Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B(4) (LTB(4)) is one of the most potent PMN chemoattractants. LTB(4) exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB(4) signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6(-/-) mice. In vitro, GRK6(-/-) PMN showed increased chemokinetic and chemotactic responses to LTB(4). GRK6(-/-) PMN respond to LTB(4) with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB(4) receptor desensitization in the absence of GRK6. However, pre-exposure to LTB(4) renders both GRK6(-/-) as well as wild-type PMN refractory to restimulation with LTB(4), indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.     

Antigens up the nose: identification of putative biomarkers for nasal tolerance induction functional studies combined with proteomics

Intranasal autoantigen delivery is the most effective means of inducing mucosal tolerance and suppression of autoimmune disease. In an effort to identify markers of the "tolerant state", we employed proteomics technology at the level of the cervical lymph node. The analysis revealed that nasal antigen administration (without adiuvant) led to modulation of various proteins among which the most prominent were haptoglobin, nonintegrin 67 kDa laminin receptor, and MRP8. The immunoregulatory haptoglobin may qualify as (bio)marker for effective immunotherapy.     

Transpeptidation and reverse proteolysis and their consequences for immunity

Reverse proteolysis and transpeptidation lead to the generation of polypeptide sequences that cannot be inferred directly from genome sequences as they are post-translational phenomena. These phenomena have so far received little attention although the physiological consequences may reach far. The protease-mediated synthesis of several immunodominant MHC class I antigens was recently reported, underscoring its importance to immunity. Reverse proteolytic and transpeptidation mechanisms as well as conditions that favor successful protease-catalyzed synthetic events are discussed here.     

G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis

Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1-125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2(+/-) mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2(+/-) mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2(+/-) animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2(+/-) mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2(+/-) animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.     

Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination

Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund’s Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund’s complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.     

Fine mapping and imprinting analysis for fatness trait QTLs in pigs

Quantitative trait loci (QTL) for fatness traits were reported recently in an experimental Meishan × Large White and Landrace F₂ cross. To further investigate the regions on pig Chr 2 (SSC2), SSC4, and SSC7, 25 additional markers from these regions were typed on 800 animals (619 F₂ animals, their F₁ parents, and F₀ grandfathers). Compared with the published maps, a modified order of markers was observed for SSC4 and SSC7. QTL analyses were performed both within the half-sib families as well as across families (line cross). Furthermore, a QTL model accounting for imprinting effects was tested. Information content could be increased considerably on all three chromosomes. Evidence for the backfat thickness QTL on SSC7 was increased, and the location could be reduced to a 33-cM confidence interval. The QTL for intramuscular fat on SSC4 could not be detected in this half-sib analysis, whereas under the line cross model a suggestive QTL on a different position on SSC4 was detected. For SSC2, in the half-sib analysis, a suggestive QTL for backfat thickness was detected with the best position at 26 cM. Imprinting analysis, however, revealed a genome-wise, significant, paternally expressed QTL on SSC2 with the best position at 63 cM. Our results suggest that this QTL is different from the previously reported paternally expressed QTL for muscle mass and fat deposition on the distal tip of SSC2p. Received: 15 October 1999 / Accepted: 21 February 2000     

Detection of anti-M. leprae antibodies in children in leprosy-endemic areas: A systematic review

