First LightCycler real-time PCR assay for the quantitative detection of Mycoplasma suis in clinical samples

Mycoplasma suis cannot be cultivated in vitro. Therefore, PCR-based methods are irreplaceable for the diagnosis of M. suis infections especially when clinical symptoms are not evident. Currently, no easy and reliable method allowing the quantitative detection of M. suis is available. This report describes the development of a quantitative LightCycler PCR assay based on the msg1 gene of M. suis (LC MSG1 PCR). No PCR signals were obtained with closely related haemotrophic and non-haemotrophic mycoplasmas, with other bacteria, and with M. suis-free blood and tissue arguing for a high analytical specificity. Test sensitivity was found to be 100%, and test specificity 96.7%. To test the diagnostic suitability of the LC MSG1 PCR, 25 pigs with clinical porcine eperythrozoonosis and 25 healthy pigs were investigated. All ill pigs revealed a positive real-time PCR result whereas only one healthy pig was detected to be M. suis-infected. M. suis was quantitatively detected in 19 blood specimens of 100 sows from Switzerland and in 17 of 160 post-weaning piglets from Germany. In conclusion, this new LC MSG1 PCR assay represents a powerful tool for the improvement of the current M. suis diagnosis and for prevalence and pathogenesis studies.     

Ultrastructural detection of proteins, lipids and carbohydrates in oocytes of Amblyomma triste (Koch, 1844) (Acari; Ixodidae) during the vitellogenesis process

The ovary of the tick Amblyomma triste is classified as panoistic, which is characterized by the presence of oogonia without nurse and follicular cells. The present study has demonstrated that the oocytes in all developmental stages (I-IV) are attached to the ovary through a pedicel, a cellular structure that synthesizes and provides carbohydrate, lipids and proteins supplies for the oocytes during the vitellogenesis process. The lipids are deposited during all oocyte stages; they are freely distributed as observed in stages II, III and IV or they form complexes with other elements. The proteins are also deposited in all stages of the oocytes, however, in lower concentration in the stage IV. There is carbohydrate deposition from oocytes in the stage II as well as in stages III and IV. In addition, the present work has demonstrated that the oocyte yolk of A. triste has a glycolipoprotein nature and the elements are deposited in the following sequence: firstly the lipids and proteins, and finally the carbohydrates.     

Endemic persistence or disease extinction: The effect of separation into sub-communities

Consider an infectious disease which is endemic in a population divided into several large sub-communities that interact. Our aim is to understand how the time to extinction is affected by the level of interaction between communities. We present two approximations of the expected time to extinction in a population consisting of a small number of large sub-communities. These approximations are described for an SIR epidemic model, with focus on diseases with short infectious period in relation to life length, such as childhood diseases. Both approximations are based on Markov jump processes. Simulations indicate that the time to extinction is increasing in the degree of interaction between communities. This behaviour can also be seen in our approximations in relevant regions of the parameter space.     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-alpha, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-kappaB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn's disease.     

Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.     

Characterization of the amino acids involved in substrate specificity of methionine sulfoxide reductase A

Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide (MetSO) back to methionine. In vivo, Msrs are essential in protecting cells against oxidative damages on proteins and in the virulence of some bacteria. There exists two structurally unrelated classes of Msrs. MsrAs are stereo-specific toward the S epimer on the sulfur of the sulfoxide, whereas MsrBs are specific toward the R isomer. Both classes of Msrs display a similar catalytic mechanism of sulfoxide reduction by thiols via the sulfenic acid chemistry and a better affinity for protein-bound MetSO than for free MetSO. Recently, the role of the amino acids implicated in the catalysis of the reductase step of Neisseria meningitidis MsrA was determined. In the present study, the invariant amino acids potentially involved in substrate binding, i.e. Phe-52, Trp-53, Asp-129, His-186, Tyr-189, and Tyr-197, were substituted. The catalytic parameters under steady-state conditions and of the reductase step of the mutated MsrAs were determined and compared with those of the wild type. Altogether, the results support the presence of at least two binding subsites. The first one, whose contribution is major in the efficiency of the reductase step and in which the epsilon-methyl group of MetSO binds, is the hydrophobic pocket formed by Phe-52 and Trp-53, the position of the indole ring being stabilized by interactions with His-186 and Tyr-189. The second subsite composed of Asp-129 and Tyr-197 contributes to the binding of the main chain of the substrate but to a lesser extent.     

Recognition of a defined region within p24 gag by CD8+ T cells during primary human immunodeficiency virus type 1 infection in individuals expressing protective HLA class I alleles

Human immunodeficiency virus type 1 (HIV-1)-specific immune responses during primary HIV-1 infection appear to play a critical role in determining the ultimate speed of disease progression, but little is known about the specificity of the initial HIV-1-specific CD8(+) T-cell responses in individuals expressing protective HLA class I alleles. Here we compared HIV-1-specific T-cell responses between subjects expressing the protective allele HLA-B27 or -B57 and subjects expressing nonprotective HLA alleles using a cohort of over 290 subjects identified during primary HIV-1 infection. CD8(+) T cells of individuals expressing HLA-B27 or -B57 targeted a defined region within HIV-1 p24 Gag (amino acids 240 to 272) early in infection, and responses against this region contributed over 35% to the total HIV-1-specific T-cell responses in these individuals. In contrast, this region was rarely recognized in individuals expressing HLA-B35, an HLA allele associated with rapid disease progression, or in subjects expressing neither HLA-B57/B27 nor HLA-B35 (P < 0.0001). The identification of this highly conserved region in p24 Gag targeted in primary infection specifically in individuals expressing HLA class I alleles associated with slower HIV-1 disease progression provides a rationale for vaccine design aimed at inducing responses to this region restricted by other, more common HLA class I alleles.     

Enzymatic conversion of dynorphin A in the rat brain is affected by administration of nandrolone decanoate

The misuse of anabolic androgenic steroids (AAS) seems to produce profound effects on the central nervous system, leading to aggressive behavior and increased sensitivity to other drugs of abuse. The present study addresses the effect on the enzymatic transformation, here called dynorphin converting enzyme-like activity. The formation of the mu/delta opioid peptide receptor-preferring Leu-enkephalin-Arg(6) from the kappa opioid peptide receptor-preferring dynorphin A was measured in rats treated with nandrolone decanoate. Significant variations in enzymatic transformation were observed in several brain regions. An altered receptor activation profile in these regions may be one contributory factor behind AAS-induced personality changes.     

Innate versus adaptive immunity in sticklebacks: evidence for trade-offs from a selection experiment

In vertebrates, the immune system consists of two arms of different characteristics: the innate and the acquired immune response. Parasites that are only shortly exposed to the immune system are most efficiently attacked by fast, constitutive innate immune mechanisms. Here, we experimentally selected within four fish families for high innate resistance versus susceptibility of three-spined sticklebacks (Gasterosteus aculeatus) against infection with the eye-fluke (Diplostomum pseudospathacaeum), a parasite whose metacercariae are protected from the immune system within the eye lens. We predicted that in families with high susceptibility, the adaptive immune system would be upregulated when challenged with infection. In accordance, we found that MHC class IIB expression is increased by approximately 50% in those lines selected for higher parasite load (i.e. low innate response). This suggests extensive genetic correlations between innate and adaptive immune system and/or crosstalk between both lines of defense. An efficient, specific innate immune response might reduce overall activation of the immune system and potentially alleviate associated effects of immunopathology.