Chromogenic, Biochemical and Proteomic Identification of Yeast and Yeast-like Microorganisms Isolated from Clinical Samples from Animals of Costa Rica.

Yeast infections are challenging human and animal medicine due to low rates of detection and the emergence of unknown ecology isolates. The aim of this study was to verify the biochemical identification of yeasts and yeast-like microorganisms obtained from animals comparing the results with chromogenic media and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). Between January and August 2023, yeast and yeast-like isolates from samples of animals with suspicion of mycosis were identified using Vitek® 2 Compact, Brilliance® Candida Agar and MALDI Biotyper® MSP. A total of 39 cases were included, and 45 isolations were obtained. Cryptococcus neoformans (15.5%, 7/45), Meyerozyma guilliermondii (13.3%, 6/45), Candida parapsilosis (11.1%, 5/45), Candida albicans and Candida tropicalis (8.9%, each one 4/45) were the most identified organisms. There was full agreement with the three identification methods in 71.1% (32/45) of the isolates, disagreement on species in 17.8% (8/45), disagreement on genus and species in 6.7% (3/45) and, in 4.4% (2/45), there was no matched pattern in MALDI-TOF to compare the results. Biochemical methods are a good option in laboratories where proteomics are not available, and chromogenic media enhances diagnostics by detecting mixed infections. Surveillance must be implemented to improve the detection of agents shared between humans and animals.

Type 1 interferons and Foxo1 down-regulation play a key role in age-related T-cell exhaustion in mice.

Foxo family transcription factors are critically involved in multiple processes, such as metabolism, quiescence, cell survival and cell differentiation. Although continuous, high activity of Foxo transcription factors extends the life span of some species, the involvement of Foxo proteins in mammalian aging remains to be determined. Here, we show that Foxo1 is down-regulated with age in mouse T cells. This down-regulation of Foxo1 in T cells may contribute to the disruption of naive T-cell homeostasis with age, leading to an increase in the number of memory T cells. Foxo1 down-regulation is also associated with the up-regulation of co-inhibitory receptors by memory T cells and exhaustion in aged mice. Using adoptive transfer experiments, we show that the age-dependent down-regulation of Foxo1 in T cells is mediated by T-cell-extrinsic cues, including type 1 interferons. Taken together, our data suggest that type 1 interferon-induced Foxo1 down-regulation is likely to contribute significantly to T-cell dysfunction in aged mice.

Profiling the lymphoid-resident T cell pool reveals modulation by age and microbiota.

Despite being implicated in non-lymphoid tissues, non-recirculating T cells may also exist in secondary lymphoid organs (SLO). However, a detailed characterization of this lymphoid-resident T cell pool has not yet been done. Here we show that a substantial proportion of CD4 regulatory (Treg) and memory (Tmem) cells establish long-term residence in the SLOs of specific pathogen-free mice. Of these SLOs, only T cell residence within Peyer's patches is affected by microbiota. Resident CD4 Treg and CD4 Tmem cells from lymph nodes and non-lymphoid tissues share many phenotypic and functional characteristics. The percentage of resident T cells in SLOs increases considerably with age, with S1PR1 downregulation possibly contributing to this altered homeostasis. Our results thus show that T cell residence is not only a hallmark of non-lymphoid tissues, but can be extended to secondary lymphoid organs.

Calcium-mediated shaping of naive CD4 T-cell phenotype and function.

Continuous contact with self-major histocompatibility complex ligands is essential for the survival of naive CD4 T cells. We have previously shown that the resulting tonic TCR signaling also influences their fate upon activation by increasing their ability to differentiate into induced/peripheral regulatory T cells. To decipher the molecular mechanisms governing this process, we here focus on the TCR signaling cascade and demonstrate that a rise in intracellular calcium levels is sufficient to modulate the phenotype of mouse naive CD4 T cells and to increase their sensitivity to regulatory T-cell polarization signals, both processes relying on calcineurin activation. Accordingly, in vivo calcineurin inhibition leads the most self-reactive naive CD4 T cells to adopt the phenotype of their less self-reactive cell-counterparts. Collectively, our findings demonstrate that calcium-mediated activation of the calcineurin pathway acts as a rheostat to shape both the phenotype and effector potential of naive CD4 T cells in the steady-state.

Macrophages Induce Long-Term Trapping of γδ T Cells with Innate-like Properties within Secondary Lymphoid Organs in the Steady State.

