal precoce altera o processo de maturação durante um periodo crítico do desenvolvimento encefálico.

Tissue-specific expression and subcellular localization of ALADIN, the absence of which causes human triple A syndrome

Triple A syndrome is a rare genetic disorder caused by mutations in the achalasia-addisonianism-alacrima syndrome (AAAS) gene which encodes a tryptophan aspartic acid (WD) repeat-containing protein named alacrima-achalasia-adrenal insufficiency neurologic disorder (ALADIN). Northern blot analysis shows that the 2.1 kb AAAS mRNA is expressed in various tissues with stronger expression in testis and pancreas. We show that human ALADIN is a protein with an apparent molecular weight of 60 kDa, and expressed in the adrenal gland, pituitary gland and pancreas. Furthermore, biochemical analysis using anti-ALADIN antibody supports the previous finding of the localization of ALADIN in the nuclear membrane. The mutations S544G and S544X show that alteration of S544 residue affects correct targeting of ALADIN to the nuclear membrane.

Forkhead box O-class 1 and Forkhead box G1 as Prognostic Markers for Bladder Cancer

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.

Forkhead box O-class 1 and Forkhead box G1 as Prognostic Markers for Bladder Cancer

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.

Expression of nestin and vimentin in gliomatosis cerebri / Expressão de nestina e vimentina na gliomatosis cerebri

Gliomatosis cerebri (GC) is a rare form of CNS neoplasia in which there is diffuse involvement of the nervous tissue with or without the presence of tumor mass. The origin of the tumor is unknown, nor whether it represents a disease with diffuse onset or infiltration from a neoplastic focus. Here we studied the histopathologic characteristics of 6 cases with a diagnosis of GC and performed an immunohistochemical analysis using glial fibrillary acidic protein (GFAP), synaptophysin, nestin and vimentin. Most tumor cells were negative for GFAP, even though there were foci of positivity for this marker in all cases. We detected the presence of many positive cells for nestin and vimentin in all studied samples. The presence of these cells may indicate origin of the tumor from undifferentiated cells with a high degree of mobility.

A gliomatosis cerebri (GC) é uma forma rara de neoplasia do sistema nervoso central em que existe o envolvimento difuso do tecido nervoso com ou sem a presença de massa tumoral. A origem do tumor é incerta, bem como se representa uma doença de início difuso ou uma infiltração a partir de um foco de neoplasia. Foram estudadas as características histopatológicas de seis casos com diagnóstico de GC e realizada imuno-histoquímica utilizando-se GFAP, sinaptofisina, nestina e vimentina. A maioria das células tumorais mostrou-se negativa para GFAP, apesar de existirem focos de positividade para este marcador em todos os casos. Observamos muitas células positivas para nestina e para vimentina em todas as amostras estudadas. Estas células poderiam indicar a origem do tumor em células multipotenciais com alto grau de mobilidade.

An immunohistochemical, clinical and electroneuromyographic correlative study of the neural markers in the neuritic form of leprosy

The nerve biopsies of 11 patients with pure neuritic leprosy were submitted to routine diagnostic procedures and immunoperoxidase staining with antibodies against axonal (neurofilament, nerve growth factor receptor (NGFr), and protein gene product (PGP) 9.5) and Schwann cell (myelin basic protein, S-100 protein, and NGFr) markers. Two pairs of non-adjacent histological cross-sections of the peripheral nerve were removed for quantification. All the fascicles of the nerve were examined with a 10X-ocular and 40X-objective lens. The immunohistochemistry results were compared to the results of semithin section analysis and clinical and electroneuromyographic data. Neurofilament staining was reduced in 100 percent of the neuritic biopsies. NGFr positivity was also reduced in 81.8 percent, PGP staining in 100 percent of the affected nerves, S100 positivity in 90.9 percent, and myelin basic protein immunoreactivity in 90.9 percent. Hypoesthesia was associated with decreased NGFr (81.8 percent) and PGP staining (90.9 percent). Reduced potential amplitudes (electroneuromyographic data) were found to be associated with reduced PGP 9.5 (63.6 percent) and nerve fiber neurofilament staining (45.4 percent) by immunohistochemistry and with loss of myelinated fibers (100 percent) by semithin section analysis. On the other hand, the small fibers (immunoreactive dots) seen amid inflammatory cells continued to be present even after 40 percent of the larger myelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression of neural markers and the integrity of nerve fiber. The association of these structural changes with the clinical and electroneuromyographic manifestations of leprosy peripheral neuropathy was also discussed.

Oligodendroglioma in a French Bulldog

A 5-year-old, male French bulldog with bradycardia, dyspnea, and decerebrate rigidity was necropsied. Macroscopic findings were restricted to the brain, and a single mass, 1.5x2.0x1.5 cm in size, was observed mainly at the right cingulum with prominently protruding into the dilated right lateral ventricle. The mass was grayish white in color, soft and gelatinous, but not clearly delineated. Microscopically, the mass consisted of diffuse proliferated neoplastic oligodendroglial cells characterized by small, round, and hyperchromatic nuclei with clear cytoplasm and the cells aggressively invaded into the adjacent parenchyma. Immunohistochemistry demonstrated that most of the neoplastic cells were positive for S-100 protein, vimentin, neuron specific enolase (NSE), and neurofilament protein (NFP). From these findings, the mass was diagnosed as oligodendroglioma.

Desmoplastic astrocytoma of infancy: a case report with histopathologic and immunohistochemistry profile

The clinicopathological features and immunohistochemistry profile of desmoplastic cerebral astrocytoma of infancy are discussed in a 4 month old male infant who presented with an increasing head circumference more pronounced in the last two weeks prior to admission to the University Pediatric Hospital. This is a rare tumor that occurs in infants within the first two years of life and it is characterized by a massive, often cystic, supratentorial lesion usually in the frontoparietal region. It has a biphasic histologic pattern with an astrocytic and desmoplastic component and a good prognosis after total or near total surgical resection. This patient represents the first case of desmoplastic cerebral astrocytoma of infancy diagnosed in the Puerto Rico Medical Center.

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina.

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11-12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photo-receptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.

A clinical checklist for fragile X syndrome: screening of Thai boys with developmental delay of unknown cause.

The aim of this study was to determine a cost-effective clinical checklist for fragile X syndrome (FXS) screening in a Thai male pediatric population with developmental delay of unknown cause. We studied 179 non-FXS male patients and 27 FXS patients from 18 families (age < or = 15 years). A six-item clinical checklist was used including family history (FH), long and narrow face (F), prominent and large ears (E), attention deficit/hyperactivity (AH), autistic-like behavior (AT) and testicular volume (T). These were scored as 0 if absent, 1 if borderline, and 2 if present. All patients were tested by using PCR and/or southern blot for the FMR1 gene. We used a logistic regression model from a computer program to analyze the data (Stata, version 5.0). We used logistic regression with cluster in the same family (average score) to eliminate bias from the related FXS cases. We found that a five-item checklist, 2FH + F + 0.5E + 2AH + T = total score, was the best model. When we used this clinical checklist with a threshold of total score of 4, 78.7 per cent of the screened cases with total scores < or = 4 could be eliminated as negative cases. In addition, all positive FXS cases had total scores > 4. We propose this five-item model for FXS screening in clinical pediatric practice, particularly from Asian population settings.

Immunohistochemical study of syntaxin-1 and snap-25 in the pituitaries of mouse, guinea pig and cat

In the present work we have investigated the presence of the membrane proteins Syntaxin-1 and synaptosomal-associated protein (SNAP-25) by immunohistochemistry in the different parts of the pituitary of mouse, guinea pig and cat. We have demostrated Syntaxin-1 and SNAP-25 immunoreactivity in the adenohypophysis as well as in the neurohypophysis but not in intermediate lobe. The results suggest that Syntaxin-1 and SNAP-25 are involved in the hormonal secretory process of adenohypophysis as well as neurohypophysis of these animals.

Immunohistochemical study of the expression of human groEL-stress protein in human nervous tissue.

Monoclonal antibody (ML-30) directed against 65 kDa stress protein of mycobacteria, is shown to identify human cellular protein homologous with the groEL heat shock protein in many prokaryotes. Immunohistochemical survey of nervous tissue, both central and peripheral, from patients dying of various inflammatory, degenerative and neoplastic conditions and from experimental animals, using this antibody showed punctate granular staining of the cells to a variable degree. The astrocytes showed strong immunolabelling. The normal neurons and oligodendroglia stained variably, while abnormal neurons were darkly labelled. Ependymal cells showed apical granular positivity. The ubiquitinated inclusion bodies in amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease were not recognised by the ML-30 antibody. In diseased and stressed nervous tissue from experimental animals, the expression of the ML-30 recognisable stress protein was variable. The epitope recognised by ML-30 was found stable in postmortem tissues collected up to 36 h after death and processed for paraffin sectioning, after fixation in formalin for many years. Enhanced expression of the human groEL stress protein homologue in mammalian nervous tissue following various forms of stress may play a role in modulating the extent of tissue damage by autoimmune mechanism because of its high immunogenic nature and constitutive presence in the cells.