The pathogenesis of Yersinia enterocolitica infection in gnotobiotic piglets

Yersinia enterocolitica is an important cause of enteritis and mesenteric adenitis in many countries. However the pathogenesis of the disease caused by this organism has not been fully elucidated. Most isolates from clinical material possess two independent properties associated with virulence whose relative contribution to the development of disease is not known. These are the ability to penetrate the intestinal wall, which is thought to be controlled by a plasmid gene, and the production of heat-stable enterotoxin, which is controlled by a chromosomal gene. In this study, we infected neonatal gnotobiotic piglets with strains of Y. enterocolitica expressing these two properties in various combinations. The suitability of the piglet model was shown in experiments in which piglets fed virulent Y. enterocolitica serogroup O3 developed a clinical illness related to the size of the inoculum, which was accompanied by intestinal lesions similar to those reported in naturally and experimentally infected people and animals. The results confirmed the key role of a 47 X 10(6)-mol. wt plasmid in the pathogenicity of Y. enterocolitica, but suggested that penetration of the intestinal wall may be governed by chromosomal rather than plasmid-borne genes. No role for enterotoxin in the pathogenesis of yersiniosis was shown, although there was evidence that enterotoxin may promote intra-intestinal proliferation of Y. enterocolitica, thus favouring increased shedding of bacteria and encouraging their spread between hosts.     

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.     

Collared crypts in irradiated small intestine

Crypts of Lieberkuhn are radiosensitive: the technique of crypt counting is an established method of assessing radiation induced changes in the small intestine. However, there has been little work done on the surface contours of the crypts, as they open into the intervillous cleft. The current paper describes the structure of control mouse crypt mouths as unobtrusive openings approximately 5 microns in diameter. After radiation with heavy ion particles, the crypt mouths are substantially larger (up to 10 microns in diameter) with a marked collar which is similar to that sometimes seen in coeliac disease. The shape and incidence of the collared crypts is described for specimens irradiated with neon, silicon and iron ions, with treatment with iron producing the most marked collars: it is suggested that the size and incidence of the collared crypts may be related to the LET of the beam used. It is of interest that the abnormal crypts are not produced after single doses of X-irradiation. The consideration of the structure of the collared crypts may require a redefinition of the terms crypt and villus with priority being given to the position of subepithelial vessels rather than surface shape. Finally, although the collared crypts can not be directly equated with 'tunnel' or 'channel' lesions, it is pointed out that they do represent localised damage with a specific position and shape.     

Continuing education for general practice and the role of the pharmaceutical industry.

A survey of the involvement in and attitudes towards continuing medical education of 101 general practitioners achieved a 95% response rate. Ninety per cent of the 96 doctors worked in practices which held meetings the content of which was organized by representatives of pharmaceutical companies but only 46% worked in practices which organized their own educational meetings. Seventy six per cent attended meetings away from their practice which were organized by drug companies and 75% had attended at some time continuing medical education activities organized by a local postgraduate centre. The promotional aspects of the drug company organized meetings were disliked by a majority of respondents (58%); more of the trainers (62%) and more of those who had entered general practice within the last seven years (71%) disliked this aspect. Nonetheless the educational content of both meetings held in the practice and those held elsewhere was the aspect most liked by over half of the respondents (59% and 53% respectively). Only 16% of all respondents thought that visits by representatives from pharmaceutical companies were educationally valuable and 37% thought that educational events organized by these companies were of value. Surprisingly 60% of those who worked in practices which held meetings organized by drug company representatives thought them to be of little or no educational value. There is clearly a need for practice based continuing medical education but the current level of dependence on drug companies for organizing these meetings must be questioned. Alternative strategies for the provision of independent non-sponsored educational activities should be sought.     

Distribution of glutathione S-transferase isoenzymes in human kidney: basis for possible markers of renal injury.

To determine whether the tissue distribution of glutathione S-transferase (GST) isoenzymes could define the precise nature of renal injury, 13 adult kidneys were studied, using specific antibodies raised against purified isoenzymes. Basic GST stained strongly proximal convoluted tubules and some medullary tubules; acidic GST stained strongly distal convoluted tubules and medullary tubules; neutral GST stained similarly to acidic GST, but weaker, and microsomal GST stained glomerular and interstitial endothelium and collecting ducts deep in the medulla, although there was considerable variation in staining intensity among cases. It is suggested that the measurement of these isoenzymes in serum and urine may help to elucidate the localisation of tissue damage, which may be particularly valuable in patients with cyclosporine toxicity following renal transplantation.     

