Extensive Staining Research Articles

Inducible nitric oxide synthase expression in N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis.

Nitric oxide (NO), an important regulatory molecule for immune response and cytotoxicity, is endogenously generated from L-arginine by NO synthase (NOS). One mechanism for NO-induced cytotoxicity is through its interaction with superoxide to produce peroxynitrite, which causes DNA damage. Three distinct isoforms of NOS have been isolated and represent the products of three different genes. The inducible form, inducible nitric oxide synthase (iNOS), is a mediator of inflammation and a regulator of epithelial cell growth. Upregulation of iNOS has been linked to epithelial tumorigenesis in various human and animal tissues. In the current investigation, normal esophagus and N-nitrosomethylbenzylamine (NMBA)-induced preneoplastic and papillomatous lesions of the rat esophagus were characterized for expression of iNOS. F344 rats were injected subcutaneously with NMBA (0.5 mg/kg body weight) three times per week for 5 wk. At 3, 6, 9, 12, 15, 18, 21, 24, 30, and 36 wk following initiation of NMBA treatment, esophagi were collected from 12 untreated and 12 NMBA treated animals. Results of reverse transcription (RT)-polymerase chain reaction (PCR) and immunohistochemistry demonstrated a correlation between the upregulation of iNOS and neoplastic progression in the rat esophagus. The expression of iNOS mRNA in preneoplastic tissues and papillomas was significantly elevated when compared to normal tissues. Immunohistochemical analysis showed more extensive cytoplasmic staining of iNOS protein in preneoplastic tissues and papillomas than in normal tissues. Our data suggest, therefore, that the production of iNOS by the epithelium of the esophagus is associated with the development of NMBA-induced esophageal tumorigenesis in rats.

Connexin 43 expression and distribution in compensated and decompensated cardiac hypertrophy in patients with aortic stenosis.

Gap junctions (GJ) are important determinants of conduction. In advanced heart failure alterations of the major ventricular GJ protein, connexin 43 (Cx43) are found. However, changes in Cx43 expression during the progression from compensated cardiac hypertrophy to heart failure, especially in humans, have not been studied extensively. The aim of the present study was to investigate changes in Cx43 expression and distribution in compensated and decompensated left ventricular (LV) hypertrophy in pressure-overloaded human hearts with valvular aortic stenosis (AS). We measured Cx43 levels by Western blot and quantitative immunoconfocal microscopy of LV septum biopsies from three groups of patients with AS (group I (n=9): ejection fraction (EF)>50%; group II (n=12): EF 30-50%; group III (n=9): EF<30%). LV biopsies from six patients with mitral valve stenosis and two donor hearts served as controls. Only the early phase of LV hypertrophy (AS-I) was characterized by extensive Cx43 lateral staining. As compared to controls, the AS-I group showed a 44.3% increase in Cx43 protein, which was reflected in an augmented number of GJs per 100 microm(2) intercalated disc area (control: 62.5+/-6.4 vs. AS-I: 79.8+/-4, p<0.001) and an increased GJ surface density (control: 0.00547 vs. AS-I: 0.00724 microm(2)/microm(3), p<0.01). Decompensated LV hypertrophy (AS-III) was specified by reduced percentage of the Cx43 signal per myocyte area (control: 1.74% vs. AS-III: 1.31%, p<0.01) or per intercalated disc (control: 18.3% vs. AS-III: 11.3%, p<0.005). Mean GJ area and GJ number per intercalated discs in the AS-III group were decreased significantly by, respectively, 42.5% and 36.4% as compared to control. In addition, decompensated LV myocardium showed a markedly heterogeneous spatial distribution of Cx43. The quantity and spatial distribution of Cx43 differs markedly between compensated and decompensated LV hypertrophy in human patients with AS. Upregulation of Cx43 in compensated hypertrophy may represent the immediate adaptive response to increased load, whereas diminished and heterogeneous Cx43 distribution in decompensated hypertrophy may play maladaptive roles culminating in heart failure and ventricular arrhythmias.

