Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously

Transcutaneous immunization (TCI) is a new method for vaccine delivery that has been shown to induce immunity relevant to enteric disease vaccines. We evaluated the clinical safety and immunogenicity of a recombinant subunit vaccine against enterotoxigenic Escherichia coli (ETEC) delivered by TCI. Adult volunteers received patches containing the recombinant ETEC colonization factor CS6, either with heat-labile enterotoxin (LT) or patches containing CS6 alone. The vaccine was administered at 0, 1, and 3 months, and serum antibodies and antibody-secreting cells (ASCs) were assessed. Among the 26 volunteers that completed the trial, there were no responses to CS6 in the absence of LT. In the groups receiving both CS6 and LT, 68 and 53% were found to have serum anti-CS6 immunoglobulin G (IgG) and IgA, respectively; 37 and 42% had IgG and IgA anti-CS6 ASCs. All of the volunteers receiving LT had anti-LT IgG, and 90% had serum anti-LT IgA; 79 and 37% had anti-LT IgG and IgA ASCs. Delayed-type hypersensitivity (DTH), suggesting T-cell responses, was seen in 14 of 19 volunteers receiving LT and CS6; no DTH was seen in subjects receiving CS6 alone. This study demonstrated that protein antigens delivered by a simple patch could induce significant systemic immune responses but only in the presence of an adjuvant such as LT. The data suggest that an ETEC vaccine for travelers delivered by a patch may be a viable approach worthy of further evaluation.     

Flutamide-metformin plus an oral contraceptive (OC) for young women with polycystic ovary syndrome: switch from third- to fourth-generation OC reduces body adiposity

BACKGROUND: Low-dose flutamide-metformin has been developed as a background therapy for non-obese adolescents and young women with hyperinsulinaemic hyperandrogenism, a variant of polycystic ovary syndrome (PCOS). We verified whether the lipolytic efficacy of flutamide-metformin in women with PCOS is enhanced by giving an oral contraceptive (OC) co-therapy that contains drospirenone, instead of gestodene, as progestin. METHODS: An open-labelled study was carried out in which non-obese women with PCOS (n = 29; age approximately 20 years), who had been on a combination of flutamide (62.5 mg/day), metformin (850 mg/day) and ethinylestradiol-gestodene for 8-15 months, were randomized for replacement of the gestodene OC by a drospirenone OC. Assessments of endocrine-metabolic state and body composition (by dual-energy X-ray absorptiometry) were performed at randomization and after 6 months. RESULTS: The switch to drospirenone OC was accompanied by a reduction of total and abdominal fat (mean -0.8 and -0.5 kg) and by an increment of lean body mass (+0.6 kg; all P < 0.01), so that body adiposity was strikingly reduced without changing body weight. CONCLUSION: In non-obese women with PCOS, low-dose flutamide-metformin reduces total and abdominal fat excess more effectively if contraceptive co-therapy contains drospirenone, instead of gestodene, as progestin.     

Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine

Alkylating agents are a structurally diverse group of compounds that cause a wide range of biological effects, including cell death, mutation and cancer. DNA damaged by these agents contains widely different amounts of 12 alkylated purines/pyrimidines and two phosphotriester isomers. The biological effects appear to be mediated predominantly by attack at the O(6) position of guanine. DNA extracted from various normal human tissues contains detectable levels of O(6)-alkylguanine, the source of which has not been defined. Given that, following DNA replication, this lesion cannot only generate point mutations but can also initiate mismatch repair-mediated DNA recombination and cell death, it seems worthwhile to consider the possible contribution of these events and cell killing to the aetiology of human cancer. There is increasing evidence that point mutations are not the only mechanism involved in malignant transformation by alkylating agents. Some cancer chemotherapeutic agents exploit the cytotoxic effects of O(6)-alkylguanine and an understanding of the processing of this lesion has allowed strategies to be developed that should increase the effectiveness of such agents.     

Worldwide genomic diversity of the human papillomaviruses-53, 56, and 66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 "high-risk" HPV types are frequently found in anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and 18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2-4.2% of the nucleotide sequences were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the "star-phylogenies" formed by HPV-16 and 18 variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6 oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV type.     

Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system

Optimization of three enzyme immunoassays of very high sensitivity using three antiprolamin monoclonal antibodies (MAbs) (13B4, 11C4 and 12A1) is presented here. These MAbs are specific for those prolamins toxic for coeliac patients, as determined by immunoblotting analysis. Biotinylated MAbs were used in two of the assays. In a competitive ELISA, the binding of each biotinylated MAb to a gliadin‐coated solid phase was inhibited by gliadin in the fluid phase. The best result was obtained using the biotinylated MAbl3B4 (detection limit: 20 ng ml^?1). With regard to capture ELISA, we tested the performance of the three MAbs. In this sandwich ELISA, the MAb used for antigenic capture was the same as that used as secondary biotinylated antibody. The MAbl2Al had the best performance (detection limit: 1 ng ml^?1). The use of biotin‐labelled gliadin in a quantitative immunoassay with a detection limit of 5 ng ml^?1 is also reported. This assay involves an antigenic capture using the MAbl2Al followed by a competition between biotinylated and non‐biotinylated gliadin. We have found the use of the streptavidin‐biotin interaction as signal amplification system to be very useful. This technique, as far as we know, has not been previously reported for gliadin quantification.     

