Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of beta-lactoglobulin

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.     

Dendritic cell (DC)-based protection against an intracellular pathogen is dependent upon DC-derived IL-12 and can be induced by molecularly defined antigens

Upon loading with microbial Ag and adoptive transfer, dendritic cells (DC) are able to induce immunity to infections. This offers encouragement for the development of DC-based vaccination strategies. However, the mechanisms underlying the adjuvant effect of DC are not fully understood, and there is a need to identify Ag with which to arm DC. In the present study, we analyzed the role of DC-derived IL-12 in the induction of resistance to Leishmania major, and we evaluated the protective efficacy of DC loaded with individual Leishmania Ag. Using Ag-pulsed Langerhans cells (LC) from IL-12-deficient or wild-type mice for immunization of susceptible animals, we showed that the inability to release IL-12 completely abrogated the capacity of LC to mediate protection against leishmaniasis. This suggests that the availability of donor LC-derived IL-12 is a requirement for the development of protective immunity. In addition, we tested the protective effect of LC loaded with Leishmania homolog of receptor for activated C kinase, gp63, promastigote surface Ag, kinetoplastid membrane protein-11, or Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a. The results show that mice vaccinated with LC that had been pulsed with selected molecularly defined parasite proteins are capable of controlling infection with L. major. Moreover, the protective potential of DC pulsed with a given Leishmania Ag correlated with the level of their IL-12 expression. Analysis of the cytokine profile of mice after DC-based vaccination revealed that protection was associated with a shift toward a Th1-type response. Together, these findings emphasize the critical role of IL-12 produced by the sensitizing DC and suggest that the development of a DC-based subunit vaccine is feasible.     

Influenza A virus infection inhibits the efficient recruitment of Th2 cells into the airways and the development of airway eosinophilia

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.     

Dendritic cells and host resistance to infection

Host defence against infection requires an integrated response of both the innate and adaptive arms of the immune system. Emerging data indicate that dendritic cells contribute an essential part to the initiation and regulation of adaptive immunity. Dendritic cells guard the sites of pathogen entry to the host and are uniquely suited to detect and capture invading microbes. Upon recognition of microbial structures and appropriate activation, a maturation programme is triggered and dendritic cells migrate to lymphoid organs to stimulate a primary cell-mediated immune response. Moreover, dendritic cells play a critical role in shaping the emerging response, thereby controlling the course of infection. They can discriminate between various types of microorganisms and are capable of producing different cytokines in response to different microbial stimuli. On the other hand, pathogens developed numerous strategies to evade and subvert dendritic cell functions. Elucidating the interactions of dendritic cells with microbial pathogens may lead to novel strategies for combating infectious diseases by dendritic cell-based vaccination and immunotherapy. This review highlights recent advances in our knowledge of the unique role of dendritic cells in counteracting microbial infections.     

Organization and formation of the tight junction system in human epidermis and cultured keratinocytes

Occludin and several proteins of the claudin family have been identiried in simple epithelia and in endothelia as major and structure-determining transmembrane proteins clustered in the barrier-forming tight junctions (TJ), where they are associated with a variety of TJ plaque proteins, including protein ZO-1. To examine whether TJ also occur in the squamous stratified epithelium of the interfollicular human epidermis we have applied several microscopic and biochemical techniques. Using RT-PCR techniques, we have identiried mRNAs encoding protein ZO-1, occludin and claudins 1, 4, 7, 8, 11, 12, and 17 in both tissues, skin and cultured keratinocytes, whereas claudins i and 10 have only been detected in skin tissue. By immunocytochemistry we have localized claudin-1, occludin and protein ZO-1 in distinct plasma membrane structures representing cell-cell attachment zones. While claudin-1 occurs in plasma membranes of all living cell layers, protein ZO-1 is concentrated in or even restricted to the uppermost layers, and occludin is often detected only in the stratum granulosum. Using electron microscopy, typical TJ structures ("kissing points") as well as some other apparently related junctional structures have been detected in the stratum granulosum, interspersed between desmosomes. Modes and patterns of TJ formation have also been studied in experimental model systems, e.g., during wound healing and stratification as well as in keratinocyte cultures during Ca2+-induced stratification. We conclude that the epidermis contains in the stratum granulosum a continuous zonula occludens-equivalent structure with typical TJ morphology and molecular composition, characterized by colocalization of occludin, claudins and TJ plaque proteins. In addition, cell-cell contact structures and certain TJ proteins can also be detected in other epidermal cell layers in specific cell contacts. The pattern of formation and possible functions of epidermal TJ and related structures are discussed.     

Differentially regulated expression and function of CD22 in activated B-1 and B-2 lymphocytes

CD22 is a B cell-restricted transmembrane protein that apparently controls signal transduction thresholds initiated through the B cell Ag receptor (BCR) in response to Ag. However, it is still poorly understood how the expression of CD22 is regulated in B cells after their activation. Here we show that the expression levels of CD22 in conventional B-2 cells are markedly down-regulated after cross-linking of BCR with anti-IgM mAb but are up-regulated after stimulation with LPS, anti-CD40 mAb, or IL-4. In contrast, treatment with anti-IgM mAb barely modulated the expression levels of CD22 in CD5(+) B-1 cells, consistent with a weak Ca(2+) response in anti-IgM-treated CD5(+) B-1 cells. Moreover, in CD22-deficient mice, anti-IgM treatment did not trigger enhanced Ca(2+) influx in CD5(+) B-1 cells, unlike CD22-deficient splenic B-2 cells, suggesting a relatively limited role of CD22 in BCR signaling in B-1 cells. In contrast, CD22 levels were markedly down-regulated on wild-type B-1 cells in response to LPS or unmethylated CpG-containing oligodeoxynucleotides. These data indicate that the expression and function of CD22 are differentially regulated in B-1 and conventional B-2 cells, which are apparently implicated in innate and adaptive immunity, respectively.     

In Vitro Degradation of Endothelial Catenins by a Neutrophil Protease

It has been recently proposed that adhesion of polymorphonuclear cells (PMNs) to human umbilical vein endothelial cells leads to the disorganization of the vascular endothelial cadherin–dependent endothelial adherens junctions. Combined immunofluorescence and biochemical data suggested that after adhesion of PMNs to the endothelial cell surface, β-catenin, as well as plakoglobin was lost from the cadherin/catenin complex and from total cell lysates. In this study we present data that strongly suggest that the adhesion-dependent disappearance of endothelial catenins is not mediated by a leukocyte to endothelium signaling event, but is due to the activity of a neutrophil protease that is released upon detergent lysis of the cells.     

The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species.

The mutant mouse strain Mpv17 carries a retroviral insert in its genome which inactivates the Mpv17 gene. At a young age these mice develop glomerulosclerosis and nephrotic syndrome which resembles human disease. We show here that the Mpv17 gene product is highly conserved and encodes a peroxisomal protein. Loss of the Mpv17 protein does not impair peroxisome biogenesis but instead leads to a reduced ability to produce reactive oxygen species (ROS). In turn, overproduction of the Mpv17 gene in transfected cells results in dramatically enhanced levels of intracellular ROS indicating a direct involvement of Mpv17 in ROS production. These data reveal a role for the Mpv17 protein in peroxisomal reactive oxygen metabolism and establish a novel link between peroxisomal ROS production and glomerulosclerosis.