The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.     

Immunohistochemical labeling of Giardia trophozoites spp. in formalin fixed paraffin embedded tissues.

An immunoperoxidase method using rabbit anti human Giardia lamblia serum for the demonstration of giardia in paraffin embedded intestinal animal tissue is described. Specificity was tested against other protozoal parasites.     

Immunohistochemical detection of parainfluenza type 3 virus antigens in paraffin sections of pneumonic caprine lungs

Pneumonia is a leading cause of loss to ruminants throughout the world. Parainfluenza type-3 virus (PI-3) is one of the most important respiratory pathogens of bovine and ovine. In this study, prevalence of PI-3 virus infection as causative agent of pneumonia in goats was investigated. For this purpose, a total of 1505 goat lungs slaughtered in Bitlis and Van slaughterhouses were grossly examined and pneumonia was detected in 74 cases (4.91%). Lesions were more frequently encountered in anteroventral lobes than caudal lobes. With the exception of verminous pneumonia observed in 32 cases, immunohistochemical examinations were performed on 42 pneumonic lungs. Formalin-fixed and paraffin-embedded lung tissue samples were immunohistochemically stained by the avidin-biotin-peroxidase complex procedure using polyclonal antibodies to detect PI-3 viral antigens. The presence of PI-3 viral antigens was detected in 28 (66.6%) of 42 pneumonic lungs. Viral antigens were found most frequently in the cytoplasm of bronchiolar epithelial cells, type II pneumocytes, and less frequently in the epithelial cells of bronchial glands, syncytial cells, alveolar macrophages, and lymphocytes and plasma cells. In conclusion, it was found that there was a close relationship between the pneumonia in goats and the presence of PI-3 viral antigens. Incidence of PI-3 virus in pneumonic lungs of goats was detected to be very high in the present study performed in the region of Bitlis and Van, Turkey.     

Peroxidase-antiperoxidase labelling of Leishmania amastigotes in formalin fixed and paraffin embedded tissue sections.

A modified PAP-Method (peroxidase-antiperoxidase) is established for laboratory use to demonstrate Leishmania spp. amastigotes in paraffin sections of experimentally and naturally infected dogs. The results demonstrate that the PAP technique is a valuable tool to detect Leishmania amastigotes in tissue sections with low parasite density.     

Demonstration of bovine virus diarrhoea virus antigen in formalin fixed, paraffin embedded tissue using a streptavidin/biotin technique

The detection of bovine virus diarrhoea virus (BVDV) antigen in sections from formalin fixed, paraffin embedded tissue is described. Pre-digestion of the sections with 0.02 per cent protease XIV for 18 hours at 4 degrees C is necessary to unmask formalin fixed antigen. A hyperimmune antiserum prepared in a pig, using a combination of BVDV and hog cholera virus inoculations, linked to a biotinylated anti-pig/streptavidin peroxidase detection system demonstrated antigen in a wide range of tissues from cases of mucosal disease and persistently viraemic animals. The inclusion of a monoclonal anti-pig immunoglobulin linked to a biotinylated anti-mouse/streptavidin peroxidase detection system greatly reduced non-specific staining.     

Individual antigens of cattle. Differentiation antigens expressed predominantly on CD4- CD8- T lymphocytes (WC1, WC2).

The 14 mAbs representing workshop cluster 1 recognise a 215/300 kDa antigen expressed on a subpopulation of lymphocytes which express low levels of CD5 but are negative for other B and T cell markers defined by workshop antibodies. Separate studies with cDNA probes for bovine CD3 and T cell receptor indicate that these lymphocytes are gamma/delta T cells. It is of note that the different mAbs react with varying proportions of this cell population, suggesting that the antigen undergoes considerable post-translational modification. A further two mAbs, designated workshop cluster 2, react with a 37/47 kDa heterodimeric molecule expressed in a subpopulation of the WC1+ cells and on an additional small population of T lymphocytes. The cell populations recognised by the two mAbs are different although they overlap in some animals. It is suggested that these mAbs may be specific for T cell receptor molecules.     

Regulation of uncoupling proteins 2 and 3 in porcine adipose tissue

This study was performed to determine whether or not uncoupling protein 2 (UCP2) and UCP3 expression in porcine subcutaneous adipose tissue are hormonally regulated in vitro and whether their expression is correlated with changes in metabolic activity. Tissue slices (approximately 100 mg) were placed in 12-well plates containing 1 mL of DMEM/F12 with 25 mM Hepes, 0.5% BSA, pH 7.4. Triplicate slices were incubated with basal medium or hormone supplemented media at 37 °C with 95% air/5% CO₂. Parallel cultures were maintained for either 2 or 24 h to evaluate metabolic viability of the tissue. Slices were transferred to test tubes containing 1 mL of DMEM/F12 with 25 mM Hepes, 3% BSA, 5.5 mM glucose, 1 μCi ¹⁴C-U-glucose/mL and incubated for an additional 2 h at 37 °C. Glucose metabolism in 2-h incubations did not differ from 24-h (chronic) incubations, indicating viability was maintained (P > 0.05). Expression of UCP2 and UCP3 was assessed in slices following 24 h of incubation with various combinations of hormones by semi-quantitative RT-PCR. Expression of UCP2 was induced by leptin (100 ng/mL; P < 0.05). Growth hormone (100 ng/mL) inhibited UCP2 expression (P < 0.05). Expression of UCP3 was inhibited by growth hormone (100 ng/mL; P < 0.05), tri-iodothyronine (10 nM; P < 0.05) or leptin (100 ng/mL; P < 0.05). Changes in UCP expression could not be associated with overall changes in glucose metabolism by adipose tissue slices in chronic culture.     

Molecular cloning, tissue expression, and subcellular localization of porcine peptidoglycan recognition proteins 3 and 4

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are present in most invertebrates and vertebrates. Mammals have four PGRPs, PGLYRP1-4. In the present study, we cloned the cDNAs encoding porcine PGLYRP3 and 4 from the esophagus of adult swine. The length of the complete open reading frames of porcine PGLYRP3 and 4 are identical and contain 1125bp encoding 374 amino acid residues. The amino acid sequences of these two proteins were more similar to their human orthologs (78.9% [PGLYRP3] and 73.9% [PGLYRP4]) than to their mouse orthologs (71.3% [PGLYRP3] and 67.9% [PGLYRP4]). Expression analysis revealed that both PGLYRP3 and 4 were more strongly expressed in digestive tract, especially the esophagus, than in immune organs such as spleen or mesenteric lymph nodes in both newborn and adult swine. To analyze the subcellular distribution of porcine PGLYRP1-4, we constructed transfectant cell lines. Western blot and flow cytometric analyses revealed that porcine PGLYRP3 and 4 are not only secreted, but also expressed on the cell surface, unlike PGLYRP1 and 2. These results should help contribute to the understanding of PGLYRP3- and 4-mediated immune responses via their recognition of intestinal microorganisms in newborn and adult swine.     

Gene expression profiles of CD4/CD8 double‐positive T cells in porcine peripheral blood

A characteristic subset of T cells, known as double positive T cells (DPTC) and expressing both cluster of differentiation 4 (CD4) and CD8, is observed in porcine peripheral blood. Previous studies suggested that DPTC might be memory cells. However, detailed phenotypes and functions of DPTC are yet to be fully elucidated and thus, the relatedness of DPTC with memory phenotypes remains unclear. In this study, DPTC gene expression profiles in peripheral blood were analyzed by DNA microarray in Experiment 1 and compared with those of CD4 single positive T cells (4SPTC) and CD8 single positive T cells (8SPTC). Expressions of IFNG, CCL5, NCK2, CCR2 and ITGB1 were higher than that of 4SPTC and 8SPTC. In contrast, expressions of CCR7 and SELL were lower than that of 4SPTC and 8SPTC. These results suggested that DPTC were either effector T cells or effector memory T cells (T_EM). Next, to determine whether DPTC were effector T cells or T_EM, differences in the response of DPTC and 8SPTC against immunized/primed antigens were compared (Experiment 2). While DPTC showed quick elevation of IL2 and CD25 gene expressions against in vitro stimulation of primed/immunized antigens, 8SPTC did not. These results suggest that at least some DPTC likely belong to T_EM.     

Immunohistochemical detection of extrinsic and intrinsic mediators of apoptosis in porcine paraffin-embedded tissues

Apoptosis is a strictly regulated mechanism of cell death that involves a complex network of biochemical pathways. Whether a cell undergoes apoptosis or not depends on a delicate balance of anti- and pro-apoptotic stimuli. This phenomenon can be induced by two different pathways: intrinsic and extrinsic pathways. The main aim of this study was to determine the ideal fixative and antigen retrieval method in porcine paraffin embedded tissues for the immunohistochemical detection of apoptosis mediators, from both extrinsic and intrinsic pathways. Tonsil, retropharyngeal lymph node and lung tissue samples were fixed in 10% neutral buffered formalin, Bouin solution and zinc salts fixative (ZSF) and different unmasking methods were carried out. Both 10% neutral buffered formalin and ZSF resulted as the fixatives of election to study apoptosis phenomena. Tween 20 (0.01% in PBS), citrate buffer (microwave, pH 6.0) and/or protease type XIV were the antigen retrieval methods which displayed better labelling. Our results allow to deep in the knowledge of apoptosis and its role in the pathogenesis of porcine diseases.     