So far, peripheral T cells have mostly been described to circulate between blood, secondary lymphoid organs (SLOs), and lymph in the steady state. This nomadic existence would allow them to accomplish their surveying task for both foreign Ags and survival signals. Although it is now well established that γδ T cells can be rapidly recruited to inflammatory sites or in certain tumor microenvironments, the trafficking properties of peripheral γδ T cells have been poorly studied in the steady state. In the present study, we highlight the existence of resident γδ T cells in the SLOs of specific pathogen-free mice. Indeed, using several experimental approaches such as the injection of integrin-neutralizing Abs that inhibit the entry of circulating lymphocytes into lymph nodes and long-term parabiosis experiments, we have found that, contrary to Ly-6C-/+CD44lo and Ly-6C+CD44hi γδ T cells, a significant proportion of Ly-6C-CD44hi γδ T cells are trapped for long periods of time within lymph nodes and the spleen in the steady state. Specific in vivo cell depletion strategies have allowed us to demonstrate that macrophages are the main actors involved in this long-term retention of Ly-6C-CD44hi γδ T cells in SLOs.

Sepsis-induced expansion of granulocytic myeloid-derived suppressor cells promotes tumour growth through Toll-like receptor 4.

Severe sepsis remains a frequent and dreaded complication in cancer patients. Beyond the often fatal short-term outcome, the long-term sequelae of severe sepsis may also impact directly on the prognosis of the underlying malignancy in survivors. The immune system is involved in all stages of tumour development, in the detection of transforming and dying cells and in the prevention of tumour growth and dissemination. In fact, the profound and sustained immune defects induced by sepsis may constitute a privileged environment likely to favour tumour growth. We investigated the impact of sepsis on malignant tumour growth in a double-hit animal model of polymicrobial peritonitis, followed by subcutaneous inoculation of MCA205 fibrosarcoma cells. As compared to their sham-operated counterparts, post-septic mice exhibited accelerated tumour growth. This was associated with intratumoural accumulation of CD11b(+) Ly6G(high) polymorphonuclear cells (PMNs) that could be characterized as granulocytic myeloid-derived suppressor cells (G-MDSCs). Depletion of granulocytic cells in post-septic mice inhibited the sepsis-enhanced tumour growth. Toll-like receptor (TLR)-4 (Tlr4) and Myd88 deficiencies prevented sepsis-induced expansion of G-MDSCs and tumour growth. Our results demonstrate that the myelosuppressive environment induced by severe bacterial infections promotes malignant tumour growth, and highlight a critical role of CD11b(+) Ly6G(high) G-MDSCs under the control of TLR-dependent signalling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Adaptive Immune-like γ/δ T Lymphocytes Share Many Common Features with Their α/ß T Cell Counterparts.

To better apprehend γ/δ T cell biological functions in the periphery, it appears crucial to identify markers highlighting the existence of distinct phenotypic and functional γ/δ T cell subsets. Interestingly, the expression of CD44 and Ly-6C subdivides murine peripheral γ/δ T cells into several subsets, with Ly-6C(-) CD44(hi) γ/δ T cells corresponding to the IL-17-producing CD27(-) γ/δ T cell subset exhibiting innate-like features. By comparing the other subsets to naive and memory CD8(+) α/ß T cells, in this study, we show that Ly-6C(- or +) CD44(lo) and Ly-6C(+)CD44(hi) γ/δ T cells greatly resemble, and behave like, their CD8(+) α/ß T cell counterparts. First, like memory CD8(+) α/ß T cells, Ly-6C(+)CD44(hi) γ/δ T cells are sparse in the thymus but largely increased in proportion in tissues. Second, similarly to naive CD8 α/ß T cells, CD44(lo) γ/δ T cells are poorly cycling in vivo in the steady state, and their proportion declines with age in secondary lymphoid organs. Third, CD44(lo) γ/δ T cells undergo spontaneous proliferation and convert to a memory-like Ly-6C(+)CD44(hi) phenotype in response to lymphopenia. Finally, CD44(lo) γ/δ T cells have an intrinsic high plasticity as, upon appropriate stimulation, they are capable of differentiating nonetheless into Th17-like and Th1-like cells but also into fully functional Foxp3(+) induced regulatory T cell-like γ/δ T cells. Thus, peripheral CD27(+) γ/δ T cells, commonly considered as a functionally related T cell compartment, actually share many common features with adaptive α/ß T cells, as both lineages include naive-like and memory-like lymphocytes with distinct phenotypic, functional, and homeostatic characteristics.

