Molecular subtyping of Vibrio cholerae O1 and O139 by pulsed-field gel electrophoresis in Hong Kong: correlation with epidemiological events from 1994 to 2002

Two hundred twenty isolates of Vibrio cholerae O1 and O139 collected from 1994 to 2002 in Hong Kong were analyzed by pulsed-field gel electrophoresis (PFGE). Chromosomal DNAs from all V. cholerae isolates in agarose plugs were digested with the restriction enzyme NotI, resulting in 20 to 27 bands. Sixty distinctive PFGE patterns in the range of 10 to 300 kb were noted among 213 isolates typeable by PFGE. By comparing the common PFGE patterns obtained from four well-defined outbreaks of V. cholerae O1 and O139 with those obtained from other, epidemiologically unrelated isolates during the study period, indistinguishable and similar PFGE patterns were identified, indicating their close relatedness, in agreement with the results of epidemiological investigations. Heterogeneous PFGE patterns (with four to six banding differences), however, were identified among strains that were imported from other parts of Asia, including Indonesia, India, and Pakistan. Correlations with epidemiological information further support the usefulness of PFGE as an epidemiological tool in laboratory investigations of suspected outbreaks. Standardization of PFGE methodology will allow international comparison of fingerprint patterns and will form the basis of a laboratory network for tracking V. cholerae.     

Association of the polymorphism in manganese superoxide dismutase gene with diabetic retinopathy in Chinese type 2 diabetic patients

OBJECTIVE: To investigate the association of the polymorphism in manganese superoxide dismutase (Mn-SOD) gene in Chinese type 2 diabetic patients with diabetic retinopathy. METHODS: The Ala(-9)Val polymorphism of the Mn-SOD gene was determined by polymerase chain reaction and direct sequencing in 198 normal control subjects and 264 patients with type 2 diabetes mellitus, among them there were 139 non-diabetic retinopathy (NDR) subjects and 125 subjects with diabetic retinopathy (DR). RESULTS: There was no statistic difference in the frequencies of VV genotype and V allele between the type 2 diabetic group and the control group. However, the frequencies of VV genotype and V allele were significantly higher in the DR group than that in the NDR group (chi-square (2)=5.015, P=0.025?chi-square (2)=10.253, P=0.001),but there was no statistic difference in the NDR group compared with the control group (P > 0.05). The presence of V allele was shown to be associated with diabetic retinopathy (OR=1.96, 95%CI: 1.29-2.97). Furthermore, the subjects carrying the VV genotype had lower serum Mn-SOD level (P=0.025) and had a tendency of higher total serum SOD activity, but this tendency had no statistic significance. CONCLUSION: The Ala(-9)Val polymorphism in the Mn-SOD gene may not be related to the etiology of type 2 diabetes, but it seems to contribute to the development of diabetic retinopathy in Chinese type 2 diabetic patients.     

Infection of the Laboratory Mouse with the Intracellular Pathogen Ehrlichia chaffeensis

To determine the basis of susceptibility and resistance to human monocytic ehrlichiosis (HME), immunocompetent and immunocompromised mice were infected with Ehrlichia chaffeensis and bacterial loads were measured by PCR and by immunohistochemistry. Immunocompetent (C.B-17 and C57BL/6) mice cleared the bacteria within 10 days, but immunocompromised SCID and SCID/BEIGE mice developed persistent infection in the spleen, liver, peritoneal cavity, brain, lung, and bone marrow and became moribund within 24 days. Both immunocompromised strains lack T and B lymphocytes, but the SCID/BEIGE strain is also deficient in natural killer (NK) cell function. During advanced stages of disease, the infections were associated with wasting, splenomegaly, lymphadenopathy, liver granulomas and necroses, intravascular coagulation, and granulomatous inflammation. Histochemical and immunohistochemical localization studies confirmed the presence of bacteria in tissues, and viable bacteria were cultured from infected animals. The data reveal that T and/or B cells play an essential role during resistance of immunocompetent mice to infection with E. chaffeensis and demonstrate the utility of immunocompromised mice as an experimental model for the study of HME.     

