Molecular dissection of TNFR-TNFα bidirectional signaling reveals both cooperative and antagonistic interactions with p75 neurotrophic factor receptor in axon patterning.

During neural development, complex organisms rely on progressive and regressive events whereby axons, synapses, and neurons are overproduced followed by selective elimination of a portion of these components. Tumor necrosis factor α (TNFα) together with its cognate receptor (Tumor necrosis factor receptor 1; TNFR1) have been shown to play both regressive (i.e. forward signaling from the receptor) and progressive (i.e. reverse signaling from the ligand) roles in sympathetic neuron development. In contrast, a paralog of TNFR1, p75 neurotrophic factor receptor (p75NTR) promotes mainly regressive developmental events in sympathetic neurons. Here we examine the interplay between these paralogous receptors in the regulation of axon branch elimination and arborization. We confirm previous reports that these TNFR1 family members are individually capable of promoting ligand-dependent suppression of axon growth and branching. Remarkably, p75NTR and TNFR1 physically interact and p75NTR requires TNFR1 for ligand-dependent axon suppression of axon branching but not vice versa. We also find that p75NTR forward signaling and TNFα reverse signaling are functionally antagonistic. Finally, we find that TNFα reverse signaling is necessary for nerve growth factor (NGF) dependent axon growth. Taken together these findings demonstrate several levels of synergistic and antagonistic interactions using very few signaling pathways and that the balance of these synergizing and opposing signals act to ensure proper axon growth and patterning.

International multidisciplinary expert panel consensus on breast reconstruction and radiotherapy.

BACKGROUND: Conflicting evidence challenges clinical decision-making when breast reconstruction is considered in the context of radiotherapy. Current literature was evaluated and key statements on topical issues were generated and discussed by an expert panel at the International Oncoplastic Breast Surgery Meeting in Milan 2017. METHODS: Studies on radiotherapy and breast reconstruction (1985 to September 2017) were screened using MEDLINE, Embase and CENTRAL. The literature review yielded 30 controversial key questions. A set of key statements was derived and the highest levels of clinical evidence (LoE) for each of these were summarized. Nineteen panellists convened for dedicated discussions at the International Oncoplastic Breast Surgery Meeting to express agreement, disagreement or abstention for the generated key statements. RESULTS: The literature review identified 1522 peer-reviewed publications. A list of 22 key statements was produced, with the highest LoE recorded for each statement. These ranged from II to IV, with most statements (11 of 22, 50 per cent) supported by LoE III. There was full consensus for nine (41 per cent) of the 22 key statements, and more than 75 per cent agreement was reached for half (11 of 22). CONCLUSION: Poor evidence exists on which to base patient-informed consent. Low-quality studies are conflicting with wide-ranging treatment options, precluding expert consensus regarding optimal type and timing of breast reconstruction in the context of radiotherapy. There is a need for high-quality evidence from prospective registries and randomized trials in this field.

ANTECEDENTES: El hecho de que la evidencia disponible sea conflictiva supone un reto para la toma de decisiones a la hora de considerar la reconstrucción mamaria en el contexto de radioterapia (radiotherapy, RT). En el seno de un panel de expertos reunidos durante el International Oncoplastic Breast Surgery Meeting celebrado en Milán en 2017, se revisó la literatura disponible y se generaron y discutieron los aspectos más relevantes. MÉTODOS: Se hizo una búsqueda bibliográfica de los estudios de RT y reconstrucción mamaria (1985-septiembre de 2017) en las bases MEDLINE, EMBASE y CENTRAL. La revisión de la literatura permitió identificar 30 cuestiones clave controvertidas. A partir de ellas, se construyeron una serie de afirmaciones, para las que se obtuvo el mayor nivel de evidencia (levels of clinical evidence, LoE) posible. El acuerdo, desacuerdo o abstención respecto a las cuestiones propuestas fueron el resultado de las discusiones de 19 expertos reunidos durante el International Oncoplastic Breast Surgery Meeting. RESULTADOS: Se identificaron 1.522 artículos publicados en revistas con peer review. Se elaboró una lista de 22 afirmaciones clave y se anotó el LoE más alto obtenido para cada una de ellas. El grado de variabilidad fue de II a IV, pero la mayoría de las afirmaciones (54,5%) obtuvieron un LoE III. Hubo un consenso total en el 41% (9/22) de las afirmaciones, mientras que se obtuvo más de un 75% de acuerdo en la mitad de las afirmaciones (11/22). CONCLUSIÓN: La evidencia en la que basar el consentimiento informado en estos pacientes es escasa. Se trata de estudios de baja calidad con gran variedad de opciones terapéuticas, que dificultan el consenso de los expertos acerca del tipo y momento óptimo para la reconstrucción mamaria en el contexto de RT. Para obtener datos de mayor calidad se precisan estudios prospectivos y ensayos clínicos en este campo.