ERAP1 Gene Expression Is Influenced by Nonsynonymous Polymorphisms Associated With Predisposition to Spondyloarthritis.

OBJECTIVE: Several polymorphisms in ERAP1 are strongly associated with susceptibility to spondyloarthritis (SpA). The combination of rs17482078, rs10050860, and rs30187 results in the construction of 3 major haplotypes that are associated with SpA (the "protective" haplotype T/T/C, the "neutral" haplotype C/C/C, and the "susceptibility" haplotype C/C/T). The aim of the present study was to determine whether such haplotypes might affect endoplasmic reticulum aminopeptidase 1 (ERAP-1) messenger RNA (mRNA) expression, protein level, and/or enzymatic activity in antigen-presenting cells, a type of cell that is potentially relevant to disease pathogenesis. METHODS: Monocyte-derived dendritic cells (DCs) were generated in 2 cohorts (a discovery cohort and a replication cohort) comprising a total of 23 SpA patients and 44 healthy controls. Lymphoblastoid B cell lines were established from individuals who were homozygous for the risk, the neutral, or the protective ERAP1 haplotype, respectively. In those samples, we investigated the relationship between ERAP1 haplotypes and mRNA expression level. We also used Western blot analysis to measure the relative protein expression of ERAP-1 and a fluorogenic assay to measure its enzymatic activity. RESULTS: In monocyte-derived DCs, there was a strong association between ERAP1 haplotypes and the ERAP-1 mRNA expression level, with higher levels in subjects harboring the susceptibility haplotype (P = 0.001 and P = 5.6 × 10(-7) in the discovery and replication cohorts, respectively). In lymphoblastoid B cell lines, we observed a significant correlation between haplotype risk score and ERAP1 transcript or protein level (P = 0.003, ρ = 0.92 for both). Enzymatic activity followed a similar trend both in monocyte-derived DCs and in lymphoblastoid B cell lines. CONCLUSION: These data provide strong evidence that SpA-associated ERAP1 polymorphisms affect the level of gene expression in antigen-presenting cells. How increased production/activity of ERAP-1 may influence susceptibility to SpA remains to be determined.

TCR signaling events are required for maintaining CD4 regulatory T cell numbers and suppressive capacities in the periphery.

CD4 regulatory T cells (Tregs) can be subdivided into two subsets according to Ly-6C expression in the periphery. Phenotypic analysis, imaging, and adoptive-transfer experiments of peripheral Ly-6C(-) and Ly-6C(+) Tregs reveal that the nonexpression of Ly-6C by ∼70% of peripheral Tregs depends on TCR signaling events. Interestingly, Ly-6C(-) Tregs express higher surface amounts of key immunosuppressive molecules than do Ly-6C(+) Tregs and produce constitutively anti-inflammatory cytokines. In line with their phenotype, Ly-6C(+) Tregs exhibit poor suppressive capacities in vitro and in vivo. Finally, although Ly-6C(-) Tregs maintain their numbers with age, Ly-6C(+) Tregs gradually disappear. Altogether, our data strongly suggest that both the survival and suppressive functions of peripheral CD4 Tregs rely on their ability to receive strong TCR signals.

Monocyte-derived dendritic cells from HLA-B27+ axial spondyloarthritis (SpA) patients display altered functional capacity and deregulated gene expression.

INTRODUCTION: This study aimed to compare the functional capacity and gene expression profile of monocyte-derived dendritic cells (MD-DCs) in HLA-B27+ axial spondyloarthritis (SpA) patients and healthy controls. METHODS: MD-DCs were differentiated with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for seven days, starting from purified CD14+ monocytes and stimulated with lipopolysaccharide (LPS) for six and twenty four hours. Their capacity to stimulate allogeneic CD4+ T cells from unrelated healthy donor was tested. Transcriptomic study was performed with Affymetrix HuGene 1.0 ST microarrays. Gene expression levels were compared between patients and controls using a multivariate design under a linear model (LIMMA). Real-time quantitative PCR (qRT-PCR) was performed for validation of the most striking gene expression differences. RESULTS: The stimulatory capacity of allogeneic CD4+ T cells by MD-DCs from SpA patients was decreased. Transcriptomic analysis revealed 81 genes differentially expressed in MD-DCs between SpA patients and controls (P <0.01 and fold-change <0.66 or >1.5). Four selected genes were validated by q RT-PCR: ADAMTS15, CITED2, F13A1 and SELL. Expression levels of ADAMTS15 and CITED2, encoding a metallopeptidase and a transcription factor, respectively, were inversely correlated with each other (R = 0.75, P = 0.0003). Furthermore, in silico analysis identified several genes of the Wnt signaling pathway having expression co-regulated with CITED2. CONCLUSION: This study revealed altered function and gene expression pattern in MD-DCs from HLA-B27+ axial SpA. Co-expression study showed an inverse correlation between ADAMTS15 and CITED2. Moreover, the Wnt signaling pathway appeared as deregulated in SpA MD-DCs, a finding which may be connected to Th17-driven inflammatory responses.

