The Influence of Two Different Foam Creams on Skin Barrier Repair of Foot Xerosis: A Prospective, Double-Blind, Randomised, Placebo-Controlled Intra-Individual Study.

BACKGROUND/AIMS: Dry skin, or xerosis, is a common condition and a key feature of skin diseases like atopic dermatitis (AD) and ichthyosis vulgaris. Foot xerosis may exist without underlying disease and could be related to very mild forms of AD or ichthyosis vulgaris. The synthesis of important skin lipids (cholesterol, free fatty acids and ceramides) is reduced in xerosis and AD, and reduced lipid synthesis is responsible for a lack of lipids and enzymes in the skin barrier. This slows down reorganisation of the lipid lamellae in the stratum corneum (SC). METHODS: Skin barrier integrity was measured by morphometric analysis of the lipid lamellae in the SC after 4 weeks of treatment with a foam cream (active agent vs. placebo). RESULTS: Significant treatment effects were shown after 2 and 4 weeks by an increasing amount of intercellular lipids in the SC. CONCLUSION: This study shows that a quick reorganisation of the SC lipids initiates a good restoration of the whole skin barrier after 4 weeks of treatment with a foam cream.

Overexpression of a single Leishmania major gene enhances parasite infectivity in vivo and in vitro.

We identified a Leishmania major-specific gene that can partly compensate for the loss of virulence observed for L. major HSP100 null mutants. The gene, encoding a 46 kD protein of unknown function and lineage, also enhances the virulence of wild type L. major upon overexpression. Surprisingly, the approximately sixfold overexpression of this protein also extends the host range of L. major to normally resistant C57BL/6 mice, causing persisting lesions in this strain, even while eliciting a strong cellular immune response. This enhanced virulence in vivo is mirrored in vitro by increased parasite burden inside bone marrow-derived macrophages. The localization of the protein in the macrophage cytoplasm suggests that it may modulate the macrophage effector mechanisms. In summary, our data show that even minor changes of gene expression in L. major may alter the outcome of an infection, regardless of the host's genetic predisposition.

The interplay between Leishmania promastigotes and human Natural Killer cells in vitro leads to direct lysis of Leishmania by NK cells and modulation of NK cell activity by Leishmania promastigotes.

NK cells represent one of the first lines of defence in the immune reaction after invasion of Leishmania parasites. Depletion of mouse natural killer (NK) cells dramatically enhances susceptibility of normally resistant mice. In this study we evaluated the fate of NK cells and parasites after contact formation. The hydrophilic fluorescent dye CMFDA (chloro-methylfluorescin diacetate) that allows analysis of cytotoxicity in flow cytometry and microscopy was used. Furthermore, these findings were confirmed with scanning and transmission electron microscopy. Direct contact points were found between Leishmania promastigotes and naïve human NK cells. These contacts were associated with transfer of cytosol by membrane bridges and cytotoxicity of NK cells against Leishmania. However, in contrast to other target cells which allow repeated exocytosis of lytic granules, contact with Leishmania causes immediate destruction of NK cells in a non-apoptotic way. Our results give a reasonable explanation for ex vivo observations of reduced NK cell numbers and impaired NK response in patients with acute cutaneous leishmaniasis. Animal models have clearly shown that NK cells play a key role in the induction and direction of the immune response. Thus inhibition of NK cells at the onset of infection would be advantageous for the survival of the parasite.

The nematode parasite Onchocerca volvulus generates the transforming growth factor-beta (TGF-beta).

