The outer membranes of Brucella spp. are resistant to bactericidal cationic peptides.

The actions of polymyxin B, rabbit polymorphonuclear lysosome extracts, 14 polycationic peptides (including defensin NP-2, cecropin P1, lactoferricin B, and active peptides from cationic protein 18 and bactenecin), EDTA, and Tris on Brucella spp. were studied, with other gram-negative bacteria as controls. Brucella spp. were comparatively resistant to all of the agents listed above and bound less polymyxin B, and their outer membranes (OMs) were neither morphologically altered nor permeabilized to lysozyme by polymyxin B concentrations, although both effects were observed for controls. EDTA and peptides increased or accelerated the partition of the hydrophobic probe N-phenyl-naphthylamine into Escherichia coli and Haemophilus influenzae OMs but had no effect on Brucella OMs. Since Brucella and H. influenzae OMs are permeable to hydrophobic compounds (G. Martínez de Tejada and I. Moriyón, J. Bacteriol. 175:5273-5275, 1993), the results show that such unusual permeability is not necessarily related to resistance to polycations. Although rough (R) B. abortus and B. ovis were more resistant than the controls were, there were qualitative and quantitative differences with smooth (S) brucellae; this may explain known host range and virulence differences. Brucella S-lipopolysaccharides (LPSs) had reduced affinities for polycations, and insertion of Brucella and Salmonella montevideo S-LPSs into the OM of a Brucella R-LPS mutant increased and decreased, respectively, its resistance to cationic peptides. The results show that the core lipid A of Brucella LPS plays a major role in polycation resistance and that O-chain density also contributes significantly. It is proposed that the features described above contribute to Brucella resistance to the oxygen-independent systems of phagocytes.     

Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells.

An important step in the pathogenesis of Neisseria meningitidis is the crossing of two cellular barriers, one in the nasopharynx and one in the brain. To approach the mechanisms by which this bacterium can achieve these goals, we studied the interactions between N. meningitidis and a monolayer of polarized tight junction-forming T84 cells grown on filter units. A capsulated, piliated, Opa-, and Opc- N. meningitidis strain is shown to be capable of adhering to and crossing this monolayer several orders of magnitude more efficiently than an isogenic nonpiliated derivative. This bacterial interaction does not affect the barrier function of tight junctions, as assessed by (i) the absence of modification of the transepithelial resistance, (ii) the lack of increase of [3H]inulin penetration across the monolayer, and (iii) the absence of delocalization of ZO-1, a tight junction protein. Electron microscopy studies and confocal examinations demonstrated that N. meningitidis (i) induces cytoskeletal rearrangements with actin polymerization beneath adherent bacteria, (ii) is intimately attached to the apical membrane of the cells, and (iii) can be internalized inside cells. Immunofluorescent staining with antipilus antibodies showed evidence that meningococcal piliation was dramatically reduced at later time points of bacterial cell interaction compared to the early phase of this interaction. In addition, adhesive bacteria recovered from an infected monolayer are piliated, capsulated, Opa-, and Opc-, a phenotype similar to that of the parental strain. Taken together, these data demonstrate that following pilus-mediated adhesion, N. meningitidis is involved in an intimate attachment which requires a bacterial component different from Opa and Opc and that meningococci cross a monolayer of tight-junction-forming epithelial cells by using a transcellular pathway rather than a paracellular route.     

Activation induces apoptosis in Herpesvirus saimiri-transformed T cells independent of CD95 (Fas,APO-1)

Signaling via the T cell receptor (TCR)/CD3 complex of pre-activated T cells induces apoptosis. Such an activation-induced cell death (AICD) is thought to play an important role in the regulation of cellular immune responses. In this study we analyzed pathways of AICD by using human T cells transformed by Herpesvirus saimiri. These growth-transformed T cells show the phenotype of activated mature T cells and continue to express a functionally intact TCR. We show that human H. saimiri-transformed T cell clones readily undergo cell death upon signaling via the TCR/CD3 complex or via phorbol 12-myristate 13-acetate (PMA) + ionomycin. The AICD in H. saimiri-transformed T cells was detectable a few hours after activation and it was not affected by the presence of interleukin (IL)-2 or by anti-CD4 cross-linking. However, AICD required tyrosine phosphorylation, since it could be blocked by herbimycin A. Cyclosporin A (CsA) did not block the development of AICD, but other consequences of activation in H. saimiri-transformed T cells like the production of interferon-γ. Surprisingly, the development of AICD was not reduced by neutralizing antibodies to tumor necrosis factor (TNF)-α or blocking antibodies directed to CD95 (Fas, APO-1), although H. saimiri-transformed T cells were sensitive to CD95 ligation. To confirm that this form of AICD is really independent of CD95, we have established an H. saimiri-transformed T cell line from a patient with a homozygous deletion in the CD95 gene. This CD95-deficient T cell line was as sensitive to AICD as other CD95-expressing H. saimiri-transformed T cells. In conclusion, we describe here a type of AICD in H. saimiri-transformed T cells that is independent of CD95 and TNF-α, not sensitive to CsA, but requires tyrosine phosphorylation. This system should be useful for the investigation of CD95-independent forms of AICD.     

