TGFβ receptor II gene deletion in leucocytes prevents cerebral vasculitis in bacterial meningitis

In bacterial meningitis, chemokines lead to recruitment of polymorphonuclear leucocytes (PMN) into the CNS. At the site of infection in the subarachnoid space, PMN release reactive oxygen species, reactive nitrogen intermediates (RNI) and interleukin-1β (IL-1β). Although these immune factors assist in clearance of bacteria, they also result in neuronal injury associated with meningitis. Transforming growth factor beta (TGFβ) is a potent deactivator of PMN and macrophages since TGFβ suppresses the production of ROI, RNI and IL-1. Here, we report that the deletion of the TGFβ receptor II gene in PMN enhances PMN recruitment into the CNS of mice with Streptococcus pneumoniae meningitis. This was associated with more efficient clearance of bacteria, and almost complete prevention of intracerebral necrotizing vasculitis. Differences in PMN in the CNS of infected control mice and mice lacking TGFβ receptor II were not explained by altered expression of chemokines acting on PMN. Instead, TGFβ was found to impair the expression of l (leucocyte)-selectin on PMN from control mice but not from mice lacking TGFβ receptor II. l-Selectin is known to be essential for PMN recruitment in bacterial meningitis. We conclude that defective TGFβ signalling in PMN is beneficial in bacterial meningitis by ameliorating migration of PMN and bacterial clearanc

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3

BACKGROUND: The transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells. RESULTS: Transgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF. CONCLUSION: Overexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog

Comparative analysis and physiological impact of different tissue biopsy methodologies used for the genotyping of laboratory mice

Genotyping of genetically modified mice and control of authenticity of the genetic background of congenic or coisogenic strains by polymerase chain reaction (PCR) is a routine procedure that can be performed with different tissue biopsies causing variable grades of trauma. In this study, some invasive and non-invasive sampling methods were compared, with the main focus on the impact on animal physiology. We compared ear punch, tail biopsy, hair plugging, mouth and rectum swabs and the simple restraint of the animals, scoring for the impact on heart rate (HR), core body temperature (BT) and motor activity by telemetry, during biopsy and for the following 6 h. Furthermore, in order to correlate the physiological impact with the practicability and reliability of the genotyping results, we performed a PCR analysis of the biopsy samples obtained by using the same collection procedures analysed by telemetry. All sampling methods and restraint induced significant increase in HR and BT and a slight increase in motor activity for 1 h, independent of the invasiveness of the method used. Genotyping of all biopsies allowed the proper identification of transgenic animals, tail biopsies, ear punches and hair follicles giving clear signals, the last method being fast, but also susceptible to cross contaminations during sampling by large numbers of animals. Restraint and all biopsy methods provoked similar physiological changes, indicating that the handling of the animals is of major importance and that the sampling procedure does not strongly influence the physiological parameters

The physiological and behavioral impact of sensory contact among unfamiliar adult mice in the laboratory

Housing mice in the laboratory in groups enables social interaction and is the way a laboratory should house mice. However, adult males show reciprocal aggression and are therefore frequently housed individually. Alternatively, a grid divider, which allows sensory contact by sight and smell but prevents fighting and injuries, can separate mice within 1 cage. This study examined the influence of this housing method on various physiological and behavioral parameters. Adult male mice housed for 10 days with sensory contact to an unfamiliar male displayed significant increases in heart rate (HR), body core temperature (BT), and motor activity (ACT). Furthermore, the mice suffered impaired nest-building behavior and significantly reduced body weight. Conversely, males housed in a similar manner with a female companion showed only a transient elevation of ACT, BT, and HR. Although no clear beneficial effect of housing males with sensory contact to females was evident, this study could not exclude it. On the other hand, housing of mature males in this way leads to sustained detrimental alterations of physiology and behavior, thus implying severe impairment of animal well-being

RA and TGF-β are involved in PP-DC mediated IgA production.

<p>Naïve splenic IgD⁺ B cells from C57BL/6 mice were cultured together with CD11c⁺ DC from the PP. Anti-CD40 mAb (5 µg/ml) was added to all cultures in the absence (A) or presence (B) of additional LPS (1 µg/ml). LE135, anti-TGF-β or the combination of both were added where indicated. At the end of 7 days of culture, supernatants were collected and IgA concentration determined by standard ELISA assay. The inhibitory effect of each reagent and their combination was calculated by comparing their IgA concentrations to control cultures containing medium alone, and are depicted as percent inhibition, medium being equivalent to 0% inhibition. Cultures were performed in triplicate and the mean±S.E.M. are shown. The data shown are from one experiment and are representative of two independent experiments.</p

Intestinal commensal bacteria are partially responsible for the ability of PP-DC to promote IgA production.

<p>Naïve splenic IgD⁺ B cells from C57BL/6 mice were cultured alone (open bars) or together with CD11c⁺ DC from the PP (black bars) or PLN (grey bars). CD11c⁺ DC were isolated from mice maintained under SPF or GF conditions as indicated. Anti-CD40 mAb (5 µg/ml) was added to all cultures in the absence or presence of additional LPS (1 µg/ml) as indicated. At the end of 7 days of culture, supernatants were collected and (A) IgA, (B) IgG1 and (C) IgM antibody concentrations determined by standard ELISA assay. Cultures were performed in triplicate and the mean±S.E.M. are shown. B.D. depicts those samples where the antibody concentration was below the detection limit of the ELISA assay. No IgA was detected in control cultures containing DC alone. The data shown are from one experiment and are representative of three independent experiments.</p

PP-DC express a distinct molecular footprint.

<p>(A) Dot plots show MACS sorted CD11c⁺ DC isolated from the PP or PLN and stained with antibodies against CD11c and CD11b. (B) Histrogram plots show cells from (A) gated for CD11c expression and stained with antibodies against MHC class II, CD40 or CD80 as indicated. All plots show expression levels of the indicated activation marker (solid line) as compared to unstained CD11c⁺ control populations (shaded area). All data shown are from one experiment and are representative of at least three independent experiments. (C) Relative mRNA levels to GAPDH for the indicated genes as determined for CD11c⁺ DC isolated from the PP or PLN by quantitative real time PCR. Results shown are from representative measurements. (D) Same as (C) shown as fold change for PP-DC (black bars) relative to PLN-DC (open bars) for which mRNA expression was normalized to a value of 1. Data represent the mean±S.E.M. of combined values from at least three independent experiments.</p