Polymorphic nucleotide within the promoter of nitrate reductase (NarGHJI) is specific for Mycobacterium tuberculosis

Mycobacterium tuberculosis rapidly reduces nitrate, leading to the accumulation of nitrite. This characteristic served for the past 40 years to differentiate M. tuberculosis from other members of the Mycobacterium tuberculosis complex (MTBC), such as Mycobacterium bovis (non-BCG [referred to here as simply "M. bovis"]), Mycobacterium bovis BCG, Mycobacterium africanum, or Mycobacterium microti. Here, a narG deletion in M. tuberculosis showed that rapid nitrite accumulation of M. tuberculosis is mediated by narGHJI. Analysis of narG mutants of M. bovis and M. bovis BCG showed that, as in M. tuberculosis, nitrite accumulation was mediated by narGHJI, and no other nitrate reductase was involved. However, in contrast to M. tuberculosis, accumulation was delayed for several days. Comparison of the narGHJI promoter revealed that, at nucleotide -215 prior to the start codon of narG, M. tuberculosis carried a thymine residue, whereas the bovine mycobacteria carried a cytosine residue. Using LightCycler technology we examined 62 strains of M. tuberculosis, M. bovis, M. bovis BCG, M. microti, and M. africanum and demonstrated that this single nucleotide polymorphism was specific for M. tuberculosis. For further differentiation within the MTBC, we included, by using LightCycler technology, the previously described analysis of oxyR polymorphism, which is specific for the bovine mycobacteria, and the RD1 polymorphism, which is specific for M. bovis BCG. Based on these results, we suggest a LightCycler format for rapid and unambiguous diagnosis of M. tuberculosis, M. bovis, and M. bovis BCG.     

ORIGINAL ARTICLE: The Persistence of Paternal Antigens in the Maternal Body is Involved in Regulatory T‐Cell Expansion and Fetal‐Maternal Tolerance in Murine Pregnancy

Citation Zenclussen ML, Thuere C, Ahmad N, Wafula PO, Fest S, Teles A, Leber A, Casalis PA, Bechmann I, Priller J, Volk H‐D, Zenclussen AC. The persistence of paternal antigens in the maternal body is involved in regulatory T‐cell expansion and fetal‐maternal tolerance in murine pregnancy. Am J Reprod Immunol 2010; 63: 200–208 Problem Mammalian pregnancy is a state of immunological tolerance and CD4⁺ CD25⁺ regulatory T cells (Treg) contribute to its maintenance. Knowing that Treg act in an antigen‐specific way during pregnancy, we hypothesized that they are generated after maternal immune cells encounter paternal antigens. Method of study We mated wild type females with transgenic green fluorescent protein (GFP) males in an allogenic setting and killed them on different days of pregnancy. Results Presence of paternal and maternal MHC class II⁺ cells in vaginal lavage on day 0.5 of pregnancy was confirmed. Thus, antigen presentation may take place early during pregnancy in the periphery either by the direct or indirect pathways. Foxp3⁺ cells known to have regulatory activity could be detected on day 2 of pregnancy in lymph nodes and shortly after implantation at the fetal‐maternal interface. Conclusion Our data suggest that paternal antigens are processed early during pregnancy, which leads to the generation of Treg. The continuous release of placental antigens into the maternal circulation allows the maintenance of a Treg population which is specific for paternal antigens and mediates tolerance toward the semi‐allogeneic fetus until the time point of birth.     

Interleukin-17A during Local and Systemic Staphylococcus aureus-Induced Arthritis in Mice

