Degenerate peptide recognition by Candida albicans adhesins Als5p and Als1p

Candida albicans and Saccharomyces cerevisiae expressing the adhesins Als5p or Als1p adhere to immobilized peptides and proteins that possess appropriate sequences of amino acids in addition to a sterically accessible peptide backbone. In an attempt to further define the nature of these targets, we surveyed the ability of yeast cells to adhere to 90- micro m-diameter polyethylene glycol beads coated with a 7-mer peptide from a library of 19(7) unique peptide-beads. C. albicans bound to ca. 10% of beads from the library, whereas S. cerevisiae expressing Als5p or Als1p bound to ca. 0.1 to 1% of randomly selected peptide-beads. S. cerevisiae expressing Als1p had a distinctly different adherence phenotype than did cells expressing Als5p. The former adhered in groups or clumps of cells, whereas the latter adhered initially as single cells, an event which was followed by the build up of cell-cell aggregates. Beads with adherent cells were removed, and the peptide attached to the bead was determined by amino acid sequencing. All adhesive beads carried a three-amino-acid sequence motif (tau phi+) that possessed a vast combinatorial potential. Adherence was sequence specific and was inhibited when soluble peptide identical to the immobilized peptide was added. The Als5p adhesin recognized some peptides that went unrecognized by Als1p. The sequence motif of adhesive peptides identified by this method is common in proteins and offers so many possible sequence combinations that target recognition by the Als proteins is clearly degenerate. A degenerate recognition system provides the fungi with the potential of adhering to a multitude of proteins and peptides, an advantage for any microorganism attempting to establish a commensal or pathogenic relationship with a host.     

Effects of a feed additive blend on broilers challenged with heat stress

ABSTRACT

We evaluated a blend of medium-chain fatty acids (MCFA), organic acids, and a polyphenol antioxidant on gut integrity. Eighty Ross Broilers were exposed to 20–22°C (control – normothermic) or to 35–39.5°C (heat stress) for eight hours a day for a period of 1 or 5 days. Birds were fed a standard diet, or a diet supplemented with the test blend. Thereafter, birds were euthanized, and intestinal sections were excised for morphological, morphometric and gene expression analyses. Blood samples were collected for glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px) activity and trolox equivalent antioxidant capacity (TEAC) determination. Heart and liver tissues were used to quantify the expression of heat shock proteins 60 and 70 (HSP60 and HSP70, respectively) and inhibitor of kappa light chain gene enhancer in B cells alpha (IKBA). The jejunum was the most sensitive intestinal section, where heat stress modulated the expression of HSP70, of the inflammatory markers IKBA, interleukin 8 (IL-8), interferon gamma (IFNγ), and toll-like receptor 4 (TLR4). Moreover, expression of tight junctions (CLDN1, ZO1 and ZO2) and nutrient transporters (PEPT1 and EAAT3) was modulated especially in the jejunum. In conclusion, the feed additive blend protected intestines during heat stress from the decrease in villus height and crypt depth, and from the increase in villus width. Especially in the jejunum, heat stress played an important role by modulating oxidative stress and inflammation, impairing gut integrity and nutrient transport, and such deleterious effects were alleviated by the feed additive blend.

RESEARCH HIGHLIGHTS

• Jejunum is the most sensitive intestinal segment during heat stress.

• Heat stress affects the expression of tight junctions and nutrient transporters.

• Feed management helps to alleviate the disturbances caused by heat stress.

• A blend of MCFA, organic acids and a polyphenol protects broilers under heat stress.

     

Endoreduplication in conjunction with tumor progression in an aneuploid laryngeal squamous cell carcinoma

We report the case of a 58-year-old man who presented with a squamous cell carcinoma pT1a G2 of the left vocal cord. Six months after histologically verified complete resection, the patient experienced an endolaryngeal and extralaryngeal local recurrence pT4 pN2b G2. We applied DNA flow cytometry (FCM) and comparative genomic hybridization (CGH) on both primary and recurrent tumor. The primary tumor and the endolaryngeal compartment of the relapse was an aneuploid cell clone with a FCM DNA index of 1.42 and 1.44, respectively. The extralaryngeal compartment showed a shift featuring a DNA index of 2.78. In the primary tumor and in both compartments of the recurrence there was an identical pattern of complex chromosomal imbalances as detected in CGH (CGH karyotype: rev ish enh [8q24.2-q24.3, 10q26.1-q26.3, 11q24-q25, 12q24.2-q23.33,X], dim [4q, 13q14.3-q31], amp[1p36.1-p36.2]). Hence, the recurrence was not associated with further gains and losses of chromosomal material. However, in the anterior part of the recurrence, the aneuploid tumor cell genome had completely doubled, obviously due to endoreduplication. Immunohistochemical analysis of several cell-cycle regulators revealed altered expression of checkpoint proteins, pointing to a complex disturbance in cell-cycle regulation.     

Cell-surface bound pertussis toxin induces polyclonal T cell responses with high levels of interferon-gamma in the absence of interleukin-12

Pertussis toxin (PTx), an exotoxin produced by Bordetella pertussis, has long been used as a mucosal adjuvant. We examined the T cell stimulatory properties of PTx in order to dissect its mechanisms of adjuvanticity. PTx or the B-oligomer of PTx (PTxB) failed to activate purified murine CD4+ or CD8+ T cells, as measured by a lack of proliferation or expression of early T cell activation markers. However, these T cells proliferated extensively in response to the toxin in the presence of syngeneic DC, and proliferation was accompanied by a high level of IFN-gamma production in the absence of IL-12. Interestingly, such responses were independent of signals mediated by MHC-TCR interaction. Both PTx and PTxB were found to bind stably to the surface of DC, and increased the adherence of DC to surrounding cells. These data suggest that polyclonal T cell responses mediated by the toxin are likely to be caused by the toxin bound on the surface of APC, either cross-linking cell surface molecules on T cells, or directly stimulating T cells together with the co-stimulatory molecules expressed on APC. B. pertussis may use this toxin as a mechanism to evade a specific immune response.     

Association of Low Concentrations of Serum Mannose-Binding Protein with Recurrent Infections in Adults

Low concentrations of mannose-binding protein (MBP; also known as mannose-binding lectin) are associated with common opsonic defect in immunodeficient children. We compared the concentrations of MBP in the sera of 47 adults with non-human immunodeficiency virus-related recurrent infections (group I) and 50 healthy adult controls. Mean serum MBP concentrations in the patient group did not differ significantly from those in the control group (P < 0.4). Nevertheless, the proportion of individuals with less than 5 ng of serum MBP per ml was significantly larger in the patient group (21%, P = 0.01) than in the control group (4%). Group II consisted of 73 pediatric and 56 adult patients with recurrent infections. Pediatric patients had significantly lower mean concentrations of serum MBP than their controls (P < 0.005), and there was no significant difference between the concentrations in sera of adult patients and adult controls (P < 0.4). Again, the proportion of individuals with less than 5 ng of serum MBP per ml was significantly larger in both pediatric (22%, P = 0.045) and adult (38%, P = 0.000016) patients than in their respective controls (4%). Our results demonstrate that, as in children, low concentrations of serum MBP can be associated with recurrent infections in adults.     

The psychologist's contribution to primary care: a reappraisal

Developments over the past few years necessitate a reappraisal of the way in which clinical psychologists can best contribute to primary care. Up until now, psychologists have concentrated on those problems which are their concern within psychiatric hospitals. In addition, they have largely adopted a specialist model of service which may not be in the best interests either of primary care or of clinical psychology. If their manner of working is to change, it will be important for psychologists to overcome the developing stereotype of their role. This might involve explicitly negotiating project-orientated commitments which respond to particular priorities in different settings.     

Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.     

Spontaneous Cytokine Production and Its Effect on Induced Production

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) by peripheral-blood B lymphocytes, T cells, CD8⁻ T cells, CD8⁺ T cells, CD3⁻ CD16/56⁺ lymphocytes (natural killer [NK] cells), CD3⁺ CD16/56⁺ lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-α production occurred most frequently, followed in descending order by IFN-γ, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-α; for 12 of these patients, TNF-α was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-α and IFN-γ, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.     

Filarial Nematode Parasites Secrete a Homologue of the Human Cytokine Macrophage Migration Inhibitory Factor

Filarial nematode parasites establish long-term chronic infections in the context of an antiparasite immunity that is strongly biased toward a Th2 response. The mechanisms that lead to this Th2 bias toward filarial antigens are not clear, but one possibility is that the parasites produce molecules that have the capacity to proactively modify their immunological environment. Here we report that filarial parasites of humans secrete a homologue of the human proinflammatory cytokine macrophage migration inhibitory factor (MIF) that has the capability of modifying the activity of human monocytes/macrophages. A cDNA clone isolated from a Brugia malayi infective-stage larva expression library encoded a 12.5-kDa protein product (Bm-MIF) with 42% identity to human and murine MIF. MIF homologues were also found to be expressed in the related filarial species Wuchereria bancrofti and Onchocerca volvulus. Bm-mif was transcribed by adult and larval parasites, and the protein product was found in somatic extracts and in the parasite’s excretory-secretory products. Immunohistocytochemistry revealed that Bm-MIF was localized to cells of the hypodermis/lateral chord, the uterine wall, and larvae developing in utero. Unexpectedly, the activities of recombinant Bm-MIF and human MIF on human monocytes/macrophages were found to be similar. When placed with monocytes/macrophages in a cell migration assay, Bm-MIF inhibited random migration. When placed away from cells, Bm-MIF induced an increase in monocyte/macrophage migration that was specifically inhibited by neutralizing anti-Bm-MIF antibodies. Bm-MIF is the first demonstration that helminth parasites produce cytokine homologues that have the potential to modify host immune responses to promote parasite survival.