Osteogenic differentiation of human bone marrow-derived mesenchymal cells cultured on alumina ceramics.

Alumina ceramics have excellent mechanical and biocompatible properties, but are bioinert and hence have no bone-bonding properties. We took a tissue-engineering approach in an attempt to modify the ceramic surface and so provide an osteogenic/osteoconductive milieu. We obtained human bone marrow mesenchymal cells from four donors and then cultured the cells for two weeks on alumina ceramic in the presence of beta-glycerophosphate, ascorbic acid and dexamethasone. The cells showed extensive alkaline phosphatase staining and mineralization, as evidenced by Alizarin Red S staining and calcein uptake. Biochemical analyses revealed high levels of alkaline phosphatase activity, osteocalcin expression and calcium content. This data indicates the appearance of active osteoblasts that are concomitant with bone matrix formation, i.e., in vitro cultured bone. The cultured bone/alumina composites should prevent the aseptic loosening of all-alumina ceramic joints or the detachment of implanted alumina ceramics, and thus could have clinical significance in orthopedic reconstructive surgery.

Opioid modulation of GABA release in the rat inferior colliculus

BackgroundThe inferior colliculus, which receives almost all ascending and descending auditory signals, plays a crucial role in the processing of auditory information. While the majority of the recorded activities in the inferior colliculus are attributed to GABAergic and glutamatergic signalling, other neurotransmitter systems are expressed in this brain area including opiate peptides and their receptors which may play a modulatory role in neuronal communication.ResultsUsing a perfusion protocol we demonstrate that morphine can inhibit KCl-induced release of [3H]GABA from rat inferior colliculus slices. DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin) but not DADLE ([D-Ala2, D-Leu5]-enkephalin or U69593 has the same effect as morphine indicating that μ rather than δ or κ opioid receptors mediate this action. [3H]GABA release was diminished by 16%, and this was not altered by the protein kinase C inhibitor bisindolylmaleimide I. Immunostaining of inferior colliculus cryosections shows extensive staining for glutamic acid decarboxylase, more limited staining for μ opiate receptors and relatively few neurons co-stained for both proteins.ConclusionThe results suggest that μ-opioid receptor ligands can modify neurotransmitter release in a sub population of GABAergic neurons of the inferior colliculus. This could have important physiological implications in the processing of hearing information and/or other functions attributed to the inferior colliculus such as audiogenic seizures and aversive behaviour.

A reappraisal of the Rb1 gene abnormalities in the diagnosis of parathyroid cancer.

Some histological features may suggest the malignant nature of a parathyroid tumour. However, the diagnosis of parathyroid cancer can only be definitively established in the presence of local invasion or metastases. We further investigated the role of the retinoblastoma gene (Rb1) and the breast cancer susceptibility gene (BRCA2) in the differential diagnosis between benign and malignant parathyroid tumours by evaluating loss of heterozygosity (LOH) at these loci and Rb protein (pRb) immunohistochemistry. Fifty-three parathyroid adenomas from patients with sporadic primary hyperparathyroidism (PHPT) and 10 parathyroid cancer specimens were studied. Microsatellite polymorphisms at the Rb1 and BRCA2 loci were polymerase chain reaction (PCR) amplified from each patient's paired tumour and leucocyte DNA samples, using oligonucleotide primers flanking the repeat sequence. Immunohistochemical staining of pRb was carried out using a monoclonal antibody. All but one of the 53 tumour-leucocyte pairs was informative for at least one of the three polymorphic markers of the Rb1 gene. Fifteen adenomas (28.8%) showed LOH. Regarding the BRCA2 gene, 46 tumour-leucocyte pairs were informative and LOH was present in eight (17.4%). All six carcinomas had LOH for at least one marker at the Rb1 locus. LOH for the BRCA2 microsatellite was found in three of the five informative primary tumour samples. Immunohistochemical analysis revealed that all adenomas were positive and the number of pRb-positive cells varied significantly among different samples. The mean percentage of stained cells was 15.7%. Eleven of the 30 (36.7%) adenomas showed sparse positive staining, 13 (43.3%) intermediate staining and six (20%) extensive staining. All parathyroid cancers were entirely negative for pRb immunostaining. Inactivation of the Rb1 gene is a common event in parathyroid tumorigenesis. Retention of heterozygosity seems to exclude parathyroid malignancy, which is suggested by the combined finding of LOH and lack of protein expression.

Histologic and Immunohistochemical Study of Bone Marrow Monocytic Nodules in 21 Cases With Myelodysplasia

Previously, 4 cases of myelodysplastic syndrome were reported that had unusual, distinct monocytic nodules in bone marrow. The monocytic nodules, predominantly composed of monocytes with CD68+ immunostaining, had no or low expression of Ki-67 and topoisomerase IIα. The purpose of the present study was to further define the associated clinical diseases, histologic features, and immunohistochemical characteristics of 21 such cases. Relevant hematopathologic slides of all cases were reviewed, and extensive immunohistochemical staining was performed. Most patients (15/21 [71%]) had monocytosis in the bone marrow, and chronic myelomonocytic leukemia was the most commonly associated clinical disease. In 4 patients, the monocytic nodules also were present in lymph node, spleen, or skin. Immunohistochemical staining results for the monocytes in the nodules were similar to those for plasmacytoid monocytes. Our study established that monocytic nodules can be present in myelodysplastic syndromes, myeloproliferative diseases, and acute myeloid leukemia; verified the monocytic lineage; and revealed the low proliferative state of these cells.

Histologic and Immunohistochemical Study of Bone Marrow Monocytic Nodules in 21 Cases With Myelodysplasia

Previously, 4 cases of myelodysplastic syndrome were reported that had unusual, distinct monocytic nodules in bone marrow. The monocytic nodules, predominantly composed of monocytes with CD68+ immunostaining, had no or low expression of Ki-67 and topoisomerase IIα. The purpose of the present study was to further define the associated clinical diseases, histologic features, and immunohistochemical characteristics of 21 such cases. Relevant hematopathologic slides of all cases were reviewed, and extensive immunohistochemical staining was performed. Most patients (15/21 [71%]) had monocytosis in the bone marrow, and chronic myelomonocytic leukemia was the most commonly associated clinical disease. In 4 patients, the monocytic nodules also were present in lymph node, spleen, or skin. Immunohistochemical staining results for the monocytes in the nodules were similar to those for plasmacytoid monocytes. Our study established that monocytic nodules can be present in myelodysplastic syndromes, myeloproliferative diseases, and acute myeloid leukemia; verified the monocytic lineage; and revealed the low proliferative state of these cells.

Immunostaining for the tumour suppressor gene p16 product is a useful marker to differentiate melanoma metastasis from lymph-node nevus.

Upon the introduction of extensive sampling protocols of sentinel node biopsies, pathologists are increasingly confronted with small melanoma metastases. Using conventional histology, it proves sometimes difficult or impossible to differentiate small melanoma metastases from lymph-node nevi. Loss of the tumour suppressor gene p16 has been shown to be associated with tumour progression of melanoma. We investigated nevus and melanoma cells for the presence of the product of the gene p16, using immunohistochemistry. All nevus cells, independent of their location (nodal or skin) displayed an extensive nuclear and cytoplasmic staining for p16. In contrast, all cells of melanoma metastases, except one skin metastasis, lacked nuclear staining for p16. These findings indicate that p16 is a reliable marker to distinguish lymph-node nevi from melanoma metastasis.

Aminophospholipids Have No Access to the Luminal Side of the Biliary Canaliculus: IMPLICATIONS FOR THE SPECIFIC LIPID COMPOSITION OF THE BILE FLUID

About 95% of the bile phospholipids are phosphatidylcholine. Although the fractions of phosphatidylcholine and of both aminophospholipids phosphatidylserine and phosphatidylethanolamine in the canalicular membrane are in the same order of about 35% of total lipids, both aminophospholipids are almost absent from the bile. To rationalize this observation, we studied the intracellular uptake of various fluorescent phospholipid analogues and their subsequent enrichment in the bile canaliculus (BC) of HepG2 cells. Diacylaminophospholipid analogues but not phosphatidylcholine analogues became rapidly internalized by an aminophospholipid translocase (APLT) activity in the plasma membrane of HepG2 cells. We observed only low labeling of BC by diacylaminophospholipids but extensive staining by phosphatidylcholine analogues. In the presence of suramin, known to inhibit APLT, a strong labeling of BC by diacylaminophospholipid analogues was found that declined to a level observed for control cells after removal of suramin. Unlike diacylphosphatidylserine, diether phosphatidylserine analogue, which is not an appropriate substrate of APLT, accumulated in the BC. The correlation between low labeling of BC and an APLT-mediated transbilayer movement suggests the presence of an APLT activity in the canalicular membrane that prevents exposure of aminophospholipids to the bile.

Cdx2 Protein Expression in Normal and Malignant Human Tissues: An Immunohistochemical Survey Using Tissue Microarrays

Cdx2 has been identified as a marker of colon cancer in RNA-profiling experiments. We show here that the detection of Cdx2 protein by immunohistochemistry correlates well with RNA transcript levels as detected by oligonucleotide microarrays. Using tissue microarrays containing most normal tissue types and an antibody to the Cdx2 protein, strong diffuse Cdx2 staining was only seen in the nuclei of small and large intestinal epithelium and portions of the pancreatic duct system. In tissue microarrays containing 745 cancers from many anatomic sites, colonic adenocarcinomas showed strong extensive staining in 90% of cases, with adenocarcinomas of the stomach, esophagus, and ovary (endometrioid and mucinous types) showing extensive staining in only 20–30% of cases. Other types of carcinomas showed extensive staining in only ≤1% of cases. Of 30 neuroendocrine tumors examined, carcinoids of the midgut and hindgut had the most cases with extensive staining (73% and 44%, respectively), thus paralleling the distribution of Cdx2 expression in adenocarcinomas. Cdx2 shows a limited range of expression in the spectrum of human tissues and neoplasia and thus may have utility in determining the site of origin of tumors in certain clinical situations.

Abnormalities in bone mineral density and bone histology in thalassemia.

This study demonstrated that there was extensive iron staining on trabecular surface and marked reduction in trabecular bone volume without significant alteration in bone formation and bone resorption rates as well as significant reduction in bone mineral density in 18 thalassemic patients. Serum IGF-I was reduced and may modulate the reduction of bone mass. Bone histomorphometric studies in thalassemia to show alterations in bone histology and their relationship to biochemical parameters are very limited. Therefore, this study was systematically conducted to determine the alterations in thalassemia patients. Serum biochemical parameters, trans-iliac crest bone biopsy, and determination of bone mineral density of femur and lumbar spine were done in 18 thalassemic patients (10 females and 8 males). Serum osteocalcin, carboxy terminal teleopeptide fragment of type I collagen, and parathyroid hormone levels were within normal limits, but serum 25(OH) vitamin D (19.3 +/- 1.6 ng/ml) and 1,25(OH)2 vitamin D (33.77 +/- 1.51 pg/ml) levels were decreased. Serum insulin-like growth factor I (IGF-I; 145.2 +/- 20 ng/ml) was suppressed, whereas serum ferritin (1366.6 +/- 253.9 ng/ml) was markedly elevated. Reduced bone mineral density was found in all studied areas. Trabecular bone volume was significantly decreased (16.65 +/- 1.12%), whereas bone formation rate, eroded surface, and other bone histomorphometric parameters were within normal limits. The trabecular bone volume varied significantly with bone mineral density of total femur (r = 0.48, p = 0.04). There was an extensive stainable iron surface on the mineral front (9-60%). Significant correlation between serum IGF-I, serum ferritin, stainable iron surface, and bone mineral density, lumbar spine, and total femur were found. Serum IGF-I correlated with trabecular bone volume (r = 0.6, p = 0.03), inversely with both serum ferritin level (r = -0.6, p < 0.01), and inversely with stainable iron surface (r = -0.53, p = 0.02). Multiple regression analysis demonstrated that IGF-I was the only independent variable that determined bone mineral density of lumbar spine and total femur. Low bone mineral density and reduced trabecular bone volume with extensive iron deposition are the predominant findings in thalassemic patients. There was no evidence of increased bone resorption or mineralization defect. A reduction in circulatory IGF-I may modulate the reduction of bone mass.

