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We evaluated Leptospira-induced renal inflammation by quantification of mRNA transcription of CxCL1 (KC), CxCL2 (MIP-2), CCL5 (RANTES), TNFα and IFNγ by qRT-PCR. Total concentration of IgM, IgG, IgG1, IgG2a and IgG3 antibodies, and B. CxCL1, CxCL2, CCL5, TNFα and IFNγ, which were quantified by ELISA two weeks post-infection. If these PMN were phagocytic, we would have expected a decrease in the number of Leptospira in the kidney of L. plantarum treated group, which was not the case. A primer list is provided in supplemental material (S1 Table). Samples were processed with xMAP Technology by MagPix Luminex using 50-bead count and analyzed with eBioscience Procarta Plex Analyst software. The slides were processed and stained in parallel and images were taken using the same illumination. here. Lactobacillus plantarum is one of the commonly-isolated species in vaginal fluid, together with other lactobacilli such as L. acidophilus, L. jensenii, L. gasseri, L. crispatus, , , . Immuno Technologies Inc., Memphis, Tennessee, United States of America, Roles Data is representative of one of two experiments. B. Histopathology was empirically quantified by scoring interstitial nephritis blindly, and glomeruli were scored by measuring size (um) in 5 fields per sample and averaging per group. A striking result observed was that, in the absence of infection, treatments with L. plantarum led to an increase of neutrophils and a decrease of macrophages and dendritic cells in spleen, and to an increase of monocytes in peripheral lymph nodes, whereas Leptospira infection of mice pre-treated with L. plantarum led to decreased populations of monocytes and increased populations of macrophages in both tissues. Future studies to analyze the effect of L. plantarum in post-exposure treatments could also shed light on the possible use of commonly available probiotics as alternative palliative care for leptospirosis. Differences in KC were significant in kidney and blood, and differences in RANTES were significant only in blood. These results are consistent with findings in other studies in which depletion or absence of T cells led to severe pathological lesions in kidney in C3H/HeJ mice [32] and worse kidney lesions in C57BL/6J [14]. Analysis of the immune cell profiles in lymphoid tissues of mice pre-treated with L. plantarum showed increased numbers of B cells as well as naïve and memory CD4+ helper T cell populations in uninfected mice that shifted towards increased numbers of effector CD4+ helper T in infected mice. Cells were labelled with anti-CD19, anti-CD3, anti-CD4 and anti-CD8 lineage surface markers. Kidney tissue was used for quantification of spirochetes, quantification of immune marker transcripts, histopathology and culture at 30°C for 7 days for determination of spirochetal viability. A. Supervision, We used Lactobacillus plantarum strain 256 (kindly gifted by Dr. Jos Seegers, Caelus Pharmaceuticals BV), a bacterium Generally Recognized As Safe, to perform oral treatments as described [9], [10] prior to Leptospira infection. Interstitial collagen deposition (fibrosis) was evaluated using Masson’s trichrome staining of kidney sections from infected mice, which was quantified by digital image analysis (Fig 3). Figure 1: Colony morphology (A) and gram stain (B) of Lactobacillus plantarum. Further analysis of immune marker transcripts in kidney (CxCL1/KC, CXCL2/MIP-2, CCL5/RANTES, TNFα and IFNγ) showed that infected mice pre-treated with L. plantarum produced less pro-inflammatory chemokines KC, MIP-2 and RANTES known to be upregulated during infection [13, 29–31][11] although transcription of cytokines TNFα and IFNγ was not affected.

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