Highly self-reactive naive CD4 T cells are prone to differentiate into regulatory T cells.

Upon activation, naive CD4 T cells differentiate into a variety of T-helper-cell subsets characterized by different cytokine production and functions. Currently, lineage commitment is considered to depend mostly on the environmental context to which naive CD4 T cells are exposed. Here we challenge this model based on the supposed homogeneity of the naive CD4 T-cell compartment. We show that peripheral naive CD4 T cells can be subdivided into two subsets according to Ly-6C expression. Furthermore, the two newly defined subsets (Ly-6C(-) and Ly-6C(+) naive CD4 T cells) are not equal in their intrinsic ability to commit into the induced regulatory T-cell lineage. Finally, phenotypic analysis, imaging and adoptive transfer experiments reveal that Ly-6C expression is modulated by self-recognition, allowing the dichotomization of the naive CD4 T-cell compartment into two cell subsets with distinct self-reactivity. Altogether, our results show that naive CD4 T cells with the highest avidity for self are prone to differentiate into regulatory T cells.

Serial evolution of TCR beta chain transcript mobilization in HIV type-1-infected patients following vaccine immune stimulation and HAART interruption.

In this article, we studied the T cell receptor (TCR)beta chain transcript mobilization in peripheral blood lymphocytes harvested from HIV-1-infected patients before and after vaccination with a mixture of six lipopeptides and at the moment and serially after highly active antiretroviral therapy (HAART) interruption. This study was performed by using a combined qualitative and quantitative assessment of Vbeta mRNA alterations at the level of complementary determining region 3 length distribution (CDR3-LD) of the TCR. Whereas healthy individuals displayed both stable CDR3-LD profiles and Vbeta transcript accumulations over time, the four HIV-1-infected patients in a quiescent disease phase under HAART have a highly significantly biased CDR3-LD. In addition, they displayed a significant further increase of alterations of their beta CDR3-LD profile after vaccination and both a more altered CDR3-LD (p < 0.05) and an increased transcript accumulation of some Vbeta families after HAART interruption. These modifications mostly concerned the CD8(+ve) T cells. Such a global approach of TCR alterations may help to follow the immune response of these patients and allow targeting of more complex in vivo studies by identifying the T cells with a selected repertoire.

Use of well-defined HIV-derived epitopes to evaluate CD4(+) and CD8(+) T cell responses in patients with chronic HIV-1 infection treated with HAART.

Highly active antiretroviral therapy (HAART) is associated with a dramatic clinical benefit to HIV-infected patients through significant plasma viremia reduction and CD4(+) T cells increase. In previous reports, HIV-specific CD4(+) and/or CD8(+) T cell responses have been studied separately during HAART; therefore the relationship between these two virus-specific populations is currently not well understood. In this study, both HIV-specific CD4(+) and CD8(+) T cell responses were investigated using a large panel of well-defined T cell epitope peptides in 24 HIV-1-infected patients undergoing HAART, with undetectable viral load and CD4(+) T cell count >/= 350/mm(3). One-third of the patients had CD4(+) T cells able to proliferate when exposed to HIV-1 protein fragments but only two patients displayed polyclonal responses. In addition the majority (78%) of HAART-treated patients displayed no or monospecific CD8(+) T cell responses and the phenotypic analysis of these HIV-specific CD8(+) T cells demonstrated the absence of terminally differentiated effectors. In conclusion, the experimental approach used in this study shows that CD4(+) T cell responses may persist during HAART but are not associated with strong CD8(+) T cell responses