Antiviral and immunomodulatory effects of a novel bacterial exopolysaccharide of shallow marine vent origin.

AIMS: To evaluate a novel exopolysaccharide (EPS1) from the recently described haloalkaliphilic, thermophilic Bacillus licheniformis strain T14, isolated from a shallow hydrothermal vent of Panarea Island (Italy), for its antiviral and immunomodulatory effects against herpes simplex virus type 2 (HSV-2). METHODS AND RESULTS: EPS1-T14 hindered the HSV-2 replication in human peripheral blood mononuclear cells (PBMC) but not in WISH cells, indicating that cell-mediated immunity was involved in the antiviral activity. High levels of Th1-type cytokines were detected in supernatants of EPS1-treated PBMC, while Th2-type cytokines were not induced. CONCLUSIONS: The novel EPS1-T14 is a water-soluble, noncytotoxic exopolymer able to stimulate the immune response and thus contribute to the antiviral immune defence, acting as immunomodulator. SIGNIFICANCE AND IMPACT OF THE STUDY: The exopolysaccharide produced by B. licheniformis strain T14, stimulator of Th1 cell-mediated immunity, could be used as therapy in immunocompromised host.

New bacilli from shallow hydrothermal vents of Panarea Island (Italy) and their biotechnological potential.

AIMS: To characterize bacilli isolated from shallow hydrothermal vents of Panarea Island (Italy) and evaluate their biotechnological potential. METHODS AND RESULTS: Fifteen isolates were characterized by culture and molecular methods. Eleven isolates were thermophilic, six isolates were alkalophilic and four of them were haloalkalophilic. After 16S rRNA gene sequencing, four strains, exhibiting sequence similarity below 95% with deposited strains, may represent novel species of bacilli. One strain was strictly related to Geobacillus subterraneus, but shared phenotypic characteristics for which it could be considered a new strain of this species. Four strains were affiliated with different Bacillus spp. Most isolates produced gelatinase, lipases and amylase, and some were mercury tolerant. Exopolysaccharides (EPS) production was tested adding different sugars (glucose, sucrose, trehalose, fructose, ribose, xylose and mannose, 1% w/v) as a carbon source in a minimal medium. The highest EPS yield (185 mg l(-1)) was reached by strain 1A70 utilizing ribose as a carbon source. CONCLUSIONS: Novel strains of Geobacillus and indigenous ribotypes of Bacillus with biotechnological potential inhabit shallow vents of Panarea Island. SIGNIFICANCE AND IMPACT OF THE STUDY: New strains of thermophilic bacilli from Panarea are producers of useful biomolecules for industrial purposes as well as environmental and biotechnological applications.

Conventional and molecular methods to detect bacterial pathogens in mussels.

AIM: To detect Aeromonas spp., Salmonella spp., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in mussels and water samples from a farming area, conventional and molecular methods were applied to enrichment cultures. METHODS AND RESULTS: The aerolysin gene (aero) of Aeromonas spp., the invasion plasmid antigen B (ipaB) gene of Salmonella spp., the enterotoxin secretion protein (epsM) gene of V. cholerae, the species-specific region of 16S rRNA gene of V. vulnificus, the 16S-23S rDNA (IGS) gene of V. parahaemolyticus and the pR72H fragment of V. parahaemolyticus were amplified by multiplex polymerase chain reaction (PCR) assays on DNA extracted from enrichment cultures. The haemolysin gene (tdh) of pathogenic V. parahaemolyticus was also amplified. Conventional culture method allowed the isolation of V. parahaemolyticus and V. vulnificus from water and mussels. The genes aero, epsM and 16S rRNA of V. vulnificus were occasionally detected in the enrichment cultures. In mussels, the ipaB and IGS genes were detected from June to September and from April to November, respectively. All genes, except aero, were amplified from mussels collected in September, when pathogenic V. parahaemolyticus (tdh+) strains were also isolated. CONCLUSIONS: Multiplex-PCR assays were more sensitive and faster than conventional procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: The results emphasize the need of an accurate and rapid detection of bacterial pathogens in mussels to protect human health.

Evaluation of anatomical landmark position differences for head computed tomography: A reliability study among technologists.

INTRODUCTION: In computed tomography (CT) imaging protocols, lack of practice standards and variability in head positioning may all yield substantial inter-study image variance in the clinical setting which may limit the diagnostic and comparative value of subsequent scans. We aimed to evaluate repeatability of multiplanar reformatting of head CT based on the tuberculum sella (TS) to internal occipital protuberance (IOP) reference line and reduce variance. METHODS: Reference lines that correspond to the TS-IOP plane on high-resolution CT scans were reviewed by technologists manually to calculate Yaw (z-rotation, rotation along the superoinferior direction), Pitch (x-rotation, rotation along the left-right direction), and Roll (y-rotation, rotation along the anteroposterior direction) angles in this pre-post design intervention study. The Yaw, Pitch, and Roll angles deviating from the reference TS-IOP in the head CT images before and after technologist training were measured with the technologists' actual graphical prescriptions, and their differences were calculated with t-tests. The intra-rater agreement was calculated using the intraclass correlation coefficient (ICC). RESULTS: Mean pitch, yaw, and roll before technologist training was 6.7° ± 5.4°, 0.9° ± 1.5°, and 1.1° ± 1.2° and after training were 3.2° ± 2.6°, 0.6° ± 1.1°, and 0.6° ± 1.1°, respectively. Technologist training resulted in a significant decrease in pitch (p < 0.001) and roll (p = 0.001) inter-subject variability with respect to the TS-IOP line, however no significant difference for the yaw correction (p = 0.065) was noted. Intra-rater agreement regarding the reproducibility of TS-IOP reformation was excellent (ICC>0.950). CONCLUSION: TS-IOP reference line corrected for direct roll, yaw, and pitch can be readily achieved by trained technologists. IMPLICATIONS FOR PRACTICE: Adoption of the TS-IOP reference line should facilitate intra- and intermodality comparisons, leading to more reproducible and readily interpretable CT images.

The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Frequency of detection of respiratory viruses in the lower respiratory tract of hospitalized adults.

BACKGROUND: Respiratory infections are the most common infections in humans. The prevalence of respiratory viruses in adults is largely underestimated, and relevant data mostly concern infants and children. OBJECTIVES: To evaluate the prevalence of respiratory viruses in adults hospitalized in Italy. STUDY DESIGN: During April 2004--May 2005, 510 consecutive lower respiratory tract samples were prospectively collected. These were evaluated with a molecular panel that detected 12 respiratory viruses. RESULTS: Two hundred and fifteen samples were positive for at least one viral pathogen, with an overall sample prevalence of 42.2%. Human rhinoviruses (HRVs) were the most commonly detected viruses (32.9%), followed by influenza virus (FLU)-A (9.0%); the other viruses were 2% or less. Multiple agents were detected in 30 samples from 29 patients, resulting in a co-infection rate of 6.7%. CONCLUSIONS: This study shows a high prevalence of viruses in the lower respiratory tract samples of hospitalized adults, mostly HRV and FLU-A. It is not possible to establish the role of viruses detected at low frequency, but our findings suggest the necessity to consider them as potential causes or precursors of lower respiratory tract infections (LRTIs).

Phylogenetic analysis of human coronavirus NL63 circulating in Italy.

BACKGROUND: Five known human coronaviruses infect the human respiratory tract: HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63 and HCoV-HKU1. OBJECTIVES: To evaluate the prevalence of HCoV-NL63 in hospitalized adult patients and to perform molecular characterization of Italian strains. STUDY DESIGN: HCoV-NL63 was sought by RT-PCR in 510 consecutive lower respiratory tract (LRT) samples, collected from 433 Central-Southern Italy patients over a 1-year period. Phylogenetic analysis was performed by partial sequencing of S and ORF1a. Additional S sequences from Northern Italy were included in the phylogenetic trees. RESULTS: HCoV-NL63 was detected in 10 patients (2.0%) with symptomatic respiratory diseases, mainly during winter. Phylogenetic analysis indicated a certain degree of heterogeneity in Italian isolates. The ORF1a gene clustering in phylogenetic trees did not match with that of the S gene. CONCLUSIONS: As observed by others, HCoV-NL63 is often associated with another virus. Phylogenetic characterization of HCoV-NL63 circulating in Italy indicates that this virus circulates as a mixture of variant strains, as observed in other countries.

Anti-cyclic citrullinated peptide antibodies in patients affected by HCV-related arthritis.

Hepatitis C Virus (HCV) infection can induce immunological disorders with different clinical expressions such as arthritis, Sjogren Syndrome and various forms of vasculitis. Retrospectively, the prevalence of anti-Cyclic Citrullinated peptide antibodies (anti-CCP) in a group of patients affected by HCV-related arthritis with positivity for Rheumatoid Factor (RF) and the eventual correlations with RF and/or Anti-Nuclear Antibodies (ANA) and articular involvement were studied. Thirty patients with arthritis were selected from a population of 380 subjects affected by HCV infection. Each patient was evaluated by clinical examination (23 denoted poliarticular and 7 mono-oligoarticular involvement), by X-graphic aspects of joint involvement (8 patients presented joint erosions), by ANA, RF and anti-CCP positivity. Ten of the HCV-related arthritis patients (33.3 percent) presented positivity for anti-CCP, without significant correlation between such parameter and ANA, RF and articular involvement. Anti-CCP resulted positive in 4 out of the 8 patients with joint erosions, and only in 6 out of the 22 patients without joint erosions. Such frequencies analyzed by chi square resulted with no significant differences. Our patients presented an interesting prevalence of the positivity for anti-CCP. These data are cause to consider the specificity recently attributed to this parameter in the diagnosis of rheumatoid arthritis.

Reduction of capsular thickness around silicone breast implants by zafirlukast in rats.

Capsular contracture is a very disappointing complication, with an overall incidence between 0.5 and 30% of breast implant operations and, if severe, requiring a further surgical procedure (capsulotomy or capsulectomy). Many frustrating attempts have been made to prevent the fibrotic reaction, mainly with steroids or antibiotics. More recently leukotriene antagonists clinically used in the asthma and lung diseases have been suggested to be potentially useful in counteracting the inflammatory pathway leading to a dense collagen membrane around the prosthesis and thus preventing contracture of the capsule. In this study we evaluated the effectiveness of zafirlukast with the following protocol. Disks of textured implant material were placed dorsally into each of the subcutaneous tissues of 40 rats that were subdivided in 2 groups: 20 rats treated with zafirlukast and 20 controls. At autopsy 77 days after, each implant with its surrounding collagenic tissue was excised, and the macroscopic measure of the membrane thickness was compared with the pathology reports, to definitely assess the foreign body reaction. The mean total thickness of the capsule around the implants was 161.97 microm in the zafirlukast-treated group compared with 345.98 microm in the control group (p < 0.001). Outstandingly, the collagen fibers and fibroblast layer were reduced in the zafirlukast-treated group compared to the controls. Our study confirms the effectiveness of this compound in preventing fibrosis and putatively also in reducing the extent of collagen reaction when a capsule has been formed.

Genome-wide DNA methylation analysis in blood cells from patients with Werner syndrome.