Influence of zinc on Pseudomonas aeruginosa susceptibilities to imipenem.

Serial dilution susceptibility testing of imipenem against 59 clinical isolates of Pseudomonas aeruginosa, conducted simultaneously on single lots of Difco and BBL Mueller-Hinton agar (MHA), resulted in MICs for 90% of strains tested of 8 and 16 micrograms/ml, respectively. MICs for Escherichia coli, Klebsiella pneumoniae, and Pseudomonas spp. were also higher on BBL MHA. Quantification of the cation content of the two MHAs by atomic absorption spectroscopy demonstrated that the zinc concentration in BBL MHA was 15 times greater than that measured in Difco MHA (2.61 and 0.17 micrograms/ml, respectively). Concentrations of calcium, magnesium, iron, manganese, and copper in the two agars were similar. Addition of zinc to Difco MHA resulted in increases in MICs of imipenem for P. aeruginosa but not in the MICs of ceftazidime or cefpirome for P. aeruginosa (P < 0.01). A lesser zinc effect was seen on the activity of imipenem against E. coli, K. pneumoniae, and Pseudomonas spp. The activities of ceftazidime and cefpirome were similar on both MHAs when tested against all gram-negative organisms in this study. Thus, the effect of zinc in MHA was clearly demonstrated by a significant increase in the MICs of imipenem for P. aeruginosa, and, to a lesser extent, for other gram-negative bacilli.     

Role of glutathione metabolism of Treponema denticola in bacterial growth and virulence expression

Hydrogen sulfide (H(2)S) is a major metabolic end product detected in deep periodontal pockets that is produced by resident periodontopathic microbiota associated with the progression of periodontitis. Treponema denticola, a member of the subgingival biofilm at disease sites, produces cystalysin, an enzyme that catabolizes cysteine, releasing H(2)S. The metabolic pathway leading to H(2)S formation in periodontal pockets has not been determined. We used a variety of thiol compounds as substrates for T. denticola to produce H(2)S. Our results indicate that glutathione, a readily available thiol source in periodontal pockets, is a suitable substrate for H(2)S production by this microorganism. In addition to H(2)S, glutamate, glycine, ammonia, and pyruvate were metabolic end products of metabolism of glutathione. Cysteinyl glycine (Cys-Gly) was also catabolized by the bacteria, yielding glycine, H(2)S, ammonia, and pyruvate. However, purified cystalysin could not catalyze glutathione and Cys-Gly degradation in vitro. Moreover, the enzymatic activity(ies) in T. denticola responsible for glutathione breakdown was inactivated by trypsin or proteinase K, by heating (56 degrees C) and freezing (-20 degrees C), by sonication, and by exposure to N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). These treatments had no effect on degradation of cysteine by the purified enzyme. In this study we delineated an enzymatic pathway for glutathione metabolism in the oral spirochete T. denticola; our results suggest that glutathione metabolism plays a role in bacterial nutrition and potential virulence expression.     

Distinct roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung injury

Acute lung injury (ALI) induced by lipopolysaccharide (LPS) is a major cause of mortality among humans. ALI is characterized by microvascular protein leakage, neutrophil influx, and expression of proinflammatory mediators, followed by severe lung damage. LPS binding to its receptors is the crucial step in the causation of these multistep events. LPS binding and signaling involves CD14 and Toll-like receptor 4 (TLR4). However, the relative contributions of CD14 and TLR4 in the induction of ALI and their therapeutic potentials are not clear in vivo. Therefore, the aim of the present study was to compare the roles of CD14 and TLR4 in LPS-induced ALI to determine which of these molecules is the more critical target for attenuating ALI in a mouse model. Our results show that CD14 and TLR4 are necessary for low-dose (300-microg/ml) LPS-induced microvascular leakage, NF-kappaB activation, neutrophil influx, cytokine and chemokine (KC, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-6) expression, and subsequent lung damage. On the other hand, when a 10-fold-higher dose of LPS (3 mg/ml) was used, these responses were only partially dependent on CD14 and they were totally dependent on TLR4. The CD14-independent LPS response was dependent on CD11b. A TLR4 blocking antibody abolished microvascular leakage, neutrophil accumulation, cytokine responses, and lung pathology with a low dose of LPS but only attenuated the responses with a high dose of LPS. These data are the first to demonstrate that LPS-induced CD14-dependent and -independent (CD11b-dependent) signaling pathways in the lung are entirely dependent on TLR4 and that blocking TLR4 might be beneficial in lung diseases caused by LPS from gram-negative pathogens.     