The whispered voice: the best test for screening for hearing impairment in general practice?

Hearing loss is an important health problem in the elderly which sometimes leads to social isolation. In a study with 62 patients, the diagnostic value of four simple tests for screening for hearing loss in general practice was examined. When paying attention to the loudness of the whispering, the whispered voice test can be a valuable test for assessment of hearing loss in general practice.     

Interleukin-6 induced at mucosal surfaces by gram-negative bacterial infection.

Interleukin-6 (IL-6) was produced in response to mucosal and systemic infection of mice with gram-negative bacteria. The IL-6 response was controlled by the lipopolysaccharide gene, Lps; in C3H/HeN mice (Lpsn/Lpsn), the urinary IL-6 levels increased within 30 min after challenge with Escherichia coli, but no response occurred in C3H/HeJ mice (Lpsd/Lpsd). In lipopolysaccharide-responder mice, the levels of local and systemic IL-6 were related to the degree of infection. The urinary response dominated after intravesical challenge, and the serum response dominated after intraperitoneal challenge. The results demonstrate that IL-6 is activated as part of lipopolysaccharide-induced mucosal and systemic responses to gram-negative bacterial infections.     

Factors in the inactivation of Encephalomyocarditis virus in aerosols.

Encephalomyocarditis virus in aerosols is inactivated rapidly at relative humidities below 50%. In glycerol-water mixtures a similar decrease of infectivity occurs when the glycerol concentration exceeds 78% (wt/wt), corresponding to a relative humidity of 50%. The decay in aerosols does not involve oxygen or surface-dependent factors. Variation of temperature shows the inactivation to be a low-energy process with an activation enthalpy of 15 kcal per mol. The damage could be ascribed to dehydration of the virion, presumably proceeding to removal of structurally essential water molecules. This might trigger irreversible changes in the protein coat, resulting in disintegration of the virion.     

Effective in vitro clearance of Porphyromonas gingivalis by Fc alpha receptor I (CD89) on gingival crevicular neutrophils

Porphyromonas gingivalis has been implicated as a causative pathogen in periodontitis. Immunotherapeutic approaches have recently been suggested to aid in the clearance of P. gingivalis from disease sites. Because antibody-Fc receptor (FcR) interactions play a role in the effector functions of polymorphonuclear neutrophils (PMN), we evaluated which FcR on PMN from gingival crevicular fluid (GCF) serves as an optimal target molecule for FcR-directed immunotherapy. GCF PMN and peripheral blood (PB) PMN from adult periodontitis patients were analyzed for their immunoglobulin G (IgG) and IgA FcR (Fc gamma R and Fc alpha R, respectively) expression and function by studying IgG- and IgA-mediated elimination of P. gingivalis. GCF PMN exhibited higher Fc alpha RI and Fc gamma RI levels and lower Fc gamma RIIa and Fc gamma RIIIb levels than PB PMN. Functional studies revealed that GCF PMN exhibited less of a capacity to phagocytose and kill IgG1-opsonized P. gingivalis than PB PMN. IgA1-mediated phagocytosis and killing capacity was, however, comparable between GCF PMN and PB PMN. In summary, these in vitro results document that Fc alpha RI represents a candidate target for FcR-directed immunotherapy for the clearance of P. gingivalis.     

Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract

BACKGROUND: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. OBJECTIVE: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. METHODS: This was a randomized, double-blind, placebo-controlled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebo-controlled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebo-controlled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG(4), and serum cytokines before and after treatment. RESULTS: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P = .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG(4) and IL-10 levels after immunotherapy in only the active group. CONCLUSION: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment.     

Use of various genetic markers in differentiation of Mycobacterium bovis strains from animals and humans and for studying epidemiology of bovine tuberculosis.

One hundred fifty-three Mycobacterium bovis strains from cattle, various animal species from zoos and wild parks, and humans were analyzed for three different genetic markers for use in the epidemiology of bovine tuberculosis. M. bovis strains isolated from cattle were found to carry a single IS6110 element, whereas the majority of strains from other animals such as antelopes, monkeys, and seals harbored multiple IS6110 elements, suggesting that the reservoirs in cattle and wild animals are separated. Because the single IS6110 element in cattle strains is located at the same chromosomal position, strain differentiation by insertion sequence fingerprinting was hampered. Therefore, we investigated the usefulness of the direct repeat and polymorphic GC-rich repeat elements for strain differentiation. Both markers allowed sufficient strain discrimination for epidemiological purposes. Evidence is presented that in Argentina, most human M. bovis infections are due to transmission from cattle, whereas M. bovis infections among humans in the Netherlands are mainly contracted from animals other than cattle. Various outbreaks of M. bovis among animals and humans are described, including a small one which likely involved transmission from human to human.