Evaluating antigen-specific cytotoxicity of CD8+ T cells in fish by granzyme B-like activity

Granzyme B plays an important role in granule-mediated apoptosis by CTL. It is a well characterized component of the cytolytic machinery in mammals and a candidate for the evaluation of cytotoxic activity of CTL as an alternative to conventional cytotoxicity assay. In this study, we examined the effects of granzyme inhibitors to assess the characteristics of fish granzymes in terms of substrate specificity and the involvement of granzyme B-like in the cytotoxic response. 3,4-dichloroisocoumarin (DCI), which inhibit the activity of serine protease including all members of the granzyme family, markedly suppressed the cytotoxic activity of CTL. However, CTL-mediated cytotoxicity was significantly but not completely suppressed by the addition of carbobenzyloxy-Ile-Glu-Thr-Asp-fluoromethyl ketone (Z-IETD-FMK) that specifically blocks granzyme B activity. These results suggest that additional serine proteases as well as granzyme B-like are involved in cytotoxicity of CTL in fish. We further compared cytotoxicity with the granzyme B-like hydrolytic activity against fluorogenic substrate acetyl-Ile-Glu-Thr-Asp-4-methylcoumaryl-7-amide (Ac-IETD-MCA) and found that granzyme B-like activity correlated well with the cytotoxicity of CTL in ginbuna crucian carp. Present results suggest that the granzyme activity assays is useful to assess cytotoxic activity of CTL in fish in which genetic information on granzymes and specific tools for cytotoxicity assay are not available because of well conserved catalytic triad residues and substrate binding sites in granzyme B throughout vertebrates.     

Inflammatory cell infiltration as an indicator of Staphylococcus aureus infection and therapeutic efficacy in experimental mouse mastitis

Staphylococcus aureus intramammary colonization of the mouse mammary gland induces migration of polymorphonuclear neutrophils (PMNs) similar to that observed during bovine mastitis. In the present study, a method combining acridine orange staining, fluorescence microscopy and computer-assisted image analysis has been developed to quantitate PMN infiltration in a mouse model of mastitis. This was carried out using paraffin embedded sections, and using this method, we showed that the presence of PMNs increased with the number of bacteria present in tissues. Nearly 400 and 1100 times more PMNs were counted in the mammary gland tissue after 12 and 24 h of infection, respectively, compared to mice infected for 6 h. Treatment with the antibiotic cephapirin at 10 or 25 mg/kg reduced PMN infiltration by 71 and 85%, respectively. In conclusion, this method can be used to quantitate PMN infiltration as a marker of inflammation and bacterial burden in infected tissue sections.     

Larval toxocarosis in sheep: the immunohistochemical characterization of lesions in some affected organs

Morphological and immunohistochemical responses of lambs following oral infection with 10,000 infective Toxocara canis (T. canis) eggs were studied up to 28 days post-infection (pi). The small intestine, liver, lungs and brain of both infected and control lambs were examined using the routine histological methods for paraffin sections and immunohistochemical techniques for frozen tissue sections. Eosinophil-rich hepatic granulomas and diffuse T. canis-induced pulmonary inflammation were the most prominent pathological features. CD2+, CD4+, CD8+, IgM bearing cells, and macrophages were demonstrated in the liver granulomas. The observed histomorphologic changes were similar to other paratenic hosts. It was concluded that larval toxocarosis followed the classical migratory path in the infected lambs.     

Immunohistochemical detection of SWC3, CD2, CD3, CD4 and CD8 antigens in paraformaldehyde fixed and paraffin embedded porcine lymphoid tissue

Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections with inherent poor tissue morphology, and are not readily adapted to formaldehyde fixed and paraffin embedded tissue with well preserved morphology. Seven well characterised monoclonal antibodies against porcine leukocyte antigens were tested on neutral buffered paraformaldehyde fixed and paraffin embedded porcine tissue sections using the highly sensitive tyramide signal amplification system. Combining this method with different antigen retrieval techniques enabled us to detect CD2, CD3, CD4, CD8 and SWC3 antigen expressing cells in porcine lymphoid tissue. Thus, we describe herein methods for the detection of several major cell types of the porcine immune system in fixed tissue with optimal preservation of histological details.     

Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples

Differentiating between inflammatory bowel disease (IBD) and small intestinal lymphoma in cats is often difficult, especially when only endoscopic biopsy specimens are available for evaluation. However, a correct diagnosis is imperative for proper treatment and prognosis. A retrospective study was performed using surgical and endoscopic intestinal biopsy specimens from 63 cats with a history of chronic diarrhea or vomiting or weight loss. A diagnosis of lymphoma or inflammation was based on microscopic examination of hematoxylin and eosin (HE)-stained sections alone, HE-stained sections plus results of immunohistochemical labeling (IHC) for CD3e and CD79a, and HE staining, immunophenotyping, and polymerase chain reaction (PCR) results for B and/or T cell clonality. In addition, various histomorphologic parameters were evaluated for significant differences between lymphoma and IBD using Fisher's exact test. The sensitivity and specificity of each parameter in the diagnosis of lymphoma were also determined. Results of Bayesian statistical analysis demonstrated that combining histologic evaluation of small intestinal biopsy specimens with immunophenotyping and analysis of clonality of lymphoid infiltrates results in more accurate differentiation of neoplastic versus inflammatory lymphocytes. Important histologic features that differentiated intestinal lymphoma from IBD included lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. Based on the results of this study, a stepwise diagnostic algorithm that first uses histologic assessment, followed by immunophenotyping and then PCR to determine clonality of the lymphocytes, was developed to more accurately differentiate between intestinal lymphoma and IBD.     

Demonstration of pseudorabies viral antigen in thick and ultrathin tissue sections of young pigs using an immunogold method

Pseudorabies (Aujeszky's) virus antigens were labeled in thick and ultrathin tissue sections of young pig brain and liver tissue, using an indirect immunogold method. Antigens were tagged with 20 nm gold particles. Controls proved the specificity of the reaction in paraffin sections and ultrathin epoxy sections. Immunogold staining was compared with immunoperoxidase staining in paraffin sections. In ultrathin sections stained with the immunogold method, the gold particles were present on viral nucleocapsids and viral envelopes, as well as on a number of other intracellular structures. These included the inner nuclear membrane, the nucleoplasm, intranuclear filaments, the endoplasmic reticulum, and free cytoplasmic polyribosomes. Gold particles were absent on mitochondria and microtubules. In paraffin sections, immunogold labeling for pseudorabies virus antigen was less sensitive than immunoperoxidase staining. Immunogold staining of ultrathin tissue sections can yield additional information on virus-host cell interactions.     

Reactivity of monoclonal antibodies to human CD antigens with cells from mink

Three hundred and seventy six monoclonal antibodies (mAbs) raised against human leukocyte surface antigens were analyzed by flow cytometry for cross reactivities against mink leukocytes. We found 53 mAbs (14%) to cross react. This study defined cross reactions to the following human markers: CD1a, CD9 (4 mAbs), CD10, CD11a (2 mAbs), CD14 (3 mAbs), CD18 (5 mAbs), CD20 (atypical reaction), CD21, CD25 (atypical reaction), CD29 (3 mAbs), CD32, CD41, CD42a, CD44 (4 mAbs), CD45, CD45RO, CD47 (2 mAbs), CD49d (3 mAbs), CD61 (2 mAbs), CD62P, CD66abcd, CD71, CD75s, CD79b (2 mAbs), CD86, CD88, CD104 (atypical reaction), CD172a, CD236R (glycophorin C, (atypical reaction)), Xg(a) carbohydrate antigen, Rhesus antigen and two unspecified PAN-reactive mAbs. In order to characterize the molecular mass of the corresponding cross reacting mink markers, the mAbs were used to immunoprecipitate the surface antigens. Fourteen mAbs out of the 53 mAbs reactive with mink leukocytes gave reproducible IP findings. The masses of the precipitated antigens were generally in good agreement with those of the homologous human markers. We also performed immunohistochemical staining analyses on formalin fixed, paraffin embedded mink tissue from lymph node and spleen, and found 7 out of 22 mAbs to give a positive signal. Generally, the immunohistological analyses resulted in expected staining patterns.     

A modified immunoperoxidase assay for detection of antibody porcine reproductive and respiratory syndrome (PRRS) virus in swine sera

MARC-145 cell monolayers infected with PRRS virus were fixed in 3% neutral buffered formalin and embedded in paraffin. The sections were stained by avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The positive reactions were detected by ABC method and indirect immunofluorescent assay (IFA). There was good correlation between ABC and IFA, and the titers in ABC were higher than those in IFA. The present results indicate that the immunohistochemical staining is a useful test for the detection and quantitation of PRRS virus antibody in swine sera as well as IFA.     