BackgroundLeprosy elimination primarily targets transmission of Mycobacterium leprae which is not restricted to patients’ households. As interruption of transmission is imminent in many countries, a test to detect infected asymptomatic individuals who can perpetuate transmission is required. Antibodies directed against M. leprae antigens are indicative of M. leprae infection but cannot discriminate between active and past infection. Seroprevalence in young children, however, reflects recent M. leprae infection and may thus be used to monitor transmission in an area. Therefore, this literature review aimed to evaluate what has been reported on serological tests measuring anti-M. leprae antibodies in children without leprosy below the age of 15 in leprosy-endemic areas.Methods and findingsA literature search was performed in the databases Pubmed, Infolep, Web of Science and The Virtual Health Library. From the 724 articles identified through the search criteria, 28 full-text articles fulfilled all inclusion criteria. Two additional papers were identified through snowballing, resulting in a total of 30 articles reporting data from ten countries. All serological tests measured antibodies against phenolic glycolipid-I or synthetic derivatives thereof, either quantitatively (ELISA or UCP-LFA) or qualitatively (ML-flow or NDO-LID rapid test). The median seroprevalence in children in endemic areas was 14.9% and was stable over time if disease incidence remained unchanged. Importantly, seroprevalence decreased with age, indicating that children are a suitable group for sensitive assessment of recent M. leprae infection. However, direct comparison between areas, solely based on the data reported in these studies, was impeded by the use of different tests and variable cut-off levels.ConclusionsQuantitative anti-PGL-I serology in young children holds promise as a screening test to assess M. leprae infection and may be applied as a proxy for transmission and thereby as a means to monitor the effect of (prophylactic) interventions on the route to leprosy elimination.     

Importance of Weight Loss Maintenance and Risk Prediction in the Prevention of Type 2 Diabetes: Analysis of European Diabetes Prevention Study RCT

Background

Prevalence of type 2 diabetes (T2D) is increasing worldwide. T2D prevention by lifestyle intervention is effective. Pragmatic scalable interventions are needed, with evidence to efficiently target and monitor such interventions. We report pooled analyses of data from three European trial cohorts: to analyse T2D incidence, sustained weight loss and utility of risk predictors.

Methods

We analysed data on 749 adults with impaired glucose tolerance (278 men and 471 women, mean age 56 years, mean BMI 31 kgm⁻²) recruited between 1993 and 2003, and randomised to intensive lifestyle intervention (I) or lifestyle advice control (C). The intervention aimed to increase physical activity, modify diet, and promote weight loss≥5%. Using Cox-regression survival analysis, we assessed T2D incidence and the impact on T2D incidence of sustained weight loss, and of baseline cut-point values of FINDRISC score, fasting plasma glucose (FPG), and HbA_1c.

Results

Mean follow-up duration was 3.1 years. T2D was diagnosed in 139 participants (I = 45/379, C = 94/370). Cumulative T2D incidence was 57% lower in the intervention compared with the control group (HR 0.42 (95% CI 0.29 to 0.60) P<0.001). Participants with ≥5% weight loss at one year had 65% lower T2D incidence (HR 0.35 (95% CI 0.22 to 0.56) P<0.001); maintaining ≥5% weight loss for two and three years further reduced T2D incidence. Recommended cut-points to identify those at high risk for T2D would have identified different proportions of European Diabetes Prevention Study (EDIPS) participants with similar hazard-ratios for intervention effect.

Conclusions

Pooled analysis of EDIPS trial data reinforces evidence for T2D prevention by lifestyle intervention. Analysis showed the preventive effect of ≥5% weight loss, especially if maintained long term, which has utility for intervention monitoring. Analysis of proposed cut-points demonstrates difficulties in balancing risk and benefit, to efficiently target interventions and suggests evidence is needed to define clinical policy.

Trial registrations

The Finnish Diabetes Prevention study, Helsinki, Finland: ClinicalTrials.gov; {"type":"clinical-trial","attrs":{"text":"NCT00518167","term_id":"NCT00518167"}}NCT00518167 The SLIM diabetes prevention study, Maastricht, The Netherlands: Clinical Trials.gov; {"type":"clinical-trial","attrs":{"text":"NCT00381186","term_id":"NCT00381186"}}NCT00381186 The EDIPS-Newcastle diabetes prevention study, Newcastle upon Tyne, UK: International Standard Randomised Controlled Trial Number; ISRCTN15670600.     

Natural Selection of Human Embryos: Decidualizing Endometrial Stromal Cells Serve as Sensors of Embryo Quality upon Implantation

Background

Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown.

Methodology/Principal Findings

We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo.

Conclusions

Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies.