Prevalence of transmitted HIV-1 drug resistance and the role of resistance algorithms: data from seroconverters in the CASCADE collaboration from 1987 to 2003

OBJECTIVES: To examine factors influencing the rate of transmitted drug resistance (TDR) among seroconverters, with particular emphasis on 3 widely used genotypic drug resistance algorithms. METHODS: The study used data from CASCADE (Concerted Action on Seroconversion to AIDS and Death in Europe), a collaboration of seroconverter cohorts in Europe and Canada. Genotypic resistance data were derived within 18 months of the last seronegative test or date of laboratory evidence of acute infection and before the initiation of antiretroviral therapy. The Stanford algorithm was used to analyze each individual's nucleotide sequence. A multivariate logistic model was used to assess independent relationships between the presence of TDR and exposure category, sex, age at seroconversion, and year of seroconversion. The paper also describes 3 alternative definitions of resistance: the Stanford algorithm, the key resistance mutations defined by the International AIDS Society, and the Agence Nationale de Recherches sur le Sida (ANRS) algorithm. RESULTS: Forty-five of 438 patients (10.3%) seroconverting between 1987 and 2003 were infected with a drug-resistant HIV-1 variant. Forty patients (9.1%) showed resistance mutations to only 1 class of antiretroviral drugs, 2 (0.5%) to 2 classes, and 3 (0.7%) to 3 classes of antiretroviral therapy. It was suggested that individuals seroconverting later in calendar time were more likely to have TDR (relative risk 3.89 and 95% CI: 0.84 to 18.02, and relative risk 4.69 and 95% CI: 1.03 to 21.31, for 1996-1999 and 2000-2003, respectively, compared with pre-1996; P trend = 0.08). This trend was apparent regardless of the definition of TDR used. The total estimated proportion of individuals with TDR varied between 10.3% and 15.5% according to which definition was used. CONCLUSIONS: Evidence was found for the rise of TDR over time. A specific definition of what constitutes TDR rather than a simple list of mutations is needed.     

Primary Immunodeficiency Diseases in Latin America: First Report from Eight Countries Participating in the LAGID

The Latin American Group for Primary Immunodeficiencies, formed in 1993, presently includes 12 countries. One goal was to study the frequency of primary immunodeficiencies in various regions of the American continent and to enhance knowledge about these diseases among primary-care physicians, as well as allergist–immunologists. Important for this purpose was the development of a registry of primary immunodeficiencies using a uniform questionnaire and computerized database. To date, eight countries have collected information on a total of 1428 patients. Predominantly antibody deficiencies were reported in 58% of patients, followed by cellular and antibody immunodeficiencies associated with other abnormalities in 18%, immunodeficiency syndromes associated with granulocyte dysfunction in 8%, phagocytic disorders in 9%, combined cellular and antibody immunodeficiencies in 5%, and complement deficiencies in 2% of patients. The information gathered from this initial analysis of data will serve to expand the patient database to more areas within participating countries and to new countries and to increase collaboration toward better diagnosis and treatment of these diseases.     

Up-regulation of tRNA biosynthesis affects translational readthrough in maf1-delta mutant of Saccharomyces cerevisiae

Maf1p is a negative effector of RNA polymerase III in yeast. The maf1-delta mutation caused an increase in the level of cellular tRNAs, but a decrease of translational readthrough at nonsense codons. Using the lacZ- luc dual gene reporter system, we detected an almost twofold diminution of UAA and UAG readthrough in maf1-delta compared with the parental strain. The maf1-delta mutation did not affect the rate of protein biosynthesis and growth at standard conditions, but resulted in temperature-sensitive growth on non-fermentable carbon sources. We examined the correlation of the temperature sensitive and antisuppression phenotypes of maf1- Delta using a colour phenotype assay in the ade2-1 SUP11 strain. Antisuppression, but not the temperature-sensitive growth defect, was compensated either by increased dosage of SUP11or by [PSI(+)], the prion form of the translation termination factor Sup35p. Summarizing, the elevated tRNA levels in maf1- Delta increase translational fidelity and, independently, affect growth under special conditions.     

