Retinoic acid induces p27Kip1 nuclear accumulation by modulating its phosphorylation

All-trans-retinoic acid (ATRA), the most biologically active metabolite of vitamin A, controls cell proliferation, apoptosis, and differentiation depending on the cellular context. These activities point to ATRA as a candidate for cancer therapy. A pivotal effect of the molecule is the modulation of p27Kip1, a cyclin-dependent kinase (CDK) inhibitor (CDKI). Here, we investigate the mechanisms by which ATRA regulates p27Kip1 level in LAN-5, a neuroblastoma cell line. When added to the cells, ATRA causes a rapid nuclear increase of p27Kip1, which clearly precedes growth arrest. The early buildup is not due to impairment of the CDKI degradation, in contrast to previous observations. Particularly, we did not detect the down-regulation of Skp2 and Cks1, two proteins involved in the nuclear ubiquitin-dependent p27Kip1 removal. Moreover, the morphogen does not impair the CDKI nuclear export and does not cause CDK2 relocalization. The characterization of CDKI isoforms by two-dimensional PAGE/immunoblotting showed that ATRA induces an early nuclear up-regulation of monophosphorylated p27Kip1. Immunologic studies established that this isoform corresponds to p27Kip1 phosphorylated on S10. The buildup of phospho(S10)p27Kip1 precedes the CDKI accumulation and increases its half-life. Finally, ATRA-treated nuclear LAN-5 extracts showed an enhanced capability of phosphorylating p27Kip1 on S10, thus explaining the nuclear up-regulation of the isoform. In conclusion, our data suggest a novel mechanism of ATRA antiproliferative activity, in which the morphogen rapidly up-regulates a nuclear kinase activity that phosphorylates p27Kip1 on S10. In turn, this event causes the stabilization of p27Kip1 and its accumulation in the nuclear compartment.     

Multicentre Evaluation of a Commercial Test for the Rapid Diagnosis of Clostridium difficile-Mediated Antibiotic-Associated Diarrhoea

 An immunoassay for the detection of Clostridium difficile toxin A in stool samples (Clearview C. DIFF A; Unipath, UK) was evaluated against the cell cytotoxicity assay using 407 stool samples from patients suspected to have, or considered at risk of, antibiotic-associated diarrhoea. Of the samples tested, 98 were positive and 280 were negative by both tests (sensitivity 83.1%, specificity 96.9%). Following resolution of the 29 discrepant results, the sensitivity and specificity of the immunoassay were 91% and 98%, respectively, and the sensitivity for the cell cytotoxicity assay was calculated as 91.5%, with a specificity of 99%. The Clearview C. DIFF A test proved to be a rapid simple assay for the detection of Clostridium difficile toxin A in stool samples. The test was equally suited to single or batch testing, required minimal sample handling, and provided results within 30 min of applying the sample to the test unit.     

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.     

Adverse impact of surgical site infections in English hospitals

Between October 1997 and June 2001, 140 English hospitals participating in the surveillance of surgical site infection (SSI) with the Nosocomial Infection National Surveillance Service (NINSS) reported 2832 SSIs following 67 410 surgical procedures in nine defined categories of surgery. Limb amputation had the highest incidence of SSI with 14.3 SSIs per 100 operations. For all categories of surgery, except knee prosthesis (P=0.128), there was a linear increase in the incidence of SSI when the American National Nosocomial Infections Surveillance risk index increased. Superficial incisional SSI was more common than deep incisional and organ/space SSI, and accounted for more than half of all SSIs for all categories of surgery. The postoperative length of stay (LOS) was longer for patients with SSI, and when adjusted for other factors influencing LOS, the extra LOS due to SSI ranged from 3.3 days for abdominal hysterectomy to 21.0 days for limb amputation, and was at least nine days for the other categories. The additional cost attributable to SSI ranged from pound959 for abdominal hysterectomy to pound6103 for limb amputation. Deep incisional and organ/space SSI combined incurred a greater extra LOS and cost than superficial incisional SSI for all categories of surgery, except limb amputation. The crude mortality rate was higher for patients with SSI for all categories of surgery but, after controlling for confounding, only patients with SSI following hip prosthesis had a mortality rate that was significantly higher than those without SSI [odds ratio (OR)=1.8, P=0.002]. However, the adjusted mortality rate for patients with deep incisional and organ/space SSI compared with those without SSI was significantly higher for vascular surgery (OR=6.8, P<0.001), hip prosthesis (OR=2.5, P=0.005) and large bowel surgery (OR=1.8, P=0.04). This study shows that the adverse impact of SSI differs greatly for different categories of surgery, and highlights the importance of measuring the impact for defined categories rather than for all SSIs and all surgical procedures.     

Device-related sources of bacteraemia in English hospitals--opportunities for the prevention of hospital-acquired bacteraemia

Between 1997 and 2001, 17 teaching and 56 non-teaching acute English hospitals conducted hospital-wide surveillance of hospital-acquired bacteraemia (HAB) using a standard protocol drawn up by the Nosocomial Infection National Surveillance Scheme (NINSS). The sources of organisms, the incidence of device-related HAB, and the distribution of HABs from individual device-related sources by specialty and type of hospital were determined for 6,956 HABs in order to identify where resources should best be targeted to reduce these infections. The overall incidence of HAB was higher in teaching than in non-teaching hospitals: 5.39 and 2.83 HABs per 1,000 patients at risk, respectively (P<0.001). Device-related sources were responsible for 52.4 and 43.2% of all HABs in teaching and non-teaching hospitals, respectively (P<0.001), and central lines were the commonest source, causing 38.3% of HABs in teaching versus 22.3% in non-teaching hospitals (P<0.001). In teaching hospitals, general intensive care units (ICUs), haematology, special care baby units (SCBUs), nephrology, and oncology accounted for only 6.1% of the population surveyed, but had the highest incidence of HAB, and contributed 47.8% of 2091 HABs and 56.9% of 1,095 device-related bacteraemias. Of 623 device-related bacteraemias in these high-risk specialties, 554 (88.9%) were from central lines. Thus, in teaching hospitals, resources should be targeted primarily at the prevention of central line-related bacteraemia in these five high-risk specialties, and the surveillance should include data on central line use. In non-teaching hospitals, nearly two thirds (63.3%) of 4,865 HABs and 60.7% of 2,103 device-related bacteraemias were from a few specialties with a low incidence of bacteraemia, but large numbers of patients, namely general medicine, general surgery, geriatric medicine and urology. These specialties accounted for 50.5% of the population surveyed. Central lines were the most common source of bacteraemia in general medicine and surgery, and together accounted for 23.3% of all device-related bacteraemias. However, in geriatric medicine and urology, central line sources were infrequent, accounting for only 1.7% of all device-related bacteraemias. On the other hand, bacteraemia from catheter-associated UTI were common in all these four specialties accounting for 20.9% of all device-related bacteraemias. Thus, in non-teaching hospitals, resources should be targeted primarily at these low-risk specialties and surveillance should include, at least, bacteraemia from central lines and from catheter-associated UTI. Further benefit can be obtained by including central line-related bacteraemias from general ICU and haematology patients, as they contributed 17.0% of all device-related bacteraemias in non-teaching hospitals.     

Transferrin‐immune complex disease: A potentially overlooked gammopathy mediated by IgM and IgG

The combination of marked hypersideremia, hypertransferrinemia, and monoclonal gammopathy of underdetermined significance (MGUS) should alert clinicians to the possible presence of an anti‐transferrin immunoglobulin, an uncommon acquired disorder also defined as transferrin‐immune complex disease (TICD). The authors have previously described a case of TICD with 100% transferrin saturation and liver iron overload. However, the findings in the few cases so far reported are heterogeneous, and the presence of high transferrin saturation and liver iron overload is not universal. In this article, the authors have described the identification of two additional patients with anti‐transferrin monoclonal gammopathy, hypersideremia, and hypertransferrinemia, but with incomplete transferrin saturation and no hepatic iron overload. The autoantibodies were purified by using transferrin as affinity bait and characterized. One subject showed a high‐titer monoclonal anti‐transferrin IgM with a κ‐type light chain. This finding is the first observation of IgM autoantibodies against transferrin. The other patient developed the disease after pregnancy. In this study, monoclonal antibody was an IgG mounting a κ‐type light chain with altered molecular weight. These results highlight that transferrin might induce the development of a monoclonal immune response of different classes and specificity. The identification, in a single hematologic center, of three different subjects with anti‐transferrin monoclonal gammopathy suggests that the disease probably represents a still underdiagnosed condition. From a clinical standpoint, these patients must be followed up both as MGUS and as hemochromatosis. Am. J. Hematol. 88:1045–1049, 2013. © 2013 Wiley Periodicals, Inc.     

The influence of the normal flora on Clostridium difficile colonisation of the gut

The normal stable flora of the gut of man and other adult animal species provides an effective barrier to infection by Clostridium difficile. Attempts to understand this mechanism have involved continuous flow and batch culture systems and colonisation of antibiotic pre-treated or germ free animals with gut flora from the same or unrelated species. In general attempts to re-create the barrier effect with the whole caecal or faecal flora have been successful both in vitro and in vivo, whereas attempts using components of that flora have not. The most recent developments in these types of studies have been studies aimed at understanding the mechanisms involved. Preliminary findings imply competition for various monosaccharides, especially those released from mucin, may be important.     

Quantitative study of antibiotic-induced susceptibility to Clostridium difficile enterocecitis in hamsters.

Commonly used antibiotics were compared for their ability to induce Clostridium difficile enterocecitis and death in hamsters. Susceptibility to infection with C. difficile was measured by calculating 50% lethal doses (in CFU) for hamsters for various intervals after antibiotic treatment. Infection occurred after very small doses of C. difficile were given to hamsters treated with clindamycin, ampicillin, flucloxacillin, and cefuroxime; there was little difference between the antibiotics in the degree of susceptibility that they induced. A large difference in the duration of susceptibility was observed, however, with susceptibility being temporary following ampicillin, flucloxacillin, and cefuroxime administration but long-lived following clindamycin administration. A larger dose of ampicillin, multiple doses of ampicillin, and a combination of antibiotics had comparatively small effects on the duration of susceptibility. C. difficile growth and toxin production in in vitro suspensions of cecal contents were found to correlate closely with in vivo hamster infectivity. A persisting loss of colonization resistance following antibiotic treatment may be a type of postantibiotic effect. Although these results cannot be applied directly to humans, they suggest lines of further investigation into how antibiotics may differ in producing risks of C. difficile infection and pseudomembranous colitis in patients.