Immunohistochemical Detection of CD4-, CD8- and MHC II-Expressing Immune Cells and Endoglin in the Canine Corpus Luteum at Different Stages of Dioestrus

Specific immunohistochemical methods were applied to detect the presence of CD4-, CD8- and major histocompatibility complex II (MHC II)-expressing immune cells and of endoglin in the canine corpus luteum between days 15 and 75, after ovulation. Corpora lutea were obtained from groups of three clinically healthy beagle bitches, ovariohysterectomized at the respective days. For all four parameters, the effect of time was highly significant. Quantitative evaluation yielded high values on day 15, followed by a decrease on day 30 (CD4, CD8 and endoglin) and day 45 (MHC II). While there were no further changes for cells staining positive for CD4 and endoglin, CD8-positive immune cells increased from day 45 to day 60 to drop again on day 75; MHC II-positive staining increased from day 45 to days 60-75. These data suggest an involvement of the immune system in control of luteal function also in the dog that may have both stimulatory and inhibiting effects.     

Histopathological findings of the nasopharynx-associated lymphoid tissue of pigs co-infected with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus

Porcine circovirus 2 (PCV2) causes porcine circovirus-associated disease, and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) severely affects the pig breeding industry. Both viruses target the macrophages in lymphoid tissues. Various porcine pathogens enter via the nasal cavity, and the nasopharynx-associated lymphoid tissue (NALT) acts as the mucosal immune system. However, the pathological analysis has not progressed. This study aimed to histologically examine the NALT of pigs with suspected PCV2 and PRRSV infections. Six pigs were subjected to necropsy, and their NALT, tonsils, and mesenteric lymph nodes were collected. Macrophages, lymphocytic depletion, multinucleated giant cells, intracytoplasmic inclusion bodies, and neutrophil infiltration increased in the NALT. In situ hybridization revealed positive signals for PCV2 in the NALT of all pigs and PRRSV in the NALT of three pigs. PCV2-positive macrophages were mainly identified in the follicles, whereas PRRSV-positive tissues were found primarily around the crypt and directly below the epithelium. Quantitative PCR revealed 10⁸–10¹⁰ copies of PCV2 DNA/µL and 10²–10⁴ copies of PRRSV DNA/µL in the NALT. Therefore, both PCV2 and PRRSV were detected in the NALT of pigs. In conclusion, the infection and replication of both viruses in the NALT and tonsils may suppress host immunity and promote co-infection with other pathogens.     

猪CD8β基因的克隆、表达及其结构与功能分析

本研究应用RT-PCR技术,从猪胸腺细胞总RNA中扩增、克隆了猪CD8β基因,序列分析表明CD8β含621 bp的开放阅读框,编码207个氨基酸,与NCBI/GeneBank已发表的参考基因的核苷酸及推导氨基酸序列的同源性分别为97.8%和96.8%,与人、小鼠和鸡CD8B蛋白的氨基酸同源性分别为75.7%、67.9%和33.3%.根据大肠杆菌密码子偏嗜性改造目的基因,构建了pET28a/PCD8 β原核表达系统,并经诱导获得高效表达的分子量为24 ku的重组蛋白(rPCD8 β),表达量占菌体蛋白总量的30%.利用生物信息学和分子生物学软件对猪CD8 β基因编码的蛋白进行结构预测,表明猪CD8 β成熟蛋白为跨膜蛋白,其中172aa在胞外区,22aa在跨膜区,10aa在胞内区;蛋白骨架内含有较多的柔性区域,而且分布不均匀.能形成结构松散的球状蛋白;猪CD8 β分子V区三维结构与鼠的具有非常相似的空间结构,与人的差别较大,这为猪CD8B蛋白结构与功能的进一步研究奠定了基础.     

Polymorphism of the CD4 and CD5 differentiation antigens in cattle.

Monoclonal antibodies (mAbs) specific for bovine CD4 and CD5 antigens have been found to identify polymorphic determinants on these molecules. In the case of CD5, mAb IL-A67 recognises one allotypic form of the antigen while four other CD5-specific mAbs in the workshop (CC17, CC29, BLT-1 and 8C11) recognise a second allotype. The CD4-specific mAbs submitted to the workshop reacted with the cells of all animals tested. However, a further two mAbs (CC26 and IL-A18) specific for CD4 were found to react with cells only from about 85% of animals tested. Sequential immuno-precipitation experiments together with family studies showed that the allotypes of CD4 and CD5 are both inherited in a simple Mendelian manner and are co-dominantly expressed. One of the CD5 allotypes was not detected in Bos taurus animals while the gene frequency of the second allotype was only about 10% in the B. indicus animals tested. The gene frequency of the CD4 allotype detected by CC26 and IL-A18 was similar in the two sub-species.