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.     

A new CARD15 mutation in Blau syndrome

The caspase recruitment domain gene CARD15/NOD2, encoding a cellular receptor involved in an NF-kappaB-mediated pathway of innate immunity, was first identified as a major susceptibility gene for Crohn's disease (CD), and more recently, as responsible for Blau syndrome (BS), a rare autosomal-dominant trait characterized by arthritis, uveitis, skin rash and granulomatous inflammation. While CARD15 variants associated with CD are located within or near the C-terminal leucine-rich repeat domain and cause decreased NF-kappaB activation, BS mutations affect the central nucleotide-binding NACHT domain and result in increased NF-kappaB activation. In an Italian family with BS, we detected a novel mutation E383K, whose pathogenicity is strongly supported by cosegregation with the disease in the family and absence in controls, and by the evolutionary conservation and structural role of the affected glutamate close to the Walker B motif of the nucleotide-binding site in the NACHT domain. Interestingly, substitutions at corresponding positions in another NACHT family member cause similar autoinflammatory phenotypes.     

Extracellular metalloproteinase activity in <Emphasis Type="Italic">Phytomonas françai</Emphasis>

Extracellular proteolytic activities were detected in Phytomonas françai culture supernatant. A 67-kDa enzyme was purified by ammonium sulfate precipitation and gel filtration in a HPLC system. This proteinase was optimally active at 28 °C and pH 5.0; and the use of proteolytic inhibitors indicated that it belongs to the metalloproteinase class. This is the first report on the purification of an extracellular metalloproteinase from a Phytomonas species.     

Looking for ferns: optimization of digestion pretreatment in fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.     

Enzyme-linked immunosorbent assay (ELISA) for antibodies to Clostridium difficile toxins in patients with pseudomembranous colitis and antibiotic-associated diarrhoea

Enzyme-linked immunosorbent assay (ELISA) was established with purified toxins from Clostridium difficile as antigene to measure antibody response in patiensts with pseudomembranous colitis (PMC) and prolonged antibiotic-associated diarrhoea (AAD). Positive ELISA titres were defined in a control population. Antibodies of IgG class against toxin B were demonstrated in 6/88 (7%) control sera and in 31/61 (51%) sera from 11/19 (58%) patients. Antibodies of IgA class were found in one patient while antibodies of IgM class were not demonstrated. ELISA antibodies against toxin A were not demonstrated. For comparison a neutralization test was performed and neutralizing antibodies to toxin B but not to toxin A were demonstrated in 10/61 (16%) sera from 4/19 (21%) patients and in none of the controls. ELISA was found to be a more sensitive assay than neutralization. ELISA antibodies were detected from the third week of the disease while neutralizing antibodies appeared after 5 weeks. Lack of an antibody response in ELISA seemed to correlate to a more severe colitis.     

Fluorescence in situ hybridization analysis of HER-2/neu in brushings of normal oral mucosa

Oncogene alterations have been clearly demonstrated to be related to the carcinogenesis and progression of oral squamous cell carcinoma (OSCC). However, the analysis of these alterations for screening and early diagnostic purposes generally requires invasive techniques for surgical removal of pathological epithelium. The aim of the present study was to assess the feasibility of fluorescence in situ hybridization (FISH) analysis of HER-2/neu amplification in oral mucosa brushings and to compare the HER-2/neu status with the history and smoking and drinking habits of healthy subjects. Cells obtained by centrifugation of oral brushings from 21 subjects (overall no. of cells: 5125) were suspended in physiological saline and fixed onto two slides for cytological evaluation and FISH analysis (dual-target, dual-color fluorescence assay) of the HER-2/neu gene and CEP17 centromere. A mean of 89.8% of the cells showed two HER-2/neu signals and a mean of 94% had two CEP17 signals at fluorescent microscopy. Finally, a mean of 96% of cells with HER-2/neu / CEP17 had a ratio equal to 1. No association between smoking and drinking habits, age and the HER-2/neu and CEP17 characteristics evaluated by FISH was found.