Increased inflammasome and caspase-1 activity in the thymus of infected mice.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Increased initiator caspase-8 gene expression on 120dpi group compared to the naive group. Increased inflammatory caspase-1 gene expression on 120dpi group compared to the naive group. Increased NLRP3 inflammasome gene expression on 120dpi group compared to the naive group. B) Increased pro-caspase-1 production and increased caspase-1 activity on 120dpi group compared to the naive group. C) Increased NLRP3 inflammasome complex assembly on 120dpi compared to the naive group. Statistical analysis was carried out with Student’s t-test. **p<0.01, ****p<0.0001. Representative data from three independent experiments with similar results. Expression levels of genes were represented as a relative copy numbers by using the method of delta threshold (2^-ΔΔCt). Image J software (NIH, MD, USA) was used for the estimation of the pro-caspase-1, the active form of caspase-1 and NLRP3 inflammasome assembly, through a GAPDH ratio.</p

Recirculating mature T cells home to infected thymuses, leading to increased frequency of cytokine producing Th17 and T CD8⁺ IFN-γ⁺.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Frequency of CD44^hiCD24^lo T cells increased among CD4⁺ and CD8⁺ subpopulations in 120dpi compared to the naive group. B) Cytokine producing T cells, Th17 and CD8⁺IFNγ⁺ was found in 120dpi, while practically absent in the naive group. Representative data from three independent experiments with similar results. At least 20000 events were analyzed with FlowJo vX.0.7 (Tree Star Inc., Ashland, OR, USA). Statistical analysis was carried out with Student’s t-test. ***p<0.001, ****p<0.0001. Representative data from three independent experiments with similar results.</p

Prolonged <i>Paracoccidioides brasiliensis</i> infection leads to thymic atrophy and intense fungal burden.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS or with only PBS (control group), intraperitoneally. One hundred and twenty days after inoculation, mice were killed and the thymus removed for analysis. A) Thymus from the 120dpi group was smaller in size and weighted less than the naive group. (B) Hematoxilin-Eosin staining showed histologic disorganization in 120dpi, and no evidence of typical cortical (C) and medullary (M) regions, while naive mice showed normal histologic distribution. In more detail below, giant cells and granuloma formation is present in 120dpi. Silver impregnation revealed massive fungal infiltration in the thymic medulla in 120dpi. Statistical analysis was carried out with Student’s t-test. **p<0.01. Results are expressed by Mean ± SEM. Images are representative of three independent experiments with similar results. The images were taken in an Olympus BX50. Magnification 40x (upper and lower panels); 100x (middle panel).</p

Increased inflammatory cytokines gene expression and protein levels in the thymus of infected mice.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Increased gene expression of IL-1β, IL-17, IL-18, IFN-γ and TNF-α from 120dpi group compared to the naive group. B) Protein levels of the inflammatory cytokines also showed significant increase in 120dpi group compared to the naive group. Statistical analysis was carried out with Student’s t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Representative data from three independent experiments with similar results. Expression levels of genes were represented as a relative copy numbers by using the method of delta threshold (2^-ΔΔCt).</p

<i>Paracoccidioides brasiliensis</i> infection leads to decreased thymocytes and TECs cellularity with increased cell death of CD4⁺CD8⁺ thymocytes and mTEC without cortisol contribution.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS as control group. One hundred and twenty days after inoculation mice were killed, the thymus and the blood were collected and processed individually for analysis. A) Total thymocytes numbers were lower in 120dpi compared to the naive group. B) Frequency of thymocytes subpopulations remained unchanged between naive and 120dpi groups. C) Frequency of thymic epithelial cells (TEC) subpopulations changed considerably in 120dpi, higher percentages of mTEC were found while cTEC frequency decreased in comparison to the naive group. D) Higher percentage of apoptotic double positive thymocytes from the 120dpi group compared to the naive group, but not in other thymocytes subpopulations. E) Higher percentage of apoptotic medullary thymic epithelial cells (mTEC) from the 120dpi group compared to the naive group, while no alterations in cTEC apoptosis was found. F) Cortisol production was not altered between the naive and 120dpi groups. At least 20000 events were analyzed with FlowJo vX.0.7 (Tree Star Inc., Ashland, OR, USA). Statistical analysis was carried out with Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. Representative data from three independent experiments with similar results.</p