BACKGROUND: Werner syndrome is a progeroid disorder characterized by premature age-related phenotypes. Although it is well established that autosomal recessive mutations in the WRN gene is responsible for Werner syndrome, the molecular alterations that lead to disease phenotype remain still unidentified. RESULTS: To address whether epigenetic changes can be associated with Werner syndrome phenotype, we analysed genome-wide DNA methylation profile using the Infinium MethylationEPIC BeadChip in the whole blood from three patients affected by Werner syndrome compared with three age- and sex-matched healthy controls. Hypermethylated probes were enriched in glycosphingolipid biosynthesis, FoxO signalling and insulin signalling pathways, while hypomethylated probes were enriched in PI3K-Akt signalling and focal adhesion pathways. Twenty-two out of 47 of the differentially methylated genes belonging to the enriched pathways resulted differentially expressed in a publicly available dataset on Werner syndrome fibroblasts. Interestingly, differentially methylated regions identified CERS1 and CERS3, two members of the ceramide synthase family. Moreover, we found differentially methylated probes within ITGA9 and ADAM12 genes, whose methylation is altered in systemic sclerosis, and within the PRDM8 gene, whose methylation is affected in dyskeratosis congenita and Down syndrome. CONCLUSIONS: DNA methylation changes in the peripheral blood from Werner syndrome patients provide new insight in the pathogenesis of the disease, highlighting in some cases a functional correlation of gene expression and methylation status.

HCV infection and chronic arthritis: Does viral replication matter?

BACKGROUND: HCV infection beside chronic hepatitis can induce immunological disorders with different clinical expressions such as chronic arthritis. AIM: To study the prevalence of arthritis in HCV-Ab positive patients and verify possible correlation with viral replication, hepatic damage and autoimmunity imbalance. STUDY DESIGN: Three hundred and eighty patients (196 M and 184 F) affected by HCV infection were examined and 38 (10%) were selected according to the presence of arthritis. Eight of them were excluded because arthritis raised before HCV infection. Each patient, once undergone liver biopsy, was evaluated for: clinical examination (articular evolution), Rx examination, serum expression of hepatic damage (mainly ALT), viral replication, and involvement of autoimmunity (ANA, RF, crioglobulins, AKA, CCP). RESULTS: Data from patients [Lamprecht P, Gause A, Gross WL. Cryoglobulinemic vasculitis. Arthritis Rheum 1999; 42:2507-16.] with AKA and CCP positivity were not considered for statistical examination because the clear correlation between rheumatoid arthritis and these parameters. The remaining 20 patients showed hepatic damage 47%, viral replication in 74%, RF 42%, ANA 16%, crioglobulins 42% (RF positive). No correlation was evident between ANA serum concentrations and viral replication; furthermore a significant negative correlation between RF positivity and viral replication only in a subgroup of patients with serologic expression of hepatic damage was found. CONCLUSIONS: These data support hypothesis that the onset of arthritis and presence of autoimmunity parameters ANA, RF are not related to the viral replication but others mechanism immunological induced by HCV might be considered.

Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.).

Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.

Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase.

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplast completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W10BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.

Dexamethasone apoptosis induction and glucocorticoid receptor levels in cultured normal and neoplastic cell lines.

The deletion with apoptotic mechanisms, of different normal and neoplastic cell lines [Jurkat leukemic cells, EUE epithelioid cells, normal (FG) and transformed rat fibroblasts (SGS/3A)] cultured in vitro in presence of dexamethasone, have been studied combining morphocytochemical (fluorescence microscopy), cytometric (flow cytometry) and biochemical (Radio Receptor Assay) analyses. It has been found that the synthetic glucocorticoid hormone induces an antiproliferative effect with accumulation of cells in the G1 phase and a cell loss by programmed death in some cell lines. The apoptotic incidence was found to be inversely proportional to the cytostatic effect of the hormone: the highest in EUE and Jurkat cells (in EUE cells not only affecting the G0/G1 phase of the cell cycle), the lowest in SGS/3A cells and absent in frbroblasts FG. The apoptotic degeneration, in all the cell lines studied, was characterized, morphologically and cytochemically by: a) decrease in stainability/content of cell DNA and proteins; b) condensation of cytoplasm; c) preservation of mitochondrial membrane functional integrity. In conclusion, in the presence of dexamethasone, programmed cell death was found to play a variable role during the maintenance of culture turnover in different cell lines and the incidence of degenerative phenomena does not appear to be related to glucocorticoid receptor levels.

