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Background: In endemic areas, Leishmania infantum and feline immunodeficiency virus (FIV) co-infection occurs in cats, and may favour a progressive course of feline leishmaniosis. Abnormalities in serum protein fractions have been reported, but inflammation markers have scarcely been studied. Erythrocyte sediment rate (ESR) is a marker of inflammation that is poorly used in veterinary medicine, but it has been evaluated in EDTA blood using a recently introduced automatic device. We studied ESR and a pool of feline markers of inflammation (MoI) in cats L. infantum (Li + ) and/or FIV antibody-positive (Li + FIV + /FIV + ) with the aims (a) to evaluate ESR as MoI in cats with the infectious and clinical conditions considered and (b) to provide data about a pool of MoI never investigated in the feline infections studied and in other cat diseases before., Methods: This prospective controlled study included 35 study group cats (Li + , n = 20; FIV +, n = 8; Li + FIV + , n = 7) and ten healthy antibody-negative control cats. Clinical findings at physical examination and selected clinical pathological abnormalities related to inflammation were statistically analysed in relation to the infectious status and ESR values., Results: ESR values were higher in Li + , FIV + , and Li + FIV + cats compared with control cats, and 40% of the study group cats had ESR values above the reference interval (RI). ESR positively correlated with some positive MoI and negatively with some negative MoI studied. Additionally, a higher prevalence of ESR values above the RI has been detected in cats with hypoalbuminemia or hypergammaglobulinemia and higher ESR values were measured in cats with serum protein electrophoresis (SPE) fraction abnormalities. Correlations were also found with erythrocytes, hemoglobin, hematocrit and some erythrocyte indices. FIV + and Li + FIV + cats had a higher prevalence of increased ESR values, and almost all had SPE abnormalities and more severe clinical presentations compared with Li + cats., Conclusions: Abnormal levels of MoI were found in almost all parameters studied, particularly in FIV + and Li + FIV + cats. Also, ESR can be used as a marker of inflammation in cats with L. infantum and/or FIV infection., (© 2024. The Author(s).)

Background: Infection with feline immunodeficiency virus (FIV) causes an immunosuppressive disease whose consequences are less severe if cats are co-infected with an attenuated FIV strain (PLV). We use virus diversity measurements, which reflect replication ability and the virus response to various conditions, to test whether diversity of virulent FIV in lymphoid tissues is altered in the presence of PLV. Our data consisted of the 3' half of the FIV genome from three tissues of animals infected with FIV alone, or with FIV and PLV, sequenced by 454 technology., Results: Since rare variants dominate virus populations, we had to carefully distinguish sequence variation from errors due to experimental protocols and sequencing. We considered an exponential-normal convolution model used for background correction of microarray data, and modified it to formulate an error correction approach for minor allele frequencies derived from high-throughput sequencing. Similar to accounting for over-dispersion in counts, this accounts for error-inflated variability in frequencies - and quite effectively reproduces empirically observed distributions. After obtaining error-corrected minor allele frequencies, we applied ANalysis Of VAriance (ANOVA) based on a linear mixed model and found that conserved sites and transition frequencies in FIV genes differ among tissues of dual and single infected cats. Furthermore, analysis of minor allele frequencies at individual FIV genome sites revealed 242 sites significantly affected by infection status (dual vs. single) or infection status by tissue interaction. All together, our results demonstrated a decrease in FIV diversity in bone marrow in the presence of PLV. Importantly, these effects were weakened or undetectable when error correction was performed with other approaches (thresholding of minor allele frequencies; probabilistic clustering of reads). We also queried the data for cytidine deaminase activity on the viral genome, which causes an asymmetric increase in G to A substitutions, but found no evidence for this host defense strategy., Conclusions: Our error correction approach for minor allele frequencies (more sensitive and computationally efficient than other algorithms) and our statistical treatment of variation (ANOVA) were critical for effective use of high-throughput sequencing data in understanding viral diversity. We found that co-infection with PLV shifts FIV diversity from bone marrow to lymph node and spleen.