Staphylococcus aureus is one of the dominant pathogens that induce septic arthritis in immunocompromised hosts, e.g., patients suffering from rheumatoid arthritis treated with immunosuppressive drugs. S. aureus-induced arthritis leads to severe joint destruction and high mortality despite antibiotic treatment. Recently, interleukin-17A (IL-17A) has been discovered to be an important mediator of aseptic arthritis both in mice and humans, but its function in S. aureus-induced arthritis is largely unknown. Here, we investigated the role of IL-17A in host defense against arthritis following systemic and local S. aureus infection in vivo. IL-17A knockout mice and wild-type mice were inoculated systemically (intravenously) or locally (intra-articularly) with S. aureus. During systemic infection, IL-17A knockout mice lost significantly more weight than the wild-type mice did, but no differences were found in the mortality rate. The absence of IL-17A had no impact on clinical arthritis development but led to increased histopathological erosivity late during systemic S. aureus infection. Bacterial clearance in kidneys was increased in IL-17A knockout mice compared to the level in wild-type mice only 1 day after bacterial inoculation. During systemic S. aureus infection, serum IL-17F protein levels and mRNA levels in the lymph nodes were elevated in the IL-17A knockout mice compared to the level in wild-type mice. In contrast to systemic infection, the IL-17A knockout mice had increased synovitis and erosions and locally decreased clearance of bacteria 3 days after local bacterial inoculation. On the basis of these findings, we suggest that IL-17A is more important in local host defense than in systemic host defense against S. aureus-induced arthritis.Patients with rheumatoid arthritis (RA) are susceptible to bacterial joint infections as a result of immunosuppressive treatments and the disease per se (24). The most common agent causing joint infections is Staphylococcus aureus, a microbe that can also cause sepsis. S. aureus-induced arthritis is a severe problem with a mortality rate of 5 to 20%, and 25 to 70% of affected patients develop permanent joint damage despite treatment (24). Although substantial efforts have been made to understand the immunological mechanisms that lead to S. aureus-induced joint destruction, it remains difficult to treat the infection (by maintaining the host''s ability to clear bacteria) while simultaneously limiting the joint destruction (by suppressing the immunological response). Thus, there is a need to identify new ways to treat RA that do not increase the severity of S. aureus-induced arthritis following infection.Recent evidence from humans and mice suggesting that the proinflammatory cytokine interleukin-17A (IL-17A) is an important player in RA (3, 19, 21) prompted an ongoing clinical trial of IL-17A-blocking antibodies to treat RA (6). Interleukin-17A was first described in 1993, but it was not until 2005, when Harrington et al. (8) described the unique Th17 subset, that the relevance of this cytokine was widely recognized among immunologists (5, 13, 15). Interleukin-17A appears to play a key role in host defense against local Gram-negative extracellular bacterial infections (4, 7, 9, 10, 17, 22, 29, 30) and local S. aureus infections (18) by inducing the production of neutrophil-mobilizing chemokines and growth factors and the subsequent mobilization of neutrophils (5, 13, 15, 16). Importantly, Ishigame et al. have recently shown that genetical knockout of IL-17A plus IL-17F (double knockout) in mice has very little impact on the general outcome of systemic S. aureus infection, measured as mortality and bacterial clearance at a single time point after bacterial inoculation compared with wild-type mice (11). However, in that study, the respective roles of IL-17A and -17F in S. aureus-induced arthritis were not specifically addressed (11), and this aspect is the main focus of this study. S. aureus-induced arthritis is a great concern in RA (24), and the first phase I study using IL-17A-blocking antibodies as a treatment in RA has recently been published (6). Thus, it is clinically important to determine whether reduced IL-17A levels in RA patients would have a detrimental effect on S. aureus-induced arthritis.It is well-known that, within the IL-17 family, IL-17F is the cytokine that shares the greatest structural and functional homology with IL-17A (5, 15). Both IL-17A and IL-17F exist as homodimers or as IL-17A-IL-17F heterodimers and bind to the IL-17 receptor A (IL-17RA)-IL-17RC receptor complex (28). Furthermore, these three IL-17 cytokines may exert similar biological effects, in particular with reference to the local mobilization of neutrophils (23). Studies of healthy mice have also shown that IL-17A is capable of inhibiting the production of IL-17F under certain conditions, through a IL-17RA-dependent mechanism (27). Thus, IL-17A and IL-17F seem to be functionally linked.In the present study, we characterized the kinetics of systemic and local S. aureus infections in the presence and absence of IL-17A in mice. For this purpose, we used IL-17A knockout mice (21) and wild-type control mice in our well-established mouse models of systemic and local S. aureus-induced arthritis (1) and assessed specific aspects of arthritis and more-general clinical outcomes. Using this approach, we obtained evidence that bacterial clearance, cytokine pattern, and degree of arthritis vary over time during systemic S. aureus infection and that IL-17A plays a more important role in local host defense than in systemic host defense against S. aureus-induced arthritis.     

Derivation of a xeno-free human embryonic stem cell line

Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [Stem Cells 2006; 24:221-229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611.     

