Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery

The motility imparted by the periplasmic flagella (PF) of Serpulina hyodysenteriae is thought to play a pivotal role in the enteropathogenicity of this spirochete. The complex PF are composed of multiple class A and class B polypeptides. Isogenic strains containing specifically disrupted flaAl or flaB1 alleles remain capable of expressing PF, although such mutants display aberrant motility in vitro. To further examine the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S. hyodysenteriae which is deficient in both FlaA1 and FlaB1. To facilitate construction of this strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed. The cloned flaAl and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and kanamycin gene cassettes, respectively. The inactivated flagellar genes were introduced into S. hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis. Immunoblots or cell lysates with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath and core proteins in this dual flagellar mutant. These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins. The resulting flaA1 flaB1 mutant exhibited altered motility in vitro. Surprisingly, it was capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. The virulence of this strain was assessed in a murine model of swine dysentery by determining the incidence of cecal lesions and the persistence of S. hyodysenteriae in the gut. Mice challenged with the wild-type strain or a passage control strain showed a dose-related response to the challenge organism. The dual flagellar mutant was severely attenuated in murine challenge experiments, suggesting that the FlaA1 and FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract. This strain represents the first spirochete engineered to contain specifically defined mutations in more than one genetic locus.     

Frequent clones of p53-mutated keratinocytes in normal human skin

The multiple genetic hit model of cancer predicts that normal individuals should have stable populations of cancer-prone, but noncancerous, mutant cells awaiting further genetic hits. We report that whole-mount preparations of human skin contain clonal patches of p53-mutated keratinocytes, arising from the dermal-epidermal junction and from hair follicles. These clones, 60-3000 cells in size, are present at frequencies exceeding 40 cells per cm2 and together involve as much as 4% of the epidermis. In sun-exposed skin, clones are both more frequent and larger than in sun-shielded skin. We conclude that, in addition to being a tumorigenic mutagen, sunlight acts as a tumor promoter by favoring the clonal expansion of p53-mutated cells. These combined actions of sunlight result in normal individuals carrying a substantial burden of keratinocytes predisposed to cancer.     

Studies on the action of interleukin-1 on term human fetal membranes and decidua

The present study describes the development of an enzyme-linked immunosorbent assay capable of quantifying serum antibody of all four canine IgG subclasses. A panel of subclass-restricted and subclass-specific monoclonal antibodies was used to measure IgG subclasses in the serum of healthy dogs, as well as in dogs with a range of clinical diseases. The subclasses have been redefined as IgG1, IgG2, IgG3 and IgG4 based on a comparison with the relative concentration and electrophoretic mobilities of human IgG subclasses. In serum samples from healthy dogs, the concentration of IgG1 (mean, 8.17 +/- 0.95 mg ml-1) and IgG2 (mean, 8.15 +/- 3.16 mg ml-1) were very similar and considerably higher than the levels of IgG3 (mean, 0.36 +/- 0.43 mg ml-1) and IgG4 (mean, 0.95 +/- 0.45 mg ml-1). There was no apparent difference in the level of subclasses between the different breeds comprising this normal population. Sera from dogs with a range of immune-mediated or inflammatory diseases all had markedly elevated levels of IgG2 (more than 13 mg ml-1), but IgG1 decreased (less than 4 mg ml-1) to levels below the normal range.     

Abnormal mucosal glycoprotein synthesis in inflammatory bowel diseases is not related to cigarette smoking

Patients with ulcerative colitis are usually non- or ex-smokers in contrast to Crohn's disease where smoking is common. Abnormalities of quantity and quality of intestinal mucus have been postulated in the pathogenesis of these diseases. It is possible that smoking habit may exert its effects via changes in mucus in inflammatory bowel disease. We have therefore studied incorporation of N-acetylglucosamine into synthesized colonic mucin in explants from 85 controls with normal colonoscopic appearances and histology, including 27 smokers and 58 nonsmokers, 36 patients with ulcerative colitis and 19 with ileocolonic Crohn's disease over 24 h in tissue culture. Incorporation of N-acetylglucosamine into normal explants was 31.3 +/- (SD) 7.1 dpm/microgram biopsy protein, incorporation was increased in patients with active Crohn's disease (mean 41.2 +/- (SD) 10.4 dpm/microgram biopsy protein, p = 0.003), decreased in inactive ulcerative colitis (mean 24.1 +/- 7.8 dpm/microgram biopsy protein, p = 0.0006) but normal in active ulcerative colitis (mean 35.0 +/- 13.8 dpm/microgram biopsy protein, p = 0.44). No significant relationship was found between cigarette smoking habits and mucus synthesis in controls with normal mucosa (nonsmokers, n = 58, mean 31.0 +/- (SD) 7.52 dpm/microgram biopsy protein; smokers, n = 27, mean 31.8 +/- (SD) 6.1 dpm/microgram biopsy protein, p = 0.9). This study shows that mucus glycoprotein synthesis is reduced in inactive ulcerative colitis, rising to normal levels in active disease and that synthesis is increased in Crohn's disease. There is no effect of smoking on mucus synthesis by control biopsies suggesting that the differences seen in inflammatory bowel disease are not related to cigarette smoking.     

Human papillomavirus-specific serologic response in vulvar neoplasia

We describe a case of positional dyspnea due to compression of the tracheobronchial tree by an extensive thoracic aneurysm. In a 77-year-old woman with long-standing systemic hypertension, intermittent anterior chest pain gradually developed over several years. She had no history of asthma or thoracic trauma. She was admitted to our hospital because of sudden, severe shortness of breath. The breathlessness was markedly worse when she lay on her back or on her right side. On physical examination, she was in acute respiratory distress with cyanosis, severe hypertension (180/110 mmHg), tachycardia, and inspiratory stridor. A chest X-ray film showed loss of volume and nearly complete radiopacity of the left hemithorax. Arterial blood gas analysis revealed an arterial oxygen partial pressure of 54.8 mmHg, a carbon dioxide partial pressure of 39.8 mmHg, and an oxygen saturation of 84.5 percent on room air. Computed tomographic examination of the thorax showed dilation of the aortic arch and descending aorta, and marked compression of the trachea and the left main bronchus. Examination with a fiberoptic bronchoscope revealed extrinsic compression of the trachea just proximal to the carina. The patient's symptoms stabilized. However, she did not undergo surgery because of her age and because of the size of the aneurysm. She died due to rupture of the aneurysm.     

The partial testing design: a less costly way to test equivalence for sensitivity and specificity

We propose a new, less costly, design to test the equivalence of digital versus analogue mammography in terms of sensitivity and specificity. Because breast cancer is a rare event among asymptomatic women, the sample size for testing equivalence of sensitivity is larger than that for testing equivalence of specificity. Hence calculations of sample size are based on sensitivity. With the proposed design it is possible to achieve the same power as a completely paired design by increasing the number of less costly analogue mammograms and not giving the more expensive digital mammograms to some randomly selected subjects who are negative on the analogue mammogram. The key idea is that subjects who are negative on the analogue mammogram are unlikely to have cancer and hence contribute less information for estimating sensitivity than subjects who are positive on the analogue mammogram. To ascertain disease state among subjects not biopsied, we propose another analogue mammogram at a later time determined by a natural history model. The design differs from a double sampling design because it compares two imperfect tests instead of combining information from a perfect and imperfect test.     

Vitamin D and calcium in the prevention of corticosteroid induced osteoporosis: a 3 year followup

OBJECTIVE: To determine the efficacy and safety of vitamin D 50,000 units/week and calcium 1,000 mg/day in the prevention of corticosteroid induced osteoporosis. METHODS: A minimized double blind, placebo controlled trial in corticosteroid treated subjects in a tertiary care university affiliated hospital. The sample was 62 subjects with polymyalgia rheumatica, temporal arteritis, asthma, vasculitis, or systemic lupus erythematosus. The primary outcome measure was the percentage change in bone mineral density (BMD) of the lumbar spine in the 2 treatment groups from baseline to 36 mo followup. RESULTS: BMD of the lumbar spine in the vitamin D and calcium treated group decreased by a mean (SD) of 2.6% (4.1%) at 12 mo, 3.7% (4.5%) at 24 mo, and 2.2% (5.8%) at 36 mo. In the placebo group there was a decrease of 4.1% (4.1%) at 12 mo, 3.8% (5.6%) at 24 mo, and 1.5% (8.8%) at 36 mo. The observed differences between groups were not statistically significant. The difference at 36 mo was-0.693% (95% CI -5.34, 3.95). CONCLUSION: Vitamin D and calcium may help prevent the early loss of bone seen in the lumbar spine as measured by densitometry of the lumbar spine. Longterm vitamin D and calcium in those undergoing extended therapy with corticosteroids does not appear to be beneficial.     

Composite finger metacarpophalangeal joint reconstruction in combined second and third free toe-to-hand transfers

Composite metacarpophalangeal joint reconstruction using the toe proximal phalanx and the metacarpal head, with a capsular repair, is an option during free toe transfer procedures for absent fingers. Eleven composite metacarpophalangeal finger joint reconstructions were performed in four patients concurrent with combined second and third toe-to-hand transfer. The average follow-up period was 5 years. Postoperative assessment included range of motion, stability, radiographic changes, and pain. The average range of motion was 52 degrees, average ulnar stress deviation was 14 degrees. No patients complained of pain. Composite metacarpophalangeal joint reconstruction should be considered in free toe-to-hand procedures when metacarpal head articular cartilage is preserved.