Transforming growth factor-beta (TGF-beta) is a highly conserved cytokine that has a well-known regulatory role in immunity, but also in organ development of most animal species including helminths. Homologous tgf-b genes and mRNA have been detected in the filaria Brugia malayi. The in situ protein expression is unknown for filariae. Therefore, we examined several filariae for the expression and localization of latent (stable) TGF-beta in adult and larval stages. A specific goat anti-human latency associated protein (LAP, TGF-beta 1) antibody, purified by affinity chromatography, was used for light and electron microscopic immunohistochemistry. Adult Onchocerca volvulus, Onchocerca gibsoni, Onchocerca ochengi, Onchocerca armillata, Onchocerca fasciata, Onchocerca flexuosa, Wuchereria bancrofti, Dirofilaria sp., B. malayi, and infective larvae of W. bancrofti reacted with the antibody. Labeling of worm tissues varied between negative and all degrees of positive reactions. Latent TGF-beta was strongly expressed adjacent to the cell membranes of the hypodermis, epithelia, and muscles and adjacent to many nuclei in all organs. TGF-beta was well expressed in worms without Wolbachia endobacteria eliminated by doxycycline treatment. Pleomorphic neoplasms in O. volvulus were also labeled. We conclude that latent TGF-beta protein is expressed by filariae independently of Wolbachia, possibly regulating worm tissue homeostasis.

Interacting protein kinases involved in the regulation of flagellar length.

A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis.

The secretory omega-class glutathione transferase OvGST3 from the human pathogenic parasite Onchocerca volvulus.

Onchocerciasis or river blindness, caused by the filarial nematode Onchocerca volvulus, is the second leading cause of blindness due to infectious diseases. The protective role of the omega-class glutathione transferase 3 from O. volvulus (OvGST3) against intracellular and environmental reactive oxygen species has been described previously. In the present study, we continue our investigation of the highly stress-responsive OvGST3. Alternative splicing of two exons and one intron retention generates five different transcript isoforms that possess a spliced leader at their 5'-end, indicating that the mechanism of mature mRNA production involves alternative-, cis- and trans-splicing processes. Interestingly, the first two exons of the ovgst3 gene encode a signal peptide before sequence identity to other omega-class glutathione transferases begins. Only the recombinant expression of the isoform that encodes the longest deduced amino acid sequence (OvGST3/5) was successful, with the purified enzyme displaying modest thiol oxidoreductase activity. Significant IgG1 and IgG4 responses against recombinantly expressed OvGST3/5 were detected in sera from patients with the generalized as well as the chronic hyperreactive form of onchocerciasis, indicating exposure of the secreted protein to the human host's immune system and its immunogenicity. Immunohistological localization studies performed at light and electron microscopy levels support the extracellular localization of the protein. Intensive labeling of the OvGST3 was observed in the egg shell at the morula stage of the embryo, indicating extremely defined, stage-specific expression for a short transient period only.

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion.

BACKGROUND: Plasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation. METHODS: Immunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process. RESULTS: Antigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown. CONCLUSION: The localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.

Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples.

The purpose of this study was to explore the turnaround times, section and image quality of a number of more "difficult" specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples. A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus, Orthopox virus), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis), suspensions of parasites (malaria Plasmodium falciparum, Leishmania major, Microsporidia cuniculi, Caenorhabditis elegans), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO4-postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 degrees C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization. The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens. Compared with conventional routine methods, microwave technology facilitates a significant reduction in sample processing time from days to hours without any loss in ultrastructural details. Microwave-assisted processing could, therefore, be a substantial benefit for the routine electron microscopic diagnostic workload. Due to its speed and robust performance it could be applied wherever a rapid electron microscopy diagnosis is required, e.g., if bioterrorism or emerging agents are suspected. Combining microwave technology with digital image acquisition, the 1-day diagnosis based on ultrathin section electron microscopy will become possible, with crucial or interesting findings being consulted or shared worldwide with experts using modern telemicroscopy tools via Internet.

Exchange of cytosolic content between T cells and tumor cells activates CD4 T cells and impedes cancer growth.

BACKGROUND: T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described. METHODS/ FINDINGS: Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes--even in CD4⁺ T cells and murine B cells--which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement. Electron microscopy disclosed 100-200 nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice. CONCLUSIONS: The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.

Acute liver failure, multiorgan failure, cerebral oedema, and activation of proangiogenic and antiangiogenic factors in a case of Marburg haemorrhagic fever.