Comparison of multiple-locus variable-number tandem repeat analysis and pulsed-field gel electrophoresis in a setting of polyclonal endemicity of vancomycin-resistant Enterococcus faecium.

In order to assess whether multiple-locus-variable number tandem repeat analysis (MLVA) could replace pulsed-field gel electrophoresis (PFGE) for genotyping vancomycin-resistant isolates of Enterococcus faecium (VREF), this study compared the typeability, discriminatory power, concordance and costs of these methods for VREF isolates obtained from patients, environmental samples and the hands of healthcare workers (HCWs) in a medical intensive care unit (ICU) where VREF was endemic. Over a 58-day period, 393 VREF isolates (373 vanA, one vanA/B, 19 vanB) were cultured from patient rectal swabs (n = 76), the environment (n = 270) and the hands of HCWs (n = 47). PFGE was able to divide 358 (91.1%) isolates into 19 PFGE types (>six bands different) and 24 subtypes (one to three bands different). MLVA was able to type 391 (99.5%) isolates into 11 genotypes. The discriminatory power of PFGE subtypes was 83%, as compared to 68% for MLVA. Concordance between the two methods, based on matched or mismatched MLVA types and PFGE types or subtypes, was 67.5% and 82.8%, respectively. Using PFGE, 13 isolates could be genotyped in 3 days; MLVA genotyped 94 isolates in 2 days. For both methods, the estimated costs were Euro 7 ($10)/isolate. PFGE and MLVA produced highly concordant results when assigning genotypes to nosocomial VREF isolates. MLVA was faster, but PFGE subtyping was more discriminatory.     

Characterization of murine monoclonal antibodies to Escherichia coli J5.

Results show that various inbred strains of mice can be segregated into two distinct groups, based on their capacity to allow a number of nontuberculous mycobacterial infections to grow in target organs following experimental intravenous infection. The first group, which allowed these infections to grow progressively, was thus designated as naturally susceptible to these infections; in contrast, those strains which were able to exert detectable bacteriostasis were designated as naturally resistant. It was then found that segregation of mouse strains based on this distinction also mirrored the capacity of these animals to generate acquired immunity to the mycobacterial infections. For example, Mycobacterium simiae grew progressively in susceptible C57BL/6 mice, subsequently triggering acquired mechanisms of immunity, whereas no evidence for acquired immunity could be found in resistant A/Tru mice infected with this organism. The possibility that acquired immunity could not be expressed in the latter strain as a result of a defect in macrophage activation was excluded. Moreover, it was found that the trait of resistance to these infections could be transferred by bone marrow cells into radiation chimeras, thus indicating that this trait was expressed by the progeny of hemopoietic precursor cells. Subsequent backcross analysis to determine the mode of inheritance of the trait of resistance to these mycobacterial infections revealed data that were consistent with the hypothesis that this resistance is controlled by more than one gene. Statistical analysis of the data by the maximum likelihood method suggested polygenic control, although in some cases the probability values suggested control by a major gene, influenced by modifier genes. These findings suggest that the previous hypothesis that the growth of mycobacterial infections in inbred strains of mice is controlled by a single gene should be reevaluated.     

Laser ablation of discs of agar gel

Discs of agar gel mixed with ink were used to study ablation effects with an argon laser as a light source. Varying amounts of ink were added resulting in a variation of the attenuation coefficient between 0.45 and 6.3 mm-1. For laser beam irradiation horizontally incident on a vertical sample, the average velocity of ablation was found to be approximately constant for thicknesses up to 1.7 mm. When the laser beam was directed vertically on a sample held horizontally, the vaporized debris present in the beam attenuated the incident laser energy to such a degree that the average ablation velocity decreased by a factor of approximately five. Horizontal beam experiments for various attenuation coefficients showed that an attenuation coefficient of about 1.7 mm-1 is optimal for fast penetration of discs thicker than 4 mm. Thus, based upon the optical properties of a given tissue, there may exist an optimum laser wavelength to maximise ablation velocity.     

Age changes in geometry and mineral content of the lower limb bones

Subperiosteal expansion and increase in second moments of area with aging of eleven femoral and tibial cross-sections are documented in a large archaeological sample from the American Southwest. In contrast to these geometric changes, we found little change with age in bone mineral density measured using photon absorptiometry. Thus, the most significant structural changes with age in bone appear to involve its geometry and material characteristics other than its density. Variation in age-related geometric remodeling between cross-section locations and populations may be caused by differences in mechanical stress and strain levels in vivo in the lower limb.     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.