Genotyping of a homogeneous group of Yersinia pestis strains isolated in the United States

Yersinia pestis, the causative agent of deadly plague, is considered a reemerging infectious disease and a significant biological terrorism threat. The present project focused on epidemiological investigation of the genetic variability of well-documented strains of Y. pestis from the United States by pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis with insertion sequences IS100 and IS285 as probes. We examined 37 U.S. Y. pestis strains and isolates of a single ribotype, ribotype B, recovered between 1939 and 1998 from patients, animals, and fleas. Our results showed that all isolates had similar PFGE patterns, but minor differences such as missing, additional, and shifted bands were found among almost all strains if they came from different parent strains. The 37 strains and isolates were divided into 26 PFGE types. RFLP analysis with IS100 as a probe divided these strains and isolates into 16 types, with 43% belonging to IS100 type 1. Typing with IS285 as a probe was less specific and led to only four RFLP types, with 81% belonging to type 1. Similarity analysis with BioNumerics software showed that all strains shared >or=80, 86, and 91% similarities on dendrograms prepared from digitized PFGE, IS100 RFLP analysis, and IS285 RFLP analysis images, respectively. Our results demonstrate that PFGE offers an increased ability to discriminate between strains (Simpson's index of diversity, 0.98) and therefore can significantly improve epidemiological studies related to the origin of new plague isolates.     

Increased expression of intercellular adhesion molecule-1 (ICAM-1) in a murine model of pulmonary eosinophilia and high IgE level.

T lymphocytes and eosinophils are probably involved in the pathogenesis of allergic bronchopulmonary aspergillosis (ABPA), a disease characterized by pulmonary eosinophilia and high serum and lavage IgE levels. We recently developed a murine model of ABPA. To investigate the mechanisms of T lymphocyte and eosinophil recruitment to the lung in this disease, we examined the expression of ICAM-1 in the lung tissue of mouse challenged with Aspergillus fumigatus (Af) antigen. C57B1/6 mice were intranasally exposed to Af (Af group) or saline (control group) three times a week for 1, 2 or 3 weeks. On days 4, 7, 14 and 21, mice were killed and lung tissue was fixed in acetone and embedded in glycol methacrylate. Serial 2-microns sections were stained with chromotrope 2R and MoAbs against ICAM-1, CD11a/CD18 (LFA-1) and CD3. Af-challenged mice presented significant increases in eosinophil, T lymphocyte and LFA-1-positive cell count and up-regulated expression of ICAM-1 in the lung tissue at all the time points examined. ICAM-1 expression intensity correlated with the number of T lymphocytes (r = 0.59, P < 0.01), LFA-1-positive cells (r = 0.68, P < 0.001), but not of eosinophils (r = -0.24, P > 0.05). These findings suggest that up-regulation of ICAM-1 expression is involved in the inflammatory process of this murine model of ABPA, and that this up-regulation may be more relevant to the the T lymphocyte accumulation in the lung.     

Autoantibody response to the Ro/La particle may predict outcome in neonatal lupus erythematosus.

This study was undertaken to determine the role of antibodies against both recombinant Ro (r-Ro) and La (r-La) proteins and polypeptides derived from the recombinant La protein in predicting fetal and neonatal outcome in children at risk to develop neonatal lupus erythematosus (NLE). All sera were obtained in the perinatal period and quantