Protective effect of the Nramp1 BB genotype against Brucella abortus in the water buffalo (Bubalus bubalis)

We tested 413 water buffalo cows (142 cases and 271 controls) for the presence of anti-Brucella abortus antibodies (by the skin test, the agglutination test, and the complement fixation test) and the Nramp1 genotype (by capillary electrophoresis). Four alleles (Nramp1A, -B, -C, and -D) were detected in the 3' untranslated region of the Nramp1 gene. The BB genotype was represented among only controls, providing evidence that this genotype confers resistance to Brucella abortus. The monocytes from the BB (resistant) subjects displayed a higher basal level of Nramp1 mRNA and a lower number of viable intracellular bacteria than did the monocytes from AA (susceptible) subjects. The higher basal level of the antibacterial protein Nramp1 most probably provides the BB animals with the possibility of controlling bacteria immediately after their entry inside the cell.     

Detection of capsule in strains of Clostridium difficile of varying virulence and toxigenicity

Nine toxigenic and six non-toxigenic strains of Clostridium difficile, of varying virulence in the hamster model of antibiotic-associated colitis, were examined for the presence of a capsule. Antibody stabilisation of the capsule with heterologous and/or homologous antiserum fixed in glutaraldehyde, or direct fixation in glutaraldehyde/diamine, were used with added ruthenium red to stain the capsular glycocalyx. All strains possessed a capsule which was either loose-knit or compact, sometimes with attached globular masses. Better capsule preservation was achieved in some strains with glutaraldehyde/diamine/ruthenium red fixative than with homologous or heterologous antibody stabilisation. The possession of a capsule following in vitro growth does not appear to correlate with the virulence status of these strains of C. difficile.     

Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms

The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 microm into the biofilms, which averaged 210 microm thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band, approximately 30 microm wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h(-1). These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.     

Von Hippel-Lindau-dependent polycythemia is endemic on the island of Ischia: identification of a novel cluster

Chuvash polycythemia (MIM 263400) is an autosomal recessive disorder characterized by a high hemoglobin level, relatively high serum erythropoietin, and early death. It results from a Von Hippel-Lindau (VHL) gene mutation (C598T) that causes increased HIF-1alpha activity and erythrocyte production in the face of normoxia. This polycythemia is endemic in Chuvashia, whereas its worldwide frequency is very low. We investigated the incidence of the Chuvash-type VHL mutation in Campania (South Italy) and identified 14 affected subjects (5 families). Twelve live on the island of Ischia (Bay of Naples). From analysis of the mutated allele, we found that the disease was more frequent on Ischia (0.070) than in Chuvashia (0.057). The haplotype of all patients matched that identified in the Chuvash cluster, thereby supporting the single-founder hypothesis. We also found that nonaffected heterozygotes had increased HIF-1alpha activity, which might confer a biochemical advantage for mutation maintenance. In conclusion, we have identified the first large cluster of Chuvash erythrocytosis outside Chuvashia, which suggests that this familial polycythemia might be endemic in other regions of the world.     

Relapse-associated worsening in a real-life multiple sclerosis cohort: the role of age and pyramidal phenotype

Background and purpose

The overall disability in patients with relapsing–remitting multiple sclerosis is likely to be partly rather than entirely attributed to relapse.

Materials and methods

The aim was to investigate the determinants of recovery from first relapse and relapse-associated worsening (RAW) in relapsing–remitting multiple sclerosis patients from the Italian MS Registry during a 5-year epoch from the beginning of first-line disease-modifying therapy. To determine recovery, the functional system (FS) score was used to calculate the difference between the score on the date of maximum improvement and the score before the onset of relapse. Incomplete recovery was defined as a combination of partial (1 point in one FS) and poor recovery (2 points in one FS or 1 point in two FSs or any other higher combination). RAW was indicated by a confirmed disability accumulation measured by the Expanded Disability Status Scale score confirmed 6 months after the first relapse.

Results

A total of 767 patients had at least one relapse within 5 years of therapy. Of these patients, 57.8% experienced incomplete recovery. Age (odds ratio [OR] 1.02, 95% confidence interval [CI] 1.01–1.04; p = 0.007) and pyramidal phenotype were associated with incomplete recovery (OR = 2.1, 95% CI 1.41–3.14; p < 0.001). RAW was recorded in 179 (23.3%) patients. Age (OR = 1.02, 95% CI 1.01–1.04; p = 0.029) and pyramidal phenotype (OR = 1.84, 95% CI 1.18–2.88; p = 0.007) were the strongest predictors in the multivariable model.

Conclusions

Age and pyramidal phenotype were the strongest determinants of RAW in early disease epochs.     

Spectrum of FANCA mutations in Italian Fanconi anemia patients: identification of six novel alleles and phenotypic characterization of the S858R variant

Fanconi anemia (FA) is an autosomal recessive disorder characterized by genomic instability, bone marrow failure, congenital malformations, and cancer predisposition. FA is a genetically heterogeneous disease with at least seven genes so far identified. The role of FA proteins is unknown although they interact in a common functional pathway. Here, we report six novel FANCA sequence changes and review all the mutations identified in Italy. Except for two missense substitutions, all are expected to cause a premature termination of the FANCA protein at various sites throughout the molecule. The premature terminations are due to nonsense and splice site mutations, as well as small insertions and deletions, and large genomic rearrangements. The expected truncated proteins were not detectable on Western blot analyses. The FANCA-S858R variant is instead expressed at lower level than that seen in normal cell lines and is associated with a non-ubiquinated FANCD2 protein, strongly suggesting that the amino acid substitution is a disease-causing mutation. The spectrum of FA mutations is widely in agreement with the heterogeneous ethnic origin of the Italian population.     

Effects of human immunodeficiency virus type 1 on CD4 lymphocyte subset activation.

The pathogenesis of the decline of CD4 lymphocyte counts accompanying the typical course of HIV-1 infection is not completely defined and might be related to a differential susceptibility of naive and memory cells to HIV-1 exposure. Here, we examined the effects induced by heat-inactivated HIV-1 virions on these lymphocyte populations. Exposure of CD45RA naive T cells to inactivated viral particles induced a marked decrease of both mitogenic responses and activation-induced apoptosis. Conversely, the growth of CD45RO cells was less severely restrained. Analysis of intracellular levels of cell cycle regulatory proteins revealed an arrest at the G1/S restriction point of the naive but not memory subset. This effect was associated with alterations in phosphotyrosine profile and with a marked decrease of ERK and NJK kinase activation. Finally, up-regulation of the cAMP-dependent protein kinase A (PKA) activity induced by mitogens was not affected by virus. Altogether, these findings show that interaction of HIV-1 with the T cell surface is sufficient to inhibit the proliferative response of the CD4CD45RA subset by disturbing proximal TCR signaling. This mechanism would affect renewal of naive lymphocytes, contributing in such a way to the impairment of T cell turnover during the course of HIV-1 infection.