Multiple colonic pseudopolyposis due to amyloidosis; presenting with lower gastrointestinal bleed

Gastrointestinal involvement (mucosal or neuromuscular) is common in patients with primary and secondary amyloidosis. Mucosal lesions, ulcers or pseudopolypi (submucosal polyps) are commonly seen in stomach or proximal small bowel and are occasional source of overt or occult gastrointestinal bleed. We present a patient with diffuse polypoid lesions in colon presenting with lower gastrointestinal bleed. A 51 year-old healthy black male presented with one year history of intermittent hematochezia without any local or systemic symptoms. Physical exam was remarkable for palor without any peripheral lymphadenopathy or hepatosplenomegaly. Laboratory evaluation revealed Hct 23.0 ml/dl, MCV 65/ m3 Ferritin 12ng/ml, BUN 12mg/dl. Creatinine 1.0mg/dl. Serum electrophoresis was normal. Colonoscopy showed multiple polypoid lesions varying from 0.5 cm to 1.5 cms distributed throughout colon. Biopsy showed extensive submucosal amorphous eosinophilic material and positive staining with Congo red consistent with amyloidosis. Enteroscopic examination revealed extensive polypoid lesions in stomach and proximal small bowel. Cardiac echocardiogram was normal. Patient continued to have intermittent hematochezia requiring frequent blood transfusions and iron replacement. Gastrointestinal amyloidosis is a rare cause of gastrointestinal bleeding and colonic pseudopolypi due to submucosal amyloid deposit may manifest with lower gastrointestinal tract bleeding.

Role of lipid peroxidation as a mechanism of liver injury after acetaminophen overdose in mice.

Mitochondrial oxidant stress and peroxynitrite formation have been implicated in the pathophysiology of acetaminophen-induced (AAP-induced) liver injury. Therefore, we tested the hypothesis that lipid peroxidation (LPO) might be involved in the injury mechanism. Male C3Heb/FeJ mice fed a diet high in vitamin E (1 g d-alpha-tocopheryl acetate/kg diet) for 1 week had 6.7-fold higher hepatic tocopherol levels than animals on the control diet (8.2 +/- 0.1 nmol/g liver). Treatment of fasted mice with 300 mg/kg AAP caused centrilobular necrosis with high plasma alanine aminotransferase (ALT) activities at 6 h (3280 +/- 570 U/l) but no evidence of LPO (hepatic malondialdehyde, 4-hydroxynonenal). Animals on the vitamin E diet had similar injury and LPO as mice on the control diet. To verify a potential effect of the vitamin E diet on drug-induced liver injury, animals were pretreated with a combination of phorone, FeSO4, and allyl alcohol. We observed, 2 h after allyl alcohol, massive LPO and liver cell injury in the livers of animals on the control diet, as indicated by a 32-fold increase in malondialdehyde levels, extensive staining for 4-hydroxynonenal, and ALT activities of 2310 +/- 340 U/l. Animals on the vitamin E diet had 40% lower hepatic malondialdehyde levels and 85% lower ALT values. Similar results were obtained when animals were treated for 3 days with alpha- or gamma-tocopherol (0.19 mmol/kg, ip). Both treatments reduced LPO and injury after allyl alcohol but had no effect on AAP hepatotoxicity. Thus, despite the previously shown mitochondrial oxidant stress and peroxynitrite formation, LPO does not appear to be a critical event in AAP-induced hepatotoxicity.