Molecular Basis of Commensalism in the Urinary Tract: Low Virulence or Virulence Attenuation?

In some patients, Escherichia coli strains establish significant bacteriuria without causing symptoms of urinary tract infection (UTI). These asymptomatic-bacteriuria (ABU) strains have been shown to express fewer virulence factors than the uropathogenic E. coli (UPEC) strains that cause severe, symptomatic UTI. Paradoxically, ABU strains carry many typical UPEC virulence genes, and the molecular basis of their low virulence therefore remains unclear. This study examined whether ABU strains might evolve from UPEC by genome loss and virulence gene attenuation. The presence of conserved E. coli K-12 genes was examined using an E. coli K-12 strain MG1655-specific DNA array and the distribution of UPEC virulence-related genes was examined with the E. coli pathoarray. Two groups of strains could be distinguished. Several ABU strains were shown by multilocus sequence typing and by comparative genomic analyses to be related to UPEC but to have smaller genome sizes. There were significant alterations in essential virulence genes, including reductive evolution by point mutations, DNA rearrangements, and deletions. Other strains were unrelated to UPEC and lacked most of the virulence-associated genes. The results suggest that some ABU strains arise from virulent strains by attenuation of virulence genes while others are nonvirulent and resemble commensal strains. We propose that virulence attenuation might constitute a general mechanism for mucosal pathogens to evolve toward commensalism.     

The effect of enzymatically degradable poly(ethylene glycol) hydrogels on smooth muscle cell phenotype

The formation of scar tissue due to dedifferentiation of smooth muscle cells (SMCs) is one of the major issues faced when engineering bladder tissue. Furthermore, cell sources for regenerating the SMC layer are also limiting. Here we explore if human mesenchymal stem cells (MCSs), cultured in enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffolds can be differentiated into SMC-like cells. We explored the degree to which a less synthetic SMC phenotype can be achieved when primary human SMCs are cultured within these scaffolds, It was observed that when both MSCs and SMCs are cultured in the PEG hydrogel scaffolds, but not on traditional tissue culture plastic, they up-regulate markers associated with the less synthetic SMC phenotype, decreased expression of alpha(5) integrin and THY-1, and increased expression of alpha-smooth muscle actin (alphaSMA) and myosin. Furthermore, we show that MSCs and SMCs cultured in the PEG hydrogels are able to proliferate and express matrix metalloproteinases for up to 21d in culture, the duration of the study. This study addresses the importance of the cellular microenvironment on cell fate, and proposes synthetic instructive biomaterials as a means to direct cell differentiation and circumvent scar tissue formation during bladder reconstruction.     

Schilddrüsenhormonsubstitution in der Schwangerschaft

Eine Schilddrüsenunterfunktion in der Schwangerschaft, meistens ausgelöst durch Autoimmunprozesse oder Jodmangel, kann sich negativ auf die Gesundheit von Mutter und Kind auswirken. Aktuelle Leitlinien empfehlen bei Vorliegen einer manifesten Hypothyreose übereinstimmend die Aufnahme einer L(Levo)-Thyroxin-Substitution. Bezüglich Diagnose und Therapie einer subklinischen Hypothyreose in der Schwangerschaft dagegen geben Fachgesellschaften in ihren aktuellen Leitlinien teilweise unterschiedliche Empfehlungen. Im vorliegenden Artikel wird eine Übersicht über die aktuellen Empfehlungen gegeben und versucht, auf dieser Basis das gegenwärtig bestmögliche diagnostische und therapeutische Vorgehen herauszuarbeiten.     

Developing equine mtDNA profiling for forensic application

Horse mtDNA profiling can be useful in forensic work investigating degraded samples, hair shafts or highly dilute samples. Degraded DNA often does not allow sequencing of fragments longer than 200 nucleotides. In this study we therefore search for the most discriminatory sections within the hypervariable horse mtDNA control region. Among a random sample of 39 horses, 32 different sequences were identified in a stretch of 921 nucleotides. The sequences were assigned to the published mtDNA types A–G, and to a newly labelled minor type H. The random match probability within the analysed samples is 3.61%, and the average pairwise sequence difference is 15 nucleotides. In a “sliding window” analysis of 200-nucleotide sections of the mtDNA control region, we find that the known repetitive central motif divides the mtDNA control region into a highly diverse segment and a markedly less discriminatory segment.