Rapid leukocyte integrin activation by chemokines

Summary: Chemokines control selective targeting of circulating leukocytes to the microvasculature by triggering inside-out signal transduction pathways leading to integrin-dependent adhesion. Integrin activation by chemokines is very rapid, is downmodulated within minutes and appears to involve both enhanced heterodimer lateral mobility on the plasma membrane, facilitating encounters with dispersed ligand, as well as induction of a high-affinity state. These two modalities of integrin activation by chemokines involve distinct signaling pathways in the cell, yet complement each other functionally, allowing binding of rolling cells under conditions of low as well as high ligand density. Recent data show that chemokines generate both pro- and anti-adhesive intracellular signaling events, whose equilibrium is likely to be relevant to the kinetics of adhesion and de-adhesion, and to cell movement during diapedesis and chemotaxis. Importantly, chemokines utilize different signaling mechanisms to modulate the activity of distinct integrin subtypes. These recent advances suggest that chemokines may regulate adhesive responses of immune cells based not only on patterns of chemokine receptor expression, but also on variable signaling pathways that can modulate the pro-adhesive responses of leukocytes as a function of their differentiated state, and of the local microenvironment.     

Parenting stress and parental bonding

Attachment experiences are thought to be important because of their implications for later development. The authors' aim with the questionnaire-based study was to investigate the differences between recalled parental bonding regarding 4 types of maternal and paternal bonding with respect to experienced parenting stress caused by child characteristics, parent attributes, and life events under the consideration of the child's gender and age. The authors gathered parental bonding behavior data with the German version of the Parental Bonding Instrument (PBI). The authors assessed parenting stress with their German version of the "Parenting Stress Index (PSI)." They found significant differences among 120 mothers grouped in the 4 maternal and the 4 paternal bonding types regarding parenting stress caused by child, maternal bonding: F(5, 113) = 4.13, p = .002, paternal bonding: F(5, 111) = 8.50, p < or = .0001, and parent characteristics, maternal bonding: F(5, 113) = 3.33, p = .008, paternal bonding: F(5, 111) = 7.80, p < or = .0001. The lowest level of parenting stress was experienced by mothers who themselves recalled the "optimal parental bonding type" with respect to the child and parental domain. The authors did not find any significant differences between the 4 maternal, F(5, 113) = 1.25, p = .29, and the 4 paternal, F(5, 111) = 1.87, p = .106, bonding types with respect to the life stress. According to the authors' findings, the representation of attachment relationships seems to have a special impact on the adult's capacity to cope with challenges and stress, either directly or indirectly as an internal working model of attachment. For the clinical practice, these findings seem to recommend the combination of both the PSI and PBI regarding the diagnostic of stressful mother-child system to plan an optimal intervention program.     

Development of DNA vaccines against hemolytic-uremic syndrome in a murine model

Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2 Delta A). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.     

Glycogen stability and glycogen phosphorylase activities in isolated skeletal muscles from rat and toad

There is increasing evidence that endogenous glycogen depletion may affect excitation–contraction (E–C) coupling events in vertebrate skeletal muscle. One approach employed in physiological investigations of E–C coupling involves the use of mechanically skinned, single fibre preparations obtained from tissues stored under paraffin oil, at room temperature (RT: 20–24°C) and 4°C for several hours. In the present study, we examined the effect of these storage conditions on the glycogen content in three muscles frequently used in research on E–C coupling: rat extensor digitorum longus (EDL) and soleus (SOL) and toad iliofibularis (IF). Glycogen content was determined fluorometrically in homogenates prepared from whole muscles, stored under paraffin oil for up to 6 h at RT or 4°C. Control muscles and muscles stored for 0.5 and 6 h were also analysed for total phosphorylase (Phos_total) and phosphorylase a (Phos a) activities. No significant change was observed in the glycogen content of EDL and SOL muscles stored at RT for 0.5 h. In rat muscles stored at RT for longer than 0.5 h, the glycogen content decreased to 67.6% (EDL) and 78.7% (SOL) of controls after 3 h and 25.3% (EDL) and 37.4% (SOL) after 6 h. Rat muscles stored at 4°C retained 79.0% (EDL) and 92.5% (SOL) of glycogen after 3 h and 75.2% (EDL) and 61.1% (SOL) after 6 h. The glycogen content of IF muscles stored at RT or 4°C for 6 h was not significantly different from controls. Phos_total was unchanged in all muscles over the 6 h period, at both temperatures. Phos a was also unchanged in the toad IF muscles, but in rat muscles it decreased rapidly, particularly in EDL (4.1-fold after 0.5 h at RT). Taken together these results indicate that storage under paraffin oil for up to 6 h at RT or 4°C is accompanied by minimal glycogen loss in toad IF muscles and by a time- and temperature-dependent glycogen loss in EDL and SOL muscles of the rat.     

Cell surface-associated elongation factor Tu mediates the attachment of Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins

The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.     