Canine intravascular lymphoma with overt leukemia

A 6-year-old spayed Labrador Retriever Mix dog was evaluated for a 2-week history of progressive generalized weakness and reluctance to stand. Physical examination revealed severe weakness with obtunded mentation, head tilt, bilateral nystagmus, and decreased vision. CBC findings included mild nonregenerative anemia, marked thrombocytopenia, and a few atypical mononuclear cells on the blood film. The cells were 15-30 μm in diameter and had round to oval to reniform centrally placed nuclei with stippled chromatin, prominent nucleoli, and abundant basophilic cytoplasm with numerous discrete vacuoles and, occasionally, small azurophilic granules. Similar cells were found in bone marrow. On histologic examination of tissues collected at necropsy, neoplastic cells were detected in bone marrow, hepatic sinusoids, cerebral and meningeal vessels, and in capillaries of the heart, renal interstitium, small intestinal submucosa, and muscularis, and alveolar septa. A small discrete mass in the right atrium consisted of similar neoplastic cells, and the spleen was diffusely infiltrated. Tissue distribution was suggestive of intravascular lymphoma. Neoplastic cells in tissue sections were immunoreactive for vimentin, CD18, CD45, and granzyme B and lacked immunoreactivity for cytokeratin. Neoplastic cells on bone marrow aspirate smears and blood films lacked immunoreactivity for CD3, CD79a, CD1c, CD11b, CD11c, CD11d, and E-cadherin. In the absence of immunophenotypic evidence for the neoplastic cells being derived from B-cell, T-cell, or histocytic/dendritic lineages and the lack of clonal antigen receptor gene rearrangement(s), along with positive immunoreactivity for granzyme B, a tumor of NK cells was considered likely. Based on current knowledge, this is the first report of canine intravascular lymphoma, of probable NK cell origin, with peripheral blood involvement.     

Viral interference with MHC class I antigen presentation pathway: the battle continues

CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in the defense against viral infections. In general, CD8+ CTLs recognize antigenic peptides in the context of the major histocompatibility complex (MHC) class I molecule. The MHC class I molecules are expressed on almost all the nucleated cells in the body. The trimolecular complex consisting of the class I heavy chain, beta2-microglobulin and the peptide are generated by the MHC class I antigen presentation pathway. This pathway is designed to sample the intracellular milieu and present the information to the CTLs trafficking the area. This rigorous sampling of intracellular environment enables the CTLs to quickly identify and eliminate the cells that synthesize non-self proteins as a result of a viral infection. Many viruses, including several viruses of veterinary importance, have evolved astounding strategies to interfere with the MHC class I antigen presentation pathway, as a means of evading the CTL response of the host. This review focuses on the diverse mechanisms of viral evasion of the MHC class I antigen presentation pathway with particular emphasis on viruses of veterinary importance.     

Immunohistochemical detection of avian pneumovirus in formalin-fixed tissues.

An immunohistochemical staining technique (IHC) was developed to detect avian pneumovirus (APV) antigen in formalin-fixed, paraffin-embedded tissue sections using streptavidin-biotin immunoperoxidase staining. Samples of nasal turbinates and infraorbital sinuses were collected from 4-week-old poults experimentally inoculated with APV and from older turkeys infected during naturally occurring outbreaks of avian pneumovirus. Tissue was fixed in 10% buffered neutral formalin, embedded in paraffin, sectioned and stained. Inflammatory changes were observed microscopically in the mucosa and submucosa of the nasal turbinates and infraorbital sinuses of both experimentally inoculated poults and naturally infected birds. Viral antigen was detected by IHC in the ciliated epithelial cells of nasal turbinates and infraorbital sinuses.     

Two cases of dermatophytic pseudomycetoma in the dog: an immunohistochemical study

Two cases of canine dermatophytic pseudomycetoma resulting in subcutaneous nodules resembling those previously reported in Persian cats are described. Culture performed from one nodule yielded dark yellow colonies consistent with Microsporum canis. Immunohistochemistry, using rabbit anti-M. canis, demonstrated specific binding to fungal elements in paraffin sections. The specificity of the antiserum was further tested by an agar gel immunodiffusion assay using a soluble extract from a feline isolate of M. canis as antigen. The antiserum did not cross-react with an Aspergillus fumigatus antigen. These are the first two reported cases of canine dermatophytic pseudomycetoma and immunohistochemical staining supported the diagnosis.     

The detection of bovine respiratory syncytial virus in formalin fixed bovine lung with commercially available monoclonal antibodies and avidin biotin complex immunohistochemistry.

Eight commercially available monoclonal antibodies directed against respiratory syncytial virus antigens were tested for ability to detect bovine respiratory syncytial virus (BRSV) antigen in formalin fixed, paraffin embedded bovine lung using avidin-biotin complex immunohistochemical staining. Monoclonal antibodies from clone 18B2 purchased from Biosoft, Paris, France and those from clone 8G12 purchased from the Department of Veterinary Sciences, University of Nebraska, Lincoln, Nebraska stained BRSV antigen in infected bovine lung with acceptable background staining of uninfected tissues. This method offers advantages over other techniques for BRSV diagnosis in that fresh tissue is not required and all reagents may be purchased commercially.     

Immunohistochemical study on the chicken adenohypophysis using monoclonal mouse anti-human FSH as a primary antibody

This experiment was conducted to investigate the use of monoclonal mouse anti-human FSH as a primary antibody for the immunohistochemical staining on chicken adenohypophysis paraffin sections. The use of monoclonal mouse anti-human primary antibody on chicken tissues has been developed. The protocol has been modified by comparing the results of the different groups. Briefly, this present protocol suggests modification in two areas; first, the antigen retrieval step, by placing the slides into the buffered citrate in microwave oven at 700 watt for 5 min, 3 times, and antigen-antibody reactions. Secondly, the incubation with the primary antibody (dilution 1/100 for anti-FSH) at 37 degrees C for 90 min.     

Changes in lymphocyte subsets in the bronchus-associated lymphoid tissue of goats naturally infected with different Mycoplasma species

The distribution of cells containing lysozyme, S-100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus-associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3-5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1-6), M. mycoides ssp. capri (goats no. 7-12) and M. capricolum ssp. capricolum (goats no. 13-18). Microscopically, infected animals showed a moderate broncho-interstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3+ T lymphocytes, and the ratio of CD4+:CD8+ cells was > 2. The BALT showed large germinal centres mainly composed of IgG+ B lymphocytes, with numerous S-100+ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG+ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.     

Multiple cutaneous histiocytomas in a cat

A 15-year-old domestic long haired cat developed nodular cutaneous masses over the right shoulder that were removed surgically. Similar masses developed in multiple cutaneous sites over the following three years. In each case, the lesions were characterized by diffuse dermal infiltration by histiocytic cells with a low mitotic rate and evidence of epidermotropism. The neoplastic population uniformly expressed class II molecules of the Major Histocompatibility Complex, but did not express the T and B lymphoid markers CD3 and CD79. Perivascular aggregates of CD3⁺ T lymphocytes were located at the deep margins of the tumour nodules. The clinical, histopathological and immunohistochemical features of these tumours are consistent with cutaneous histiocytoma. This tumour has not been previously well-documented in the cat.     

What is your diagnosis? Cervical mass in a cat

An 8-year-old female spayed domestic shorthair cat was presented for several months of weight loss, decreased appetite, and 2 bilateral, ventral cervical masses. Initial cytologic samples were interpreted as reactive lymphoid hyperplasia. Evaluation of subsequent mass aspirates revealed small numbers of large binucleated and multinucleated cells resembling Reed-Sternberg cells admixed with more numerous small and intermediate-sized lymphocytes. In histopathologic sections, the normal architecture of the lymph node was largely effaced by a slightly heterogeneous mass composed of round cells arranged in densely cellular sheets with a minor population of large (25-mum diameter) mononuclear cells and a few very large (30-40-mum diameter) binucleated or multinucleated cells interpreted as Reed-Sternberg-like cells. Immunohistochemically, the large neoplastic (Reed-Sternberg-like) cells were negative for CD18, CD3, CD20, and CD79a while the background population consisted of about 70% T cells and 30% B cells. This pattern of immunohistochemical staining along with cytologic and histopathologic findings supported a diagnosis of Hodgkin's-like lymphoma, specifically, the lymphocyte-rich subtype. Hodgkin's-like lymphoma has been reported previously in cats and should be suspected when Reed-Sternberg-like cells are observed in cytologic preparations of lymph node aspirates. Histopathology and immunohistochemistry are necessary for a definitive diagnosis.     

A retrospective study of the clinical, histological, and immunohistochemical manifestations of 5 dogs originally diagnosed histologically as necrotizing scleritis

Purpose To describe the clinical, histological, and immunohistochemical manifestations of canine necrotizing scleritis. Methods A retrospective examination of the clinical records and samples of ocular tissues from five dogs with a histological diagnosis ‘necrotizing scleritis’ was completed. Archived, formalin‐fixed, paraffin‐embedded samples and two control globes were stained with hematoxylin and eosin, Gram, periodic acid–Schiff (PAS) and Masson trichrome stains, and they were immunohistochemically labeled for CD3, CD18, and CD20. Results Of the five cases reviewed, only two could be confirmed as idiopathic necrotizing scleritis. The other three cases were retrospectively diagnosed as unilateral focal, non‐necrotizing scleritis, one as episcleritis and the third was scleritis secondary to a proptosed globe based on our retrospective clinical, histological, and immunohistochemical evaluations. In these two cases, idiopathic necrotizing scleritis manifested as a bilateral, progressive, inflammatory disease of the sclera and cornea that induces significant uveitis. Light microscopic examination confirmed collagen degeneration and granulomatous inflammation. There was no evidence for an infectious etiology based on Gram’s and PAS stainings. Immunohistochemical labeling revealed a predominance of B cells in idiopathic, bilateral necrotizing scleritis. Tinctorial staining abnormalities with Masson’s trichrome stain were present in scleral collagen of the two cases with idiopathic necrotizing scleritis as well as a case of secondary traumatic scleritis. Conclusions Based on a limited number of cases, idiopathic canine necrotizing scleritis shares similar histopathological features with non‐necrotizing scleritis and episcleritis; however, necrotizing scleritis is B‐cell‐dominated and bilateral, and significant collagen alterations manifest with Masson’s trichrome stain.     

Anti-CD71 antibody immunohistochemistry in the diagnosis of acute myeloid leukemia,subtype acute erythroid leukemia with erythroid dominance (AML M6-Er), in a retrovirus-negative cat

CD71 is an immunohistochemical marker used in diagnosing acute myeloid leukemia (AML) M6-Er in humans; however, to our knowledge, it has not been reportedly used for immunohistochemistry in veterinary medicine. We evaluated the pathologic features of AML M6-Er in a retrovirus-negative cat and used CD71 to support the diagnosis. A 4-y-old spayed female Scottish Fold cat was presented with lethargy, anorexia, and fever. Whole-blood PCR assay results for pro feline leukemia virus/pro feline immunodeficiency virus and feline vector-borne diseases were negative. Early erythroid precursors were observed in the peripheral blood smear. Fine-needle aspiration of the enlarged spleen and splenic lymph node showed many early erythroid precursors. Bone marrow aspirate smears revealed erythroid hyperplasia with 68.4% erythroid lineage and 3.6% rubriblasts. Dysplastic cells infiltrated other organs. The patient was diagnosed with myelodysplastic syndrome, progressing to the early phase of AML M6-Er. The patient died on day 121 despite multidrug treatments. Postmortem examination revealed neoplastic erythroblasts infiltrating the bone marrow and other organs. Neoplastic cells were immunopositive for CD71 but immunonegative for CD3, CD20, granzyme B, von Willebrand factor, CD61, myeloperoxidase, and Iba-1. Although further studies are necessary for the application of CD71, our results supported the morphologic diagnosis of AML M6-Er.     

Distribution of viral antigen in uterus, placenta and foetus of cattle persistently infected with bovine virus diarrhoea virus

The tissue distribution and cellular localisation of bovine virus diarrhoea virus (BVDV) was investigated in the uterus, placentomes, intercotyledonary foetal membranes and foetal organs of three persistently infected (PI) pregnant heifers. The uterus and ovaries of a non-pregnant PI heifer were also included in the study. Cryostat sections were examined using immunohistochemical techniques and monoclonal antibodies against BVDV. A double immunofluorescence technique was used to identify BVDV positive cells that also showed staining for either the leukocyte common antigen CD45 or the cytoskeletal filament vimentin. BVDV antigen was detected in all the organs examined, and was present in both epithelial and non-epithelial cells. In all organs many of the virus-positive cells also showed reactivity for vimentin. In the foetal liver and spleen a small, scattered population of virus-positive cells showed reactivity for CD45. A few cells showed reactivity both for BVDV antigen and for CD45 in the placentomes and intercotyledonary foetal membranes. In contrast to earlier reports, only scattered cells in the foetal part of the placentomes, the cotyledons, showed reactivity for BVDV antigen. However, in the chorion of the intercotyledonary foetal membranes, a larger proportion of the trophoblast cells showed reactivity for BVDV, especially the binuclear trophoblast cells. In the uterus, pregnancy appeared to favour virus replication, as the section from the pregnant heifers showed much stronger staining and a higher proportion of viral antigen-positive cells than sections from the non-pregnant PI heifer.