SopB effector protein of Salmonella Typhimurium is translocated in mesenteric lymph nodes during murine salmonellosis

Salmonella Typhimurium harbors two Salmonella pathogenicity islands (SPIs), each encoding a type three secretion system for virulence proteins. Although there is increasing evidence of postinvasion roles for SPI-1, it has been generally accepted that SPI-1 genes are downregulated following the invasion process. Here, we analyzed the expression and translocation of SopB in vitro, in cell culture and in vivo. To this end, a sopB-FLAG-tagged strain of Salmonella Typhimurium was obtained by epitope tagging. Tagged proteins were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. SopB expression was observed in vitro under cultured conditions that mimic the intestinal niche and different intracellular environments. In agreement, bacteria isolated from infected monolayers expressed and translocated SopB for at least 24?h postinoculation. For in vivo experiments, BALB/c mice were inoculated intraperitoneally with the tagged strain of Salmonella Typhimurium. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes. Our results showed that SopB continues to be synthesized in vivo during 5 days after inoculation. Interestingly, translocation of SopB was detected in the cytosol of cells isolated from lymph nodes 1 day after infection. Altogether, these findings indicate that the expression and translocation of SopB during Salmonella infection is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

Salmonella enterica Serovar Enteritidis Enterocolitis during Late Stages of Gestation Induces an Adverse Pregnancy Outcome in the Murine Model

Foodborne diseases caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) are a significant health problem. Pregnancy, state of immunological tolerance, is a predisposing condition for the development of infections with intracellular pathogens. Salmonella species can cause pregnancy complications such as chorioamnionitis, transplacental fetal infection, pre term labor, abortions, neonatal and maternal septicemia. However, the specific mechanisms by which Salmonella infections trigger these alterations are not clear. In the present work, using a self-limiting enterocolitis murine model, we show that the ingestion of a low dose of S. Enteritidis at late stages of pregnancy (day 15 of gestation) is sufficient to induce massive maternal infection. We found that Salmonella infection leads to 40% of pre term delivery, 33% of abortion and fetal growth restriction. Placental dysfunction during S. Enteritidis enterocolitis was confirmed through cellular infiltration and hypoxia markers (MPO activity and COX-1 and COX-2 expression, respectively). Apoptosis in placental tissue due to Salmonella infection was also evident at day 18 of gestation when investigated by morphometric procedure, DNA fragmentation and Fas/FasL expression. Also, the expression of IFN-γ, TNF-α, IL-17 and IL-10 was up regulated in response to Salmonella not only in placenta, but also in amniotic fluid and maternal serum. Altogether, our results demonstrate that S. Enteritidis enterocolitis during late stages of gestation causes detrimental effect on pregnancy outcome.     

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORγt in γδ T cells

Introduction  

Interleukin (IL)-23 is essential for the development of various experimental autoimmune models. However, the role of IL-23 in non-autoimmune experimental arthritis remains unclear. Here, we examined the role of IL-23 in the non-autoimmune antigen-induced arthritis (AIA) model. In addition, the regulatory potential of IL-23 in IL-17A and retinoic acid-related orphan receptor gamma t (RORγt) expression in CD4⁺ and TCRγδ⁺ T cells was evaluated systemically as well as at the site of inflammation.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

Intranasal immunization with temperature-sensitive mutants protects granulocytopenic mice from lethal pulmonary challenge withPseudomonas aeruginosa

Intranasal (i.n.) immunization with two temperature-sensitive (ts) mutants ofPseudomonas aeruginosa protected, in a dose-related manner, granulocytopenic (GCP) mice challenged with a lethal dose of the wild-type (wt) organism. The number of ts mutants in oronasopharyngeal lavage fluids and stools decreased steadily in both normal and GCP mice after i.n. immunization. Intranasal immunization with 10⁷ colony-forming units (CFU) of either mutant induced significant protection, whereas intraperitoneal (i.p.) immunization with similar doses induced lower protection. Protection induced by i.n. immunization was accompanied by increased levels of anti-P. aeruginosa IgA in lung lavage fluids. The results of this study demonstrate the usefulness of ts mutants ofP. aeruginosa for local immunization to protect GCP hosts from fatalP. aeruginosa pneumonia.