Background: We and others have previously reported that cell membrane-bound TGFβ (mTGFβ) on activated T regulatory (Treg) cells mediates suppressor function. Current findings suggest that a novel protein known as Glycoprotein A Repetitions Predominant (GARP) anchors mTGFβ to the Treg cell surface and facilitates suppressor activity. Recently, we have described that GARP+TGFβ+ Treg cells expand during the course of FIV infection. Because Treg cells are anergic and generally exhibit poor proliferative ability, we asked how Treg homeostasis is maintained during the course of feline immunodeficiency virus (FIV) infection., Results: Here, we report that Treg cells from FIV+ cats express GARP and mTGFβ and convert T helper (Th) cells into phenotypic and functional Treg cells. Th to Treg conversion was abrogated by anti-TGFβ or anti-GARP treatment of Treg cells or by anti-TGFβRII treatment of Th cells, suggesting that Treg cell recruitment from the Th pool is mediated by TGFβ/TGFβRII signaling and that cell-surface GARP plays a major role in this process., Conclusions: These findings suggest Th to Treg conversion may initiate a cascade of events that contributes to the maintenance of virus reservoirs, progressive Th cell immunosuppression, and the development of immunodeficiency, all of which are central to the pathogenesis of AIDS lentivirus infections.

Background: Immune activity during pregnancy must be tightly regulated to ensure successful pregnancy. This regulation includes the suppression of inflammatory activity that could target the semi-allogeneic fetus. Tregs are immunosuppressive; Th17 cells are pro-inflammatory. A precise balance in the two cell populations is critical to pregnancy maintenance, while dysregulation in this balance accompanies compromised pregnancy in humans and mice. FIV is known to target Tregs preferentially in the infected cat. Therefore, it may be hypothesized that FIV infection alters the placental Treg/Th17 cell balance resulting in aberrant immunomodulator expression by these cells and consequent pregnancy perturbation., Methods: RNA was purified from random sections of whole placental tissues collected from both uninfected and FIV-infected queens at early pregnancy, including tissues from viable and nonviable fetuses. Real time qPCR was performed to quantify expression of intranuclear markers of Tregs (FoxP3) and Th17 cells (RORγ); cytokine products of Tregs (IL-10 and TGF-β), Th17 cells (IL-2, IL-6, and IL-17a), and macrophages (IL-1β); and the FIV gag gene. Pairwise comparisons were made to evaluate coexpression patterns between the cytokines and between the cytokines and the virus., Results: Both FoxP3 and RORγ were reduced in placentas of infected animals. Neither infection status nor fetal viability affected placental expression of IL-1β. However, fetal nonviability was associated with reduced levels of all other cytokines. Infection and fetal nonviability impacted coexpression of various cytokine pairs. No obvious bias toward Treg or Th17 cells was observed., Conclusions: FIV infection coupled with fetal nonviability alters expression patterns of T cell cytokines. These data suggest that functionally altered placental T cell leukocyte populations may occur in the infected queen and possibly contribute to fetal nonviability.

Background: An appropriate balance in placental regulatory T cells (Tregs), an immunosuppressive cell population, and Th17 cells, a pro-inflammatory cell population, is essential in allowing tolerance of the semi-allogeneic fetus. TGF-β and IL-6 are cytokines that promote differentiation of Tregs and Th17 cells from a common progenitor; aberrant expression of the cytokines may perturb the balance in the two cell populations. We previously reported a pro-inflammatory placental environment with decreased levels of FoxP3, a Treg marker, and increased levels of IL-6 in the placentas of FIV-infected cats at early pregnancy. Thus, we hypothesized that FIV infection in the pregnant cat causes altered placental Treg and Th17 cell populations, possibly resulting in placental inflammation., Methods: We examined the effect of FIV infection on Treg and Th17 populations in placentas at early pregnancy using quantitative confocal microscopy to measure FoxP3 or RORγ, a Th17 marker, and qPCR to quantify expression of the key cytokines TGF-β and IL-6., Results: FoxP3 and RORγ were positively correlated in FIV-infected placentas at early pregnancy, but not placentas from normal cats, indicating virus-induced alteration in the balance of these cell populations. In control cats the expression of IL-6 and RORγ was positively correlated as predicted, but this relationship was disrupted in infected animals. TGF-β was reduced in infected queens, an occurrence that could dysregulate both Treg and Th17 cell populations. Co-expression analyses revealed a highly significant positive correlation between IL-6 and TGF-β expression in control animals that did not occur in infected animals., Conclusion: Collectively, these data point toward potential disruption in the balance of Treg and Th17 cell populations that may contribute to FIV-induced inflammation in the feline placenta.