NADPH oxidase-mediated oxidative stress: genetic studies of the p22(phox) gene in hypertension

Increased vascular production of reactive oxygen species, especially superoxide anion, significantly contributes to the oxidative stress associated with hypertension. An enhanced superoxide production causes an increased inactivation of nitric oxide that diminishes nitric oxide bioavailability, thus contributing to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that NADPH oxidases play a major role as the most important sources of superoxide anion in phagocytic and vascular cells. Several experimental observations have described an enhanced superoxide generation as a result of NADPH oxidase activation in hypertension. Although these enzymes respond to stimuli such as vasoactive factors, growth factors, and cytokines, recent data suggest a significant role of the genetic background in the modulation of the expression of its different components. Several polymorphisms have been identified in the promoter and in the coding region of CYBA, the gene that encodes the essential subunit of the NADPH oxidase p22phox, some of which seem to influence significantly the activity of these enzymes in the context of cardiovascular diseases. Among CYBA polymorphisms, genetic investigations have provided a novel marker, the -930(A/G) polymorphism, which determines the genetic susceptibility of hypertensive patients to oxidative stress.     

Recurrence of bronchioloalveolar carcinoma in donor lung after lung transplantation: microsatellite analysis demonstrates a recipient origin

Bronchioloalveolar carcinoma is a distinctive subtype of pulmonary adenocarcinoma, without effective therapy, although there have recently been some attempts to use lung transplantation. However, a high post-transplantation local recurrence rate is described with some controversy regarding the possible involved mechanisms, the main possibilities being the lymphatic spread and aerosolization. Presented herein is a case of a bilateral lung transplantation for a bilateral and pneumonic form of non-mucinous bronchioloalveolar carcinoma in a 43-year-old woman. The histological analysis of mediastinal lymph nodes during surgery did not show neoplastic cells. Thirty-five months after transplantation several nodular opacities in donor lungs were detected. Three pulmonary wedge resections were performed showing a non-mucinous bronchioloalveolar carcinoma with the same histological characteristics as the primary. Again, the mediastinal lymph nodes were tumor free. A complete microsatellites molecular analysis was performed to compare the primary and recurrent carcinoma using capillary electrophoresis, showing that the recurrent tumor was generated in a recipient cellular clone. The absence of lymph node metastasis and the molecular evidence of the recipient origin of the neoplasm supports the contamination of the new lungs at the time of implantation as being the reason for the high incidence of recurrence after lung transplantation in this kind of disease.     

Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-8, a novel metallo-beta-lactamase, in a tertiary care center in Cali, Colombia

The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates at a 195-bed tertiary care medical center in Cali, Colombia, rose from 2% in 1996 to 28% in 1997 and to over 40% in 2003. Many isolates showed high-level multiresistance, and phenotypic characterization suggested the spread of a predominant strain with minor variants. Sixty-six resistant isolates collected between February 1999 and July 2003 from hospitalized patients (n = 54) and environmental samples (n = 12) were subjected to a fuller analysis. Genetic fingerprints were compared by pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA, and bla(IMP) and bla(VIM) genes were sought by PCR. PFGE and serotyping indicated that 52 of the 66 isolates belonged to a single strain, with 82% similarity; the PFGE pattern for this organism was designated pattern A. Two further pairs of isolates represented single strains; the remaining nine isolates were unique, and in the case of one isolate, no satisfactory PFGE profile could be obtained. The pattern A isolates were mostly of serotype O12 and were highly resistant to imipenem (MICs, 32 to >256 microg/ml), with this resistance decreased eightfold or more in the presence of EDTA. They yielded amplicons with bla(VIM)-specific primers, and sequencing of DNA from a representative isolate revealed bla(VIM-8), a novel allele with three polymorphisms compared with the sequence of bla(VIM-2). Two of these nucleotide changes were silent, but the third determined a Thr139Ala substitution. Only 4 of 13 resistant isolates (2 clinical isolates and 2 environmental isolates) assigned to other PFGE types carried bla(VIM) alleles, whereas the others were less multiresistant and mostly had lower levels of imipenem resistance (MICs, < or =32 microg/ml) which was not significantly reduced by EDTA. No bla(IMP) alleles were detected. During 2003, when the environmental study was undertaken, serotype O12 isolates with bla(VIM) were recovered from sinks and stethoscopes in the most-affected units, although not from the hands of staff; the problem declined once these reservoirs were disinfected and hygienic precautions were reinforced.     

Martin-Gruber anastomosis revisited

Based on a study of 70 human cadavers (31 male, 39 female) and on cases described previously, we propose a new classification of the Martin-Gruber anastomosis, a neural connection between the median and ulnar nerves in the forearm. The anastomosis was found in 16 (22.9%) cadavers, being bilateral in three (18.7%) and unilateral in 13 (81.3%), five right and eight left. It occurred in eight (25.8%) of the 31 male cadavers and in eight (20.5%) of the 39 females. Therefore, the anastomosis was found in 19 (13.6%) of the 140 forearms. In Pattern I (89.5%) the anastomosis was made by only one branch, whereas in Pattern II (10.5%) it was made by two. The individual branches were classified as Types a, b, and c based on the nature of their origin from the median nerve. Type a (47.3%) arose from the branch to the superficial forearm flexor muscles, Type b (10.6%) from the common trunk, and Type c (31.6%) from the anterior interosseous nerve. Pattern II was a duplication of Type c (10.5%). The anastomotic branch took an oblique or arched course before joining the ulnar nerve, undivided in 15 cases, but divided into two branches in four cases. The anastomosis passed in front of the ulnar artery in four cases, behind it in six, and in nine cases it was related to the anterior ulnar recurrent artery.