Manfred Henrich

A new subgroup of immunoglobulin heavy chain variable region genes for the assessment of clonality in feline B-cell lymphomas

In human medicine, PCR-amplification of the complementarity determining region 3 of the immunoglobulin heavy chain genes followed by polyacrylamide gel electrophoresis (PAGE) is an accepted method to assess clonality in B-cell lymphomas and thereby facilitates the differentiation of neoplasias from benign hyperplasias or reactive infiltrates. To generate a basis for the development of a PCR-based assay for the assessment of clonality in feline B-cell lymphomas we analyzed 28 transcripts (cDNA) of the feline immunoglobulin heavy chain variable region genes (IGHV). Transcripts were generated using techniques for the amplification of unknown sequences (i.e. the SMART RACE and the CapFishing technique) as well as primers derived from sequences of the NCBI Trace archive of the cat. Analysis of this archive revealed traces similar to the human IGHV-1 and IGHV-3 subgroups of genes. By identification of the subgroup-specific leader sequence within the traces, two subgroup-specific primers for this region were designed and used to amplify the heavy chain variable region genes. Using all amplification techniques, transcripts of both subgroups were created and the subgroups were denominated according to their human counterparts as feline IGHV-1 and feline IGHV-3. By aligning previously described transcripts of the feline IGHV genes to our transcripts we were able to assign these to the IGHV-3 subgroup; therefore, this study provides the first description of the feline IGHV-1 subgroup of genes. On the basis of the IGHV-1 and IGHV-3 transcripts we developed a PCR-based assay. For each of the two subgroups we used one sense primer derived from the first and one sense primer derived from the third framework region each in combination with a mixture of three antisense primers derived from the fourth framework region. With these four sets of primers, the assay was able to detect monoclonality in 7/10 (70%) cats with histologically and immunohistochemically diagnosed B-cell lymphomas. In two of these cases, monoclonal rearrangement of the IGHV genes was only detectable with IGHV-1 subgroup-specific primers. Amplification of feline hyperplastic lymphatic tissue only gave results indicative of polyclonal populations. The use of a PCR-based assay in combination with standard techniques for the diagnosis of feline lymphoma is helpful and the characterization of the additional subgroup of feline variable regions genes puts this assay on a broader basis.

Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma.

BackgroundIn a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses.ResultsThe FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence.ConclusionsOur results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus.

Stimulation of duodenal biopsies and whole blood from dogs with food-responsive chronic enteropathy and healthy dogs with Toll-like receptor ligands and probiotic Enterococcus faecium.

The composition of the microbiome plays a significant role in the pathogenesis of inflammatory bowel disease (IBD) in humans and chronic enteropathies (CE) in dogs. The administration of probiotic micro‐organisms is one way of modulating the microbiome, but experiments elucidating mechanisms of action of probiotics in the intestine of healthy and CE dogs are lacking. The aim of our study was to investigate the effects of different Toll‐like receptor (TLR) ligands and Enterococcus faecium (EF) on ex vivo cultured duodenal samples and whole blood (WB) from dogs with food‐responsive chronic enteropathy (FRE) when compared to healthy dogs. Biopsy stimulation was performed in 17 FRE and 11 healthy dogs; WB stimulation was performed in 16 FRE and 16 healthy dogs. Expression of TLR2, 4, 5 and 9, IL‐17A, IL‐22, IFNy, TNFα, IL‐4, IL‐10, TGFβ and PPARy was determined in biopsies by quantitative polymerase chain reaction (PCR). In addition, production of TNFα, IL‐10, IFNy and IL‐17A protein in WB and biopsy supernatants was assessed by ELISA. Treatment with individual TLR ligands or EF induced a variety of changes in the expression of different TLRs and cytokines, but not necessarily a consistent change with a single stimulating agent. Even though cytokine protein could not be detected in supernatants from ex vivo stimulated biopsies, we found TNFα protein responses in blood to be opposite of the transcriptional responses seen in the biopsies. Stimulation of canine duodenal biopsies with TLR ligands can potentially induce anti‐inflammatory gene expression, especially in healthy tissue, whereas the effects of EF were limited.

Comparison of TNFα responses induced by Toll-like receptor ligands and probiotic Enterococcus faecium in whole blood and peripheral blood mononuclear cells of healthy dogs

The assessment of in vitro responses of blood-derived cells has traditionally been performed with peripheral blood mononuclear cells (PBMCs). However, stimulation of whole blood (WB) has advantages: ease of experimental setup, avoidance of blood cell manipulation and lower assay cost and time. WB stimulation is widely used in human research, but only infrequently in small animals. The aim of this study was to compare the response generated in canine WB and PBMCs with Toll-like receptor ligands and probiotic bacteria using TNFα as measured endpoint. WB and PBMCs were derived from a total of 15 healthy dogs. Stimulations were performed with LPS (1ngml(-1)), Pam3CSK4 (100ngml(-1)), flagellin (1μgml(-1)) and Enterococcus faecium (EF; 1×10(7)cfuml(-1)). In 4 of the dogs, PBMC numbers were matched to the numbers of PBMCs found in WB. TNFα was detected in supernatants via ELISA. TNFα production from WB was generally higher than from PBMCs (repeated measures ANOVA p<0.0128). PBMCs produced TNFα inconsistently for all stimulants apart from EF. There was no correlation between results of WB or PBMC stimulation, similar to studies that found that humanWB cytokine production correlates with stimulating monocytes, but not PBMCs. In conclusion, WB stimulation should be considered a valid alternative to PBMC stimulation in the canine system.