C-reactive protein correlates with macrophage accumulation in coronary arteries of hypercholesterolemic pigs.

A growing body of evidence supports the hypothesis that C-reactive protein (CRP) is a marker of inflammation in coronary artery disease. The purpose of the present study was to test the hypothesis that CRP correlates with macrophage accumulation during the initial stages of coronary vascular disease. Adult male pigs were fed a normal chow (NF) or a high-fat high-cholesterol (HF) diet for 20 wk. After 20 wk, blood was collected for analyses of interleukin-6 (IL-6), CRP, and lipids. After blood collection, the pigs were euthanized and the right coronary arteries (RCA) were harvested and fixed in neutral buffered formalin. Paraffin-embedded sections of RCA were stained immunohistochemically for CRP, scavenger receptor A (SRA), and monocyte chemoattractant protein-1 (MCP-1). All cholesterol fractions were elevated in the HF vs. the NF group (P < 0.05). There was little or no positive staining for CRP, SRA, or MCP-1 in the RCA of NF pigs, but there was extensive staining in lipidladen macrophage foam cells in the HF pigs. Double staining revealed colocalization of CRP with SRA and CRP with MCP-1 in foam cells. Serum IL-6 was below the assay detection limit in all pigs. Serum CRP correlated directly with plasma total cholesterol (R = 0.727, P = 0.041) and accumulation of SRA-positive macrophages (R = 0.938, P < 0.001) in RCA of HF pigs. We conclude that serum CRP correlates with macrophage accumulation and coronary artery disease in hypercholesterolemic pigs.

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs.

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.

Role of catenins in the development of gap junctions in rat cardiomyocytes

Gap junctions are intercellular communicating channels responsible for the synchronized activity of cardiomyocytes. Recent studies have shown that the membrane-associated guanylate kinase protein, zonula occludens-1 (ZO-1) can bind to catenins in epithelial cells and act as an adapter for the transport of the connexin isotype, Cx43 during gap junction formation. The significance of catenins in the development of gap junctions and whether complexes between catenins and ZO-1 are formed in cardiomyocytes are not clear. In this study, immunofluorescence and confocal microscopy showed sequential redistribution of alpha-catenin, beta-catenin, ZO-1, and Cx43 to the plasma membrane when rat cardiomyocytes were cultured in low Ca(2+) (<5 microM) medium, then shifted to 1.8 mM Ca(2+) medium (Ca(2+) switch). Diffuse cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen in the cytoplasm when cardiomyocytes were cultured in low Ca(2+) medium. Staining of alpha-catenin, beta-catenin, and ZO-1 was detected at the plasma membrane of cell-cell contact sites 10 min after Ca(2+) switch, whereas Cx43 staining was first detected, colocalized with ZO-1 at the plasma membrane, 30 min after Ca(2+) switch. Distinct junctional and extensive cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen 2 h after Ca(2+) switch. Immunoprecipitation of Triton X-100 cardiomyocyte extracts using anti-beta-catenin antibodies showed that beta-catenin was associated with alpha-catenin, ZO-1, and Cx43 at 2 h after Ca(2+) switch. Intracellular application of antisera against alpha-catenin, beta-catenin, or ZO-1 by electroporation of cardiomyocytes cultured in low Ca(2+) medium inhibited the redistribution of Cx43 to the plasma membrane following Ca(2+) switch. These results suggest the formation of a catenin-ZO-1-Cx43 complex in rat cardiomyocytes and that binding of catenins to ZO-1 is required for Cx43 transport to the plasma membrane during the assembly of gap junctions.

The expression of excitatory amino acid transporter 2 in traumatic brain injury

It is well recognized that glutamate is the major excitatory neurotransmitter, which is removed from the synaptic cleft by excitatory amino acid transporter 2 (EAAT2) located on the perisynaptic astrocytes and that neuronal death has been associated with an increased extracellular glutamate concentration. In this study, we have immunohistochemically demonstrated the expression of EAAT2 protein in the human brain after traumatic brain injury (TBI). The EAAT2 expression patterns can be divided into three types: continuous and highly extensive staining (E); continuous but sporadic staining (M); and sporadic pattern staining (S). In six of the nine short survival cases studied (1 h to 1 day), continuous and highly extensive staining for EAAT2 (E type) was observed in the ipsilateral cerebral cortex. On the other hand, we were able to demonstrate weak staining (S and M types) in 5 of the 7 long survival cases (≥1 day) and in 12 of the 14 very short survival cases (<1 h) studied. Similar findings were obtained in the contralateral cerebral cortex and also in the ipsilateral hippocampus. In addition, positive staining for glial fibrillary acidic protein was detected around the cerebral contusion, but the EAAT2-positive expression was not observed in the same region for all of the six short and long survival cases (≥1 h) after TBI. These findings clearly showed the differences in EAAT2 expression in the cerebral cortex according to the survival time and severity of cerebral contusion after TBI. Therefore, we emphasized that EAAT2 might play an important role in contributing to extracellular glutamate concentrations and secondary brain injury after TBI.

The role of the Candida albicans histidine kinase [CHK1) gene in the regulation of cell wall mannan and glucan biosynthesis.

The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.

Immunolocalization of vesicular stomatitis virus in black flies (Simulium vittatum).

Vesicular stomatitis, a disease of cattle, horses, and swine, is caused by either vesicular stomatitis virus, New Jersey serotype (VSV-NJ), or vesicular stomatitis virus, Indiana serotype, which are related viruses in the genus Vesiculovirus, family Rhabdoviridae. Although recognized for at least 160 years, the epidemiology and pathogenesis of this disease remains undefined. Black flies have been suggested as a vector for VSV-NJ. In this study we infected three- to four-week-old female black flies with VSV-NJ via feeding of virus-spiked ox blood or intrathoracic inoculation, and demonstrated the location of virus by immunohistochemistry. These preliminary findings suggest that VSV-NJ initially infects the gut in the natural situation but that subsequent spread to the salivary gland may be blocked in older flies, decreasing their ability to transmit the virus. The pattern of staining was different in intrathoracic inoculated flies. In these flies, salivary gland involvement was more likely, and extensive staining of eye, brain, and hemolymph suggested a more generalized infection that apparently circumvented the gut. We conclude that intrathoracic inoculation may be an inappropriate method of infection for determining vector competence and that the age of the vector should be considered when conducting competency studies.

Tissue distribution of avian infectious bronchitis virus following in ovo inoculation of chicken embryos examined by in situ hybridization with antisense digoxigenin-labeled universal riboprobe.

Chicken embryos were inoculated with 8 different strains of infectious bronchitis virus (IBV) representing 7 different serotypes at 17 days of embryonation. At 2 and 5 days postinfection (dpi), tissues were collected for in situ hybridization using an antisense digoxigenin-labeled riboprobe corresponding to the sequence of the mRNA coding for the membrane protein. Extensive antigen staining in the cytoplasm of epithelial cells in the trachea, lung, bursa, and intestine was detected at 2 dpi with all 8 strains of IBV. At 5 dpi, little or no positive staining was observed in these tissues. However, tubular cells of the kidney showed multifocal positive staining with the Wolgemuth strain-, Gray strain-, JMK strain-, and Mass41 strain-infected chickens. No viral RNA was detected in the spleen at any time point. The results demonstrated strict epitheliotropic nature and wide tissue tropism of strains of IBV in the chicken embryo and the universality of our riboprobe. In situ hybridization with this probe will be useful for understanding the tissue tropism and the pathogenesis of IBV in vivo.