Chronic hypoxia induced down-regulation of angiotensinogen expression in rat epididymis

The presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis has been previously established by showing the expression of several key RAS components, and in particular angiotensinogen, the indispensable element for the intracellular generation of angiotensin II. In this study, the possible involvement of this local epididymal RAS in the testicular effects of chronic hypoxia was investigated. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and by in situ hybridization histochemistry of the rat epididymis were used to show changes in localization and expression of angiotensinogen. Results from RT-PCR analysis demonstrated that chronic hypoxia caused a marked decrease (60%) in the expression of angiotensinogen mRNA, when compared with that in the normoxic epididymis. Western blot analysis demonstrated a less decrease (35%) in the expression of angiotensinogen protein. In situ hybridization histochemistry showed that the reduced angiotensinogen mRNA in chronic hypoxia was specifically localized to the epididymal epithelium from the cauda, corpus and caput regions of the epididymis; a distribution similar to that of normoxic rats. It was concluded that chronic hypoxia decreases the transcriptional and translational expression of angiotensinogen, and thus local formation of angiotensin II, in the rat epididymis. (C) 2001 Elsevier Science B.V. All rights reserved.

Fate of swallowed interleukin 8 and TNF alpha and effect on the wistar rat gut

To evaluate the passage of cytokines through the gastrointestinal tract, we investigated the digestion of interleukin-8 (IL-8) and tumour necrosis factor α (TNFα), in vitro and in vivo, and their propensity to induce intestinal inflammation. We serially immuno-assayed IL-8 and TNFα solutions co-incubated with each of three pancreatin preparations at pH 4.5 and pH 8. We gavaged IL-8, TNFα and marker into 15 Wistar rats, and measured their faecal cytokine concentrations by ELISA and histologically examined their guts. IL-8 immunoreactivity was extinguished by all pancreatin preparations after 1 h of incubation at 37 °C. TNFα concentration progressively fell from 1 to 4 h with all enzyme preparations. Buffer control samples maintained their cytokine concentrations throughout incubation. No IL-8 or TNFα was detected in any rat faecal pellets. There was no significant proinflammatory effect of the gavaged cytokines on rat intestine. IL-8 and TNFα in aqueous solution could well be fully digested in the CF gut when transit time is normal and exogenous enzymes are provided, although cytokines swallowed in viscous sputum may be protected from such digestion. Copyright © 2011 Elsevier B.V. All rights reserved

Accumulation of human heat shock protein 60-reactive T cells in the gingival tissues of periodontitis patients

Heat shock protein 60s (hsp60) are remarkably immunogenic, and both T-cell and antibody responses to hsp60 have been reported in various inflammatory conditions. To clarify the role of hsp60 in T-cell responses in periodontitis, we examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 and Porphyromonas gingivalis GroEL. To confirm the infiltration of hsp60-reactive T-cell clones into periodontitis lesions, nucleotide sequences within complementarity-determining region 3 of the T-cell receptor (TCR) beta-chain were compared between hsp60-reactive peripheral blood T cells and periodontitis lesion-infiltrating T cells. Periodontitis patients demonstrated significantly higher proliferative responses of PBMC to human hsp60, but not to P. gingivalis GroEL, than control subjects. The response was inhibited by anti-major histocompatibility complex class 11 antibodies. Analysis of the nucleotide sequences of the TCR demonstrated that human hsp60-reactive T-cell clones and periodontitis lesion-infiltrating T cells have the same receptors, suggesting that hsp60-reactive T cells accumulate in periodontitis lesions. Analysis of the cytokine profile demonstrated that hsp60-reactive PBMC produced significant levels of gamma interferon (IFN-gamma) in periodontitis patients, whereas P. gingivalis GroEL did not induce any, skewing toward a type1 or type2 cytokine profile. In control subjects no significant expression of IFN-gamma or interleukin 4 was induced. These results suggest that periodontitis patients have human hsp60-reactive T cells with a type I cytokine profile in their peripheral blood T-cell pools.

Differential expression and distribution of syndecan-1 and-2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell-cell and cell-matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell-cell and cell-matrix interactions, while syndecan-2 showed a predilection to associate with cell-matrix interactions during hard tissue formation.