Background: FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection may cause dysregulation of trophoblast immunomodulator expression, and aberrant expression of these molecules may potentiate inflammation and compromise pregnancy. The purpose of this project was to evaluate the expression of representative pro-(TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, and GM-CSF) and anti-inflammatory cytokines (IL-4, IL-5, and IL-10); CD134, a secondary co-stimulatory molecule expressed on activated T cells (FIV primary receptor); the chemokine receptor CXCR4 (FIV co-receptor); SDF-1α, the chemokine ligand to CXCR4; and FIV gag in trophoblasts from early-and late-term pregnancy., Methods: We used an anti-cytokeratin antibody in immunohistochemistry to identify trophoblasts selectively, collected these cells using laser capture microdissection, and extracted total RNA from the captured cell populations. Real time, reverse transcription-PCR was used to quantify gene expression., Results: We detected IL-4, IL-5, IL-6, IL-1β, IL-12p35, IL-12p40, and CXCR4 in trophoblasts from early-and late-term pregnancy. Expression of cytokines increased from early to late pregnancy in normal tissues. A clear, pro-inflammatory microenvironment was not evident in trophoblasts from FIV-infected queens at either stage of pregnancy. Reproductive failure was accompanied by down-regulation of both pro-and anti-inflammatory cytokines. CD134 was not detected in trophoblasts, and FIV gag was detected in only one of ten trophoblast specimens collected from FIV-infected queens., Conclusion: Feline trophoblasts express an array of pro-and anti-inflammatory immunomodulators whose expression increases from early to late pregnancy in normal tissues. Non-viable pregnancies were associated with decreased expression of immunomodulators which regulate trophoblast invasion in other species. The detection of FIV RNA in trophoblasts was rare, suggesting that the high rate of reproductive failure in FIV-infected queens was not a direct result of viral replication in trophoblasts. The influence of placental immune cells on trophoblast function and pregnancy maintenance in the FIV-infected cat requires additional study.

Background: Using the FIV model, we reported previously that CD4+CD25+ T regulatory (Treg) cells from FIV+ cats are constitutively activated and suppress CD4+CD25- and CD8+ T cell immune responses. In an effort to further explore Treg-mediated suppression, we asked whether Treg cells induce anergy through the alteration of production of cyclins, cyclin-dependent kinases and their inhibitors., Results: Lymphocytes were obtained from control or FIV+ cats and sorted by FACS into CD4+CD25+ and CD8+ populations. Following co-culture with CD4+CD25+ cells, CD8+ targets were examined by Western blot for changes in cyclins D3, E and A, retinoblastoma (Rb) protein, as well as the cyclin dependent kinase inhibitor p21cip1. Following co-culture with CD4+CD25+cells, we observed up-regulation of p21cip1 and cyclin E, with down-regulation of cyclin D3, in CD8+ cells from FIV+ cats. As expected, CD8+ targets from control cats were quiescent with little up-regulation of p21cip1 and cyclin E. There was also a lack of Rb phosphorylation in CD8+ targets consistent with late G1 cell cycle arrest. Further, IL-2 mRNA was down regulated in CD8+ cells after co-culture with CD4+CD25+ Treg cells. Following CD4+CD25+ co-culture, CD8+ targets from FIV+ cats also had increased Foxp3 mRNA expression; however, these CD8+Foxp3+ cells did not exhibit suppressor function., Conclusions: Collectively, these data suggest that CD4+CD25+ Treg cells from FIV+ cats induce CD8+ anergy by disruption of normal G1 to S cell cycle progression.

Background: Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV) model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control) and then cats were vaginally challenged with cell-associated or cell-free FIV., Results: Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN) expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls., Conclusions: The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus) can be modulated by mucosal exposure to uninfected heterologous cells.

Background: In the acute phase of infection with feline immunodeficiency virus (FIV), the virus targets activated CD4+ T cells by utilising CD134 (OX40) as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs) 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo., Results: Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134., Conclusions: The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.

Background: Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major histocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry., Results: The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies., Conclusion: Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

Immunotherapy of feline immunodeficiency virus (FIV)-infected cats with monocyte-derived dendritic cells (MDCs) loaded with aldrithiol-2 (AT2)-inactivated homologous FIV was performed. Although FIV-specific lymphoproliferative responses were markedly increased, viral loads and CD4+ T cell depletion were unaffected, thus indicating that boosting antiviral cell-mediated immunity may not suffice to modify infection course appreciably.