Classical Hodgkin lymphoma arising in the rectum

We report a case of an 81-year-old immunocompetent Mexican man who underwent an abdominal-perineal rectal resection for a mass clinically thought to be carcinoma. Histopathologic diagnosis revealed classical Hodgkin lymphoma, nodular sclerosis type, involving the rectum. The diagnosis was confirmed by immunohistochemical studies that showed that the neoplastic cells were positive for CD15 and CD30 and negative for CD45 (LCA). In situ hybridization for Epstein-Barr virus small-encoded RNA was also positive in the neoplastic cells. Hodgkin lymphoma arising in the rectum of immunocompetent patients is rare, with only 12 cases (including this one) reported in the literature. Of these, the diagnosis was confirmed by immunohistochemical studies in only two cases, and this is the first case assessed and shown to be positive for Epstein-Barr virus.     

CCL22-induced responses are powerfully enhanced by synergy inducing chemokines via CCR4: evidence for the involvement of first beta-strand of chemokine

In an attempt to clarify how cells integrate the signals provided by multiple chemokines expressed during inflammation, we have uncovered a novel mechanism regulating leukocyte trafficking. Our data indicate that the concomitant exposure to CCR4 agonists and CXCL10/IP-10 strongly enhances the chemotactic response of human T lymphocytes. This enhancement is synergistic rather than additive and occurs via CCR4 since it persists after CXCR3 blockade. Besides chemotaxis, other cellular responses are enhanced upon stimulation of CCR4-transfected cells with CCL22/MDC plus CXCL10. Several other chemokines in addition to CXCL10 were able to increase CCL22-mediated chemotaxis. The first beta-strand of the chemokine structure is highly and specifically implicated in this phenomenon, as established using synergy-inducing and non-synergy-inducing chimeric chemokines. As shown in situ for skin from atopic and allergic contact dermatitis patients, this organ becomes the ideal environment in which skin-homing CCR4(+) T lymphocytes can accumulate under the stimulus offered by CCR4 agonists, together with the synergistic chemokines that are concomitantly expressed. Overall, our results indicate that chemokine-induced synergism strengthens leukocyte recruitment towards tissues co-expressing several chemokines.     

Gene therapy for pituitary tumors

Pituitary tumors are the most common primary intracranial neoplasms. Although most pituitary tumors are considered typically benign, others can cause severe and progressive disease. The principal aims of pituitary tumor treatment are the elimination or reduction of the tumor mass, normalization of hormone secretion and preservation of remaining pituitary function. In spite of major advances in the therapy of pituitary tumors, for some of the most difficult tumors, current therapies that include medical, surgical and radiotherapeutic methods are often unsatisfactory and there is a need to develop new treatment strategies. Gene therapy, which uses nucleic acids as drugs, has emerged as an attractive therapeutic option for the treatment of pituitary tumors that do not respond to classical treatment strategies if the patients become intolerant to the therapy. The development of animal models for pituitary tumors and hormone hypersecretion has proven to be critical for the implementation of novel treatment strategies and gene therapy approaches. Preclinical trials using several gene therapy approaches for the treatment of anterior pituitary diseases have been successfully implemented. Several issues need to be addressed before clinical implementation becomes a reality, including the development of more effective and safer viral vectors, uncovering novel therapeutic targets and development of targeted expression of therapeutic transgenes. With the development of efficient gene delivery vectors allowing long-term transgene expression with minimal toxicity, gene therapy will become one of the most promising approaches for treating pituitary adenomas.     

Casein is an essential cofactor in autoantibody reactivity directed against the C-terminal SmD1 peptide AA 83-119 in systemic lupus erythematosus

The C-terminal peptide SmD1(83-119) has been identified as an important autoantigen in systemic lupus erythematosus (SLE). ELISA studies have shown that roughly 70% of all sera from patients with SLE react with this peptide. Previous findings revealed that the addition of blocking agents and sample dilution buffers influences the discrimination between positive and negative anti-SmD1(83-119) sera in SLE. The aim of the present study was to identify possible cofactors in the anti-SmD1(83-119) reactivity. We therefore tested SLE sera (n=6) for anti-SmD1(83-119) reactivity by ELISA and analysed the effects of different blocking agents (1% skim milk, 1% gelatin, and 1% BSA). In our investigation, lipids were extracted from skim milk using dichlomethane, and the putative fraction was tested to assess the assay's ability to discriminate between positive and negative sera. The effects of enzymatic digestion by casein were analyzed, and different concentrations of casein were used to determine the role of this protein in the detection of anti-SmD1(83-119) antibodies by ELISA. Furthermore, rabbits were immunized with SmD1(83-119) adsorbed to casein and control proteins. One percent skim milk was the most effective blocking agent and sample dilution buffer for the discrimination between positive and negative sera. As demonstrated by SDS electrophoresis, the discriminative capacity was influenced by enzymatic digestion of skim milk proteins, but not by lipid extraction. Differences in anti-SmD1(83-119) reactivity upon variation of the casein concentration suggest that the protein plays a significant role in the detection of anti-SmD1(83-119) antibodies. However, our immunisation studies did not show any effect of casein on anti-SmD1(83-119) reactivity, suggesting that it has no immunogenic effect on the anti-SmD1(83-119) response. In conclusion, casein seems to be an important cofactor in autoantibody reactivity directed against the C-terminal SmD1(83-119) peptide and probably functions by changing the conformation of the peptide's critical epitope.