Antigen-presenting cells in human periodontal disease tissues

T cells are present in the inflammatory infiltrates of periodontal disease lesions and require antigen presentation by antigen-presenting cells (APCs). While it is still not known whether Th1 or Th2 cells predominate in these lesions, it has been reported that different APCs may induce activation of different T-cell subsets. An immunoperoxidase technique was used to investigate the presence of CD1a+, CMRF-44+, CMRF-58+ and CD83+ dendritic cells, CD14+ macrophages or dendritic cell precursors and CD19+ B cells in gingival biopsies from 21 healthy or gingivitis and 25 periodontitis subjects. The samples were divided into three groups according to the size of infiltrate (group 1, small infiltrates; group 2, medium infiltrates; group 3, extensive infiltrates). The presence of numerous CD1a+ Langerhans cells was noted in the epithelium with no differences between the healthy/gingivitis and periodontitis groups. The percentage of CD83+ dendritic cells in the infiltrates was higher than the percentage of CD1a+, CMRF-44+ or CMRF-58+ dendritic cells. Endothelial cells positive for CD83 were found predominantly in areas adjacent to infiltrating cells, CD83+ dendritic cells being noted in the region of CD83+ endothelium. The percentage of CD14+ cells in the inflammatory infiltrates was similar to that of CD83+ dendritic cells. B cells were the predominant APC in group 2 and 3 tissues. The percentage of B cells in group 3 periodontitis lesions was increased in comparison with group 1 periodontitis tissues and also in comparison with group 3 healthy/gingivitis sections. Functional studies are required to determine the roles of different APC subpopulations in periodontal disease.

Quantitative evaluation of altered hepatic spaces and membrane transport in fibrotic rat liver

Four animal models were used to quantitatively evaluate hepatic alterations in this study: (1) a carbon tetrachloride control group (phenobarbital treatment only), (2) a CCl4-treated group (phenobarbital with CCl4 treatment), (3) an alcohol-treated group (liquid diet with alcohol treatment), and (4) a pair-fed alcohol control group (liquid diet only). At the end of induction, single-pass perfused livers were used to conduct multiple indicator dilution (MID) studies. Hepatic spaces (vascular space, extravascular albumin space, extravascular sucrose space, and cellular distribution volume) and water hepatocyte permeability/surface area product were estimated from nonlinear regression of outflow concentration versus time profile data. The hepatic extraction ratio of H-3-taurocholate was determined by the nonparametric moments method. Livers were then dissected for histopathologic analyses (e.g., fibrosis index, number of fenestrae). In these 4 models, CCl4-treated rats were found to have the smallest vascular space, extravascular albumin space, H-3-taurocholate extraction, and water hepatocyte permeability/surface area product but the largest extravascular sucrose space and cellular distribution volume. In addition, a linear relationship was found to exist between histopathologic analyses (fibrosis index or number of fenestrae) and hepatic spaces. The hepatic extraction ratio of H-3-taurocholate and water hepatocyte permeability/surface area product also correlated to the severity of fibrosis as defined by the fibrosis index. In conclusion, the multiple indicator dilution data obtained from the in situ perfused rat liver can be directly related to histopathologic analyses.

GABA(A) receptor sites in the developing human foetus

GABA(A) receptor sites were characterised in cerebral cortex tissue samples from deceased neurologically normal infants who had come to autopsy during the third trimester of pregnancy. Pharmacological parameters were obtained from homogenate binding studies which utilised the 'central-type' benzodiazepine ligands [H-3]diazepam and [H-3]flunitrazepam, and from the GABA activation of [H-3]diazepam binding. It was found that the two radioligands behaved differently during development. The affinity of [H-3]flunitrazepam for its binding site did not vary significantly between preparations, whereas the [H-3]diazepam K-D showed marked regional and developmental variations: infant tissues showed a distinctly lower affinity than adults for this ligand. The density of [H-3]flunitrazepam binding sites increased similar to35% during the third trimester to reach adult levels by term, whereas [H-3]diazepam binding capacity declined slightly but steadily throughout development. The GABA activation of [H-3]diazepam binding was less efficient early in the trimester, in that the affinity of the agonist was significantly lower, though it rose to adult levels by term. The strength of the enhancement response increased to adult levels over the same time-frame. The results strongly suggest that the subunit composition of cortical GABA(A) sites changes significantly during this important developmental stage. (C) 2002 Elsevier Science B.V. All rights reserved.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Dry matter production and boron concentrations of vegetative and reproductive tissues of canola and sunflower plants grown in nutrient solution

Canola (Brassica napus L.) and sunflower (Helianthus annuus L.), two important oilseed crops, are sensitive to low boron (B) supply. Symptoms of B deficiency are often more severe during the reproductive stage, but it is not known if this is due to a decreased external B supply with time or an increased sensitivity to low B during this stage. Canola and sunflower were grown for 75 days after transplanting (DAT) in two solution culture experiments using Amberlite (IRA-743) B-specific resin to maintain constant B concentration in solution over the range 0.6 - 53 muM. Initially, the vegetative growth of both crops was good in all treatments. With the onset of the reproductive stage, however, severe B deficiency symptoms developed and growth of canola and sunflower was reduced with less than or equal to 0.9 and less than or equal to 0.7 muM B, respectively. At these concentrations, reproductive parts failed to develop. The critical B concentration (i.e. 90% of maximum shoot dry matter yield) in the youngest opened leaf was 18 mg kg(-1) in canola and 25 mg kg(-1) in sunflower at 75 DAT. The results of this study indicate that the reproductive stage of these two oilseed crops is more sensitive than the vegetative stage to low B supply.

Long-lasting synaptic modification in the rat hippocampus resulting from NMDA receptor blockade during development

Recent reports have suggested that proper maturation of synapses in the hippocampus requires activation of NMDA receptors. We previously demonstrated that neonatal ethanol exposure results in a lasting reduction in synaptic strength in the hippocampus. To determine if this reduction was due to ethanol's effects on NMDA receptors, we investigated long-term changes in synaptic properties resulting from administration of NMDA receptor antagonists to neonatal animals. Rats were injected daily from PND 4-9 with either the noncompetitive NMDA receptor antagonist MK-801, the competitive NMDA receptor antagonist CPP, or the AMPA receptor antagonist NBQX. Control rats were either injected daily with physiological saline during the same period or left to develop normally. Hippocampal slices were prepared from nembutal-anesthetized animals between PND 35 and PND 40. The maximum pEPSP and PS values were not significantly different between controls and NMDA antagonist-treated animals. However, slices from animals injected with NMDA receptor antagonists required higher stimulus currents to attain comparable pEPSPs. The ratio of the slope of the pEPSP to the amplitude of the presynaptic volley was also reduced, as were pEPSP responses to specific stimulus currents. None of these effects were observed in slices prepared from animals treated with the AMPA receptor antagonist NBQX. Glutamate receptor antagonism did not produce lasting changes in long-term potentiation or paired-pulse facilitation. These results indicate activation of NMDA receptors during development is necessary for proper development of synapses. (C) 2001 Wiley-Liss, Inc.

Kainic acid induces 14-3-3 zeta expression in distinct regions of rat brain

Areas of the limbic system of adult male Wistar rats were screened for kainic-acid-induced gene expression. Polymerase-chain-reactionbased differential display identified a 147-bp cDNA fragment, which represented an mRNA that was upregulated in the entorhinal cortex and hippocampus in the kainic-acid-treated animals. The sequence was 97.8% homologous to rat 14-3-3 zeta isoform mRNA. Detailed Northern analysis revealed increased mRNA levels in the entorhinal cortex I h after kainic acid exposure and continued elevation 24 h post-injection in both the entorhinal cortex and hippocampus. Western blot analyses confirmed that the protein product of this gene was also present in increased amounts over the same time period. Immunohistochemistry and terminal transferase-mediated dUTP nick end labelling (TUNEL) detected expression of 14-3-3 protein exclusively in the entorhinal cortex and hippocampus, and only in TUNEL-positive neuronal cells. Expression of the tumor suppressor protein, p53 was also induced by kainate injection, and was co-localized with 14-3-3 zeta protein in selected cells only in the affected brain regions. The increase gene expression of 14-3-3 represents a transcription-mediated response associated with region selective neuronal damage induced by kainic acid. (C) 2002 Elsevier Science B.V. All rights reserved.