Identification of T cell receptor signaling pathway proteins in a feline large granular lymphoma cell line by liquid chromatography tandem mass spectrometry

Tryptic peptides of a feline large granular lymphoma cell line were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Seventeen proteins of the T cell receptor signaling pathway could be identified by this approach. So far the existence of these proteins has only been postulated in the protein databases while experimental proof of their expression is predominantly pending. This article suggests where these proteins are located within the T cell receptor signaling pathway, thereby giving a short overview of the structure and function of this cascade.

High resolution melting analysis (HRM) for the assessment of clonality in feline B-cell lymphomas

Analysis of clonality is gaining importance in diagnosing lymphomas in veterinary medicine. Usually, PCR for the analysis of antigen receptor rearrangement (PARR) is followed by electrophoretic separation of the PCR products. Aim of this study was to test the feasibility of HRM for the assessment of clonality in B-cell lymphomas of cats. High resolution melting analysis differentiates PCR products by their different melting point using the decrease in fluorescence of an intercalating dye during melting of the PCR product. Additionally, the method is easy to use with no post-PCR manipulation of the samples. Forty-seven feline B-cell lymphomas and 31 reactive lymphatic proliferations of cats were investigated by PARR followed either by capillary electrophoresis or an HRM assay. To objectify the interpretation of the HRM results a recently published mathematical approach was applied to the melting curve. To overcome discrepancies between the visual interpretation and the mathematical approach, the latter was modified to include testing of reproducibility and recognition of pseudoclonality. In 11 of 47 lymphoma cases clonal populations were detectable by HRM assay compared to 14 of 47 lymphomas in which clonal populations were detected by capillary electrophoresis assay. Neither of the methods showed a clonal pattern in any of the reactive samples. However, the HRM assay showed a unique pattern in cases of follicular lymphatic hyperplasia that had no corresponding pattern in capillary electrophoresis. CONCLUSION The capillary electrophoresis assay could identify 3 lymphomas that were not detected by the HRM assay and is therefore regarded superior to the HRM assay. The comparison however, was hampered by the overall bad performance of the PARR, that might be the consequence of insufficient primer binding due to somatic hypermutation of the binding sites during antigen stimulated proliferation of the B lymphocytes.

Favourable long-term outcome of granulomatous colitis involving two Escherichia coli strains with multiple antimicrobial resistances in a French bulldog in Germany

A two-year-old female French bulldog was presented for chronic large bowel diarrhoea. The dog had been suspected to have granulomatous colitis, but no improvement was noted with enrofloxacin treatment. Diagnosis was confirmed by histology and fluorescent in situ hybridisation. Isolated Escherichia coli strains showed multiple resistances to antimicrobials, including enrofloxacin. Treatment with potentiated amoxicillin and cefovecin resulted in complete resolution of clinical signs within two weeks and treatment was continued for a total of eight weeks. The dog had no signs of large bowel diarrhoea and gained 2 kg of bodyweight within 10 months. This is the first case report of a French bulldog with granulomatous colitis with a favourable long-term outcome despite colonisation with multiresistant adherent-invasive E coli. This case highlights the importance of antimicrobial sensitivity testing from colonic mucosal biopsy samples in canine granulomatous colitis. It should raise awareness of this disease in continental Europe.

Identification of a novel feline large granular lymphoma cell line (S87) as non-MHC-restricted cytotoxic T-cell line and assessment of its genetic instability

Feline large granular lymphocyte lymphomas are rare but very aggressive tumors with a poor prognosis. In this study, a cell line from an abdominal effusion of a cat with large granular lymphoma was characterized. Immunophenotype staining was positive for CD3 and CD45R, and negative for CD4, CD8, CD56, CD79α, BLA.36 and NK1. A TCR γ gene rearrangement was detectable by PARR. Neither FeLV antigen nor exogenous FeLV provirus could be detected. A chromosomal instability associated with a centrosome hyperamplification could also be determined. The cell line is able to lyse target cells without antigen presentation or interaction with antigen presenting cells. Therefore, these cells were classified as genetically instable non-MHC-restricted cytotoxic T cells with large granular lymphocyte morphology.