A woman developed Marburg haemorrhagic fever in the Netherlands, most likely as a consequence of being exposed to virus-infected bats in the python cave in Maramagambo Forest during a visit to Uganda. The clinical syndrome was dominated by acute liver failure with secondary coagulopathy, followed by a severe systemic inflammatory response, multiorgan failure, and fatal cerebral oedema. A high blood viral load persisted during the course of the disease. The initial systemic inflammatory response coincided with peaks in interferon-γ and tumour necrosis factor-α concentrations in the blood. A terminal rise in interleukin-6, placental growth factor (PlGF), and soluble vascular endothelial growth factor receptor-1 (sVEGF-R1) seemed to suggest an advanced pathophysiological stage of Marburg haemorrhagic fever associated with vascular endothelial dysfunction and fatal cerebral oedema. The excess of circulating sVEGF-R1 and the high sVEGF-R1:PlGF ratio shortly before death resemble pathophysiological changes thought to play a causative part in pre-eclampsia. Aggressive critical-care treatment with renal replacement therapy and use of the molecular absorbent recirculation system appeared able to stabilise--at least temporarily--the patient's condition.

Isolation and phylogenetic analysis of Batai virus, Germany.

A molecular survey including 16,057 mosquitoes captured in Southwest Germany during the summer of 2009 showed the presence of Batai virus (BATV) in Anopheles maculipennis sensu lato. Until this survey, there was no evidence for circulation of BATV in Germany. Analysis of partial S, M, and L segments showed that the sequences from all three segments were most closely related to BATV, indicating that the virus has not undergone reassortment. Phylogenetic analysis revealed a close relationship of the isolated BATV strain from Germany with strains from Slovakia, Ukraine, and Russia.

Onchocerca volvulus: expression and immunolocalization of a nematode cathepsin D-like lysosomal aspartic protease.

The N-terminal region of the cathepsin D-like aspartic protease from the human filarial parasite Onchocerca volvulus was expressed as His-tag fusion protein. Light and electron microscopic immunohistology using antibodies against the recombinant protein showed labeling of lysosomes in the hypodermis and epithelia of the intestine and the reproductive organs of Onchocerca. While developing oocytes were negative, mature oocytes and early morulae showed strong labeling. In older embryos and mature microfilariae, stained lysosomes were only found in a few cells. Cell death in degenerating microfilariae of patients untreated and treated with microfilaricidal drugs was associated with strong expression of aspartic protease. IgG1, IgG4, and IgE antibodies reactive with the recombinant protein were demonstrated in sera from onchocerciasis patients indicating exposure and recognition of the enzyme by the host's defence system. The aspartic protease of O. volvulus appears to function in intestinal digestion and tissue degradation of the filaria.

Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites.

In search of Wolbachia in human parasites, Wolbachia were identified in the sand flea Tunga penetrans. PCR and DNA sequencing of the bacterial 16S rDNA, the ftsZ cell division protein, the Wolbachia surface protein (wsp) and the Wolbachia aspartate aminotransferase genes revealed a high similarity to the respective sequences of endosymbionts of filarial nematodes. Using these sequences a phylogenetic tree was generated, that indicates a close relationship between Wolbachia from T. penetrans and from filarial parasites, but possibly as a member of a new supergroup. Ultrastructural studies showed that Wolbachia are abundant in the ovaries of neosomic fleas, whereas other, smaller and morphologically distinct, bacteria were observed in the lumen of the intestine. Wolbachia were labeled by immunohistology and immunogold electron microscopy using polyclonal antibodies against wsp of Drosophila, of the filarial parasite Dirofilaria immitis, or against hsp 60 from Yersinia enterocolitica. These results show that as in filariasis, humans with tungiasis are exposed to Wolbachia. Furthermore, antisera raised against proteins of Wolbachia from arthropods or from filarial parasites can be immunologically cross-reactive.