Direct in situ PCR allows rapid and sensitive detection of high risk human papillomavirus in cytologic specimens and formalin-fixed paraffin tissues by fluorescent labelling.

We developed a rapid, sensitive and robust high risk human papillomavirus (HR HPV) detection protocol based on direct in situ PCR technology and fluorochrome-modified nucleotides on cytologic specimens (cell smears) and on HPV infected tissues (CIN III). Reproducible results on both cytologic specimens and paraffin-embedded tissues were obtained, providing a powerful tool for clinical investigation on HR HPV infection. Quantitative PCR performed on the same tissue sections adjacent to those used for in situ techniques allowed us to establish the sensitivity of our methods, able to detect rare copies (about 15 in our paraffin-embedded tissues) of HPV.

A fatal postpartum Clostridium sordellii associated toxic shock syndrome.

Clostridium sordellii is an infrequent human pathogen. It has been demonstrated to be occasionally responsible for myonecrosis or gas gangrene. Interestingly, in the obstetric literature, some cases of postpartum maternal deaths have been associated with C sordellii infection causing a rapidly lethal toxin mediated syndrome. This is the first reported case of postpartum death in a 29 year old woman, in which a toxigenic C sordellii was isolated from the patient's blood antemortem during the fatal toxic shock, strongly indicating its role in this rare syndrome.

Are there differences in methotrexate kinetics between responding and nonresponding patients with rheumatoid arthritis?

OBJECTIVE AND STUDY DESIGN: The purpose of this study was to investigate the presence of a correlation between methotrexate pharmacokinetics and clinical efficacy in patients with rheumatoid arthritis. PATIENTS AND METHODS: The study was carried out in 29 patients with rheumatoid arthritis. The patients received intramuscular methotrexate (MTX) 7.5mg once a week for 8 weeks. Before and 0.5, 1, 2, 3, 4, 6, 9, 12 and 24 hours after the first administration, MTX serum concentrations were measured and pharmacokinetic investigations were carried out. The clinical status of the disease was evaluated before and after 8 weeks of therapy. In addition, before and after 2 and 8 weeks of treatment, the patients were monitored for a complete biochemical profile. After 8 weeks of treatment, on the basis of improvement in clinical parameters, the patients were designated responders or nonresponders. RESULTS: A clinical response was obtained in 62% of patients (18 patients responded and 11 did not) and was associated with a low incidence of adverse effects. There were no differences in the pharmacokinetic parameters of MTX between the 2 groups of patients (responders vs nonresponders), except that t(max) was significantly higher in nonresponders than in responders (p < 0.05). CONCLUSIONS: These data confirm the efficacy and tolerability of low dose MTX in patients with rheumatoid arthritis in the short term, but appear to exclude a relationship between MTX kinetics and clinical response.

Activation signals of T lymphocytes in microgravity.

Human peripheral blood lymphocytes and monocytes were activated with concanavalin A with or without exogenous recombinant interleukin 1 (IL-1) alone or IL-1 + interleukin 2 (IL-2) under microgravity conditions to test the hypothesis that lack of production of IL-1 by monocytes is the cause of the near total loss of activation observed earlier on several Spacelab flights. The 60 min failure of the on-board 1 x g reference centrifuge at the time of the addition of the activator renders the in-flight data at 1 x g unreliable. However, the data from a previous experiment on SLS-1 show that there is no difference between the results from the in-flight 1 x g centrifuge and 1 x g on ground. The comparison between the data of the cultures at 0 x g in space and of the synchronous control at 1 x g on ground show that exogenous IL-1 and IL-2 do not prevent the loss of activity (measured as the mitotic index) at 0 x g; production of interferon-gamma, however, is partially restored. In contrast to a previous experiment in space, the production of IL-1 is not inhibited.