Treatment with Vitamin D/MOG Association Suppresses Experimental Autoimmune Encephalomyelitis

<div><p>Experimental autoimmune encephalomyelitis (EAE) is an animal model to study multiple sclerosis (MS). Considering the tolerogenic effects of active vitamin D, we evaluated the therapeutic effect of myelin oligodendrocyte glycoprotein (MOG) associated with active vitamin D in EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG emulsified with Complete Freund’s Adjuvant plus <i>Mycobacterium tuberculosis</i>. Animals also received two intraperitoneal doses of <i>Bordetella pertussis</i> toxin. One day after immunization, mice were treated with 0,1μg of 1α,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) every other day during 15 days (on days 1, 3, 5, 7, 9, 11, 13 and 15). MOG (150μg) was co-administered on days 3 and 11. The administration of 1,25(OH) ₂D₃ or MOG determined significant reduction in EAE incidence and in clinical scores. When MOG was associated with 1,25(OH) ₂D₃ the animals did not develop EAE. Spleen and central nervous system (CNS) cell cultures from this group produced less IL-6 and IL-17 upon stimulation with MOG in comparison to the EAE control group. In addition, this treatment inhibited dendritic cells maturation in the spleen and reduced inflammatory infiltration in the CNS. The association of MOG with 1,25(OH) ₂D₃ was able to control EAE development.</p></div

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage-1

<p><b>Copyright information:</b></p><p>Taken from "Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage"</p><p>http://www.gvt-journal.com/content/5/1/12</p><p>Genetic Vaccines and Therapy 2007;5():12-12.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2222600.</p><p></p>a); IL-4 (b) and IL-5 (c) by splenic cells stimulated with ConA and serum levels of specific anti-hsp65 antibodies (d) were determined two weeks later. Results represent the geometric mean ± SEM of 4 to 8 individually tested animals per group. *p < 0.05 in comparison to vector group

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage-0

<p><b>Copyright information:</b></p><p>Taken from "Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage"</p><p>http://www.gvt-journal.com/content/5/1/12</p><p>Genetic Vaccines and Therapy 2007;5():12-12.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2222600.</p><p></p>er intramuscular injection of 50 ug of pVAXhsp65. Total RNA (10 ug) isolated from each tissue was treated with DNase and subjected to RT-PCR amplification with specific primers. β-actin was amplified as an RNA quality control. All RT-PCR products were analysed by agarose gel electrophoresis and visualized by ethidium bromide staining. Similar results were observed in two animals analysed for each period. No products were seen (hsp65 and β-actin) when total RNA was subjected to PCR amplification in the absence of a previous reverse transcription

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage-3

<p><b>Copyright information:</b></p><p>Taken from "Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage"</p><p>http://www.gvt-journal.com/content/5/1/12</p><p>Genetic Vaccines and Therapy 2007;5():12-12.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2222600.</p><p></p>perimental groups were identified as DNA/DNA and BCG/DNA respectively. A non-immunized group and a group immunized with 3 pVAXhsp65 doses delivered at 5, 12 and 19-day-old were identified as control and neonate, respectively. Two weeks after last dose, the serum levels of IgG1 (a) and IgG2a (b) anti-hsp65 antibodies were evaluated by ELISA. Results represent the geometric mean ± SEM of 6 – 8 individually tested animals per group. *p < 0.05 in comparison to neonate group

Effect of treatment with 1,25(OH)₂D₃+MOG association on EAE development.

<p>Kinetics of clinical scores (<b>A</b>), maximum clinical score (<b>B</b>) and weight variation (<b>C</b>). Comparisons between groups were made by one way ANOVA followed by Tukey’s test for parametric variables (<b>A</b> and <b>C</b>) and by Kruskal-Wallis followed by Dunn’s test for non-parametric variables (<b>B</b>). Results were expressed as mean or medians (25–75% ranges) of 12 animals per group. * p<0.05 compared with EAE+MOG, EAE+vitD and EAE/vitD+MOG groups. Data are representative of two independent experiments.</p