Oxidative stress induced by Cremophor EL is not accompanied by changes in NF-kappaB activation or iNOS expression

The effects of polyoxyethylenglycerol triricinoleate 35 (Cremophor EL, CrEL) on markers of oxidative stress, nuclear factor kappa B (NF-kappaB) activation and inducible nitric oxide synthase (iNOS) expression were studied in the liver of male Wistar rats. Animals were randomly divided into three groups. Group Cr1 received, i.p., CrEL at 0.046ml/kg daily for 7 days, group Cr2 received CrEL at 0.33ml/kg and the controls were injected with CrEL vehicle (saline solution with 25% ethanol). Both alanine transaminase (ALT) and aspartate transaminase (AST) serum activities were significantly increased in the Cr2 group (+16% and +25%, respectively). AST activity was also higher in the Cr1 group when compared to control animals (+20%). The cytosolic concentration of thiobarbituric acid reactive substances (TBARS) increased in both groups of rats receiving CrEL (Cr1: +24%; Cr2: +33%). Reduced glutathione (GSH) concentration was not significantly modified at any of the CrEL doses, but both the hepatic concentration of oxidised glutathione (GSSG) (Cr1: +37%; Cr2: +84%) and the GSH/GSSG ratio (Cr1: -21%; Cr2: -45%) were significantly modified. CrEL induced no significant NF-kappaB activation, changes in p50 and p65 NF-kappaB subunits or induction of iNOS protein. Data obtained indicate that although high doses of CrEL cause oxidative stress, this is not enough to induce changes in NF-kappaB activation or iNOS expression.     

A 45-Nucleotide Insertion in the NS2 Gene is Responsible for the Cytopathogenicity of a Bovine Viral Diarrhoea Virus Strain

Cytopathogenicity (cp) markers have recently been investigated in the genomes of field isolates of bovine viral diarrhoea virus (BVDV). Most of the isolates originated from mucosal disease (MD) cases observed after vaccination with a live attenuated vaccine, termed here BVDV-X. The NS2-3 genes of these isolates and of the vaccine proved to be identical, including a 45-nucleotide (nt) viral insertion at nt position 4355. The insertion originated from the NS4B/5A junction region of the BVDV genome. Interestingly, in BVDV strain CP7 a 27-nt insertion originating from the NS2 is located exactly at the same position. Complete genome analysis of BVDV-X did not reveal further potential cp markers. Furthermore, expression studies indicated that the insertion promotes NS2-3 cleavage. In order to examine the possible role of the 45-nt insertion in viral cytopathogenicity in details, a full-length infectious cDNA clone of BVDV-X was generated, and bovine turbinate (BT) cells were transfected with RNA transcribed from the clone. The recovered virus, termed BVDV-XR, showed slight retardation in growth in comparison with the original BVDV-X, and induced cytopathogenic effect (CPE). Since the natural non-cytopathogenic (ncp) counterpart of the vaccine virus was not available, an insertion-negative mutant cDNA clone was generated from BVDV-XR by PCR-directed mutagenesis. The recovered virus, termed BVDV-XR-INS-, showed the same growth characteristics as its cp counterpart BVDV-XR, but caused no CPE. These findings provide a direct proof that the 45-nt insertion at position 4355 has a basic role in the cytopathogenic character of this BVDV strain.     

Mycobacterium lentiflavum como causa principal de linfadenitis en población pediátrica

Introduction

Cervical lymphadenitis is the most common nontuberculous mycobacteria (NTM) infection in immunocompetent children, mainly in those under 5 years. For many years Mycobacterium lentiflavum (M. lentiflavum) has been considered a rare NTM causing lymphadenitis.

Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations

A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH4) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194T>C (p.I65T), c.204A>T (p.R68S), c.731C>T (p.P244L), c.782G>A (p.R261Q), c.926C>T (p.A309V), c.1162G>A (p.V388M), and c.1162G>A (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH4 to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH4. The possible stabilizing effect of the cofactor was explored using a cell-free in vitro synthesis assay combined with pulse-chase methodology. BH4 prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild-type PAH and all mild PKU mutants analyzed in this study, BH4 increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH4 substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH4 on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype.     

Particular profile of the zymodemes of Leishmania infantum causing visceral leishmaniasis in Tunisia

The isoenzymatic typing of 16 stocks of Leishmania, isolated from Tunisian visceral leishmaniasis (VL) cases, revealed that all strains belonged to Leishmania (L.) infantum species. Although zymodeme MON-1 was predictably the most frequent (9 cases), it came as a surprise that L. infantum MON-24 was responsible of about third of cases. This latter zymodeme, while previously reported in Tunisia, Algeria and Spain, was assumed to be a dermotropic and the few cases of VL that it caused occurred always in HIV infected patients. L. infantum MON-80, occasionally reported during both cutaneous and VL of immunocompetent infants was identified in 2 patients. This report confirms that in addition to the more common L. infantum MON-1, zymodeme MON-24 has a substantial role in generating VL in immunocompetent infants in Tunisia.     

Restriction enzyme analysis of ribosomal DNA shows that Candida inconspicua clinical isolates can be misidentified as Candida norvegensis with traditional diagnostic procedures

We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were Candida norvegensis and one was C. inconspicua. Although C. norvegensis is considered a pseudohypha-producing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C. inconspicua.     

Clinical and nutritional evaluation of phenylketonuric patients on tetrahydrobiopterin monotherapy

The clinical, nutritional, and neuropsychological data of 11 mild/moderate PKU patients after one year of treatment with BH4 are evaluated. BH4 monotherapy was introduced at 5 mg/kg/day in 14 PKU patients. In 11/14 patients, Phe tolerance increased significantly from 356+/-172 to 1546+/-192 mg/day (p=0.004), and special PKU formula was gradually reduced until complete removal. In them, mean plasma Phe concentrations remained below 360 micromol/L at 5 mg BH4/kg/day (7 mg/kg/day in one patient). BH4 therapy was stopped in three patients (V388M/P362T and R243Q/IVS10-11G>A genotypes) because it was not possible to improve Phe tolerance and to remove formula intake. Serum micronutrients were not significantly different at the start of treatment and at one year follow-up, except for selenium, which increased significantly after one year of therapy (p=0.017). Anthropometric, and nutritional measurements were within the age- and sex-specific percentiles for a healthy population after one year therapy. Neuropsychological follow-up indicated that intelligence scores persisted within normal limits. In terms of patients' genotype, we confirmed that the P275S mutation combined with R408W was associated with long-term BH4 responsiveness, while the combination of P362T/V388M, and R243Q/IVS10-11G>A resulted in poor metabolic control in long-term BH4 therapy. In summary, our data confirm that BH4 is a safe, and effective therapy in a selected group of mild, and moderate PKU patients who respond to the BH4 loading test. Low doses of BH4 in monotherapy permit withdrawal of the special formula and guarantee a good clinical and nutritional outcome with no adverse side effects in PKU patients.     

Subtypes of bovine adenovirus type 2 exhibit major differences in region E3

The genomes of two adenovirus type 2 strains which were isolated from different hosts have been investigated. One of these strains designated ORT-111 was originally isolated from a lamb in Hungary during an outbreak of pneumoenteritis. This isolate was typed as bovine adenovirus type 2 (Ad bos 2) in a neutralization assay. The genome of ORT-111 was compared to that of the prototype strain of Ad bos 2, a virus which exclusively has been isolated from cattle. Electron microscopic heteroduplex analysis showed that 95% of the genomes were well matched, forming stable duplexes at Tm -6 degrees. Two distinct substitution loops were, however, seen which were approximately 0.5 and 1.0 kbp long. The centers of the two loops were located 5.3 and 7.7 kbp from one end of the Ad bos 2 genome. In order to map these regions relative to the gene map of human adenovirus type 2 (Ad2), restriction enzyme cleavage fragments of the two bovine viruses were cloned and hybridized to different sets of restriction fragments of human Ad2. From these results it was apparent that the centers of the two substitution loops were located at coordinates 76 and 83, respectively; thus at positions which fall within region E3 and the adjacent gene for polypeptide VIII of human Ad2. The observed differences between the genomes of the two Ad bos 2 strains are in sharp contrast to those previously observed when the genomes of different human adenovirus serotypes were compared. In the latter case the hexon and the fiber genes showed the most pronounced variation.     

Persistent airflow limitation in adult-onset nonatopic asthma is associated with serologic evidence of Chlamydia pneumoniae infection

BACKGROUND: Persistent airflow limitation may develop in patients with asthma, particularly in adults with nonatopic (intrinsic) disease. Although the underlying mechanisms are still unknown, respiratory infections might be involved. OBJECTIVE: We investigated the annual loss of lung function in relation to seropositivity to Chlamydia pneumoniae in different subgroups of patients with severe asthma according to age at onset of asthma and atopic status. METHODS: One hundred one nonsmoking outpatients with a pulmonologist's diagnosis of severe asthma (32 men and 69 women; mean age, 46.0 years; range, 18-75 years) were included in a cross-sectional study. C pneumoniae-specific serum IgG and IgA were measured by means of ELISA. The estimated decline in lung function was calculated from the relationship between postbronchodilator FEV(1)/vital capacity (percent predicted) and the duration of asthma and expressed as the slope of the regression line. RESULTS: Patients with adult-onset nonatopic asthma and positive IgG antibodies to C pneumoniae had a significantly steeper slope of the regression line compared with the other subgroups of asthmatic patients (P =.001), being indicative of a 4-fold greater estimated decline in postbronchodilator FEV(1)/vital capacity (2.3% vs 0.5% predicted per year of asthma duration). CONCLUSION: These results suggest that C pneumoniae infection might promote the development of persistent airflow limitation in patients with nonatopic adult-onset asthma. It remains to be established whether viable pathogens that are accessible for therapeutic intervention are still present in the lower airways.     

IL-1 alpha (-889) promoter polymorphism is a risk factor for osteomyelitis

As osteomyelitis (OM) induces the synthesis of inflammatory cytokines and IL-1 mediates bone resorption by osteoclasts we determined if there is an association between certain common polymorphisms of the genes encoding proinflammatory cytokines (IL-1 alpha and beta, IL-6, TNF-alpha) and OM in adults. The IL-1 alpha (-889) TT genotype was significantly more frequent among 52 OM patients than in 109 healthy controls (13/52, [25.0%] vs. 9/109, [8.3%], P = 0.0081, chi(2) = 7.01, OR = 3.7, 95% CI, 1.35-10.34). Patients who were homozygous for the T allele were younger than the rest of the OM patients (mean age 35.7 +/- 11.5 vs. 58.1 +/- 18.6 years, P = 0.001). IL-1 beta TT (+3953) polymorphism was also more frequent in OM patients (P = 0.014, chi(2) = 5.12, OR = 5.1, 95% CI, 1.21-52.14), but IL-1 beta is in linkage disequilibrium with the IL-1 alpha *T (P < 0.001). Route of infection, chronicity of the infection, type of microorganism isolated, and frequency of relapses were similar in patients with and without the IL-1 alpha TT genotype. There were no associations between OM and polymorphisms of other cytokines genes. IL-1 alpha serum levels were significantly increased in all the OM patients independently of their IL-1 genotype compared to the controls (P = 0.021). Although IL-1 alpha serum levels were not significantly higher in patients with the IL-1 alpha (-889) polymorphism, this does not exclude a difference in production of IL-1 alpha by osteoclasts or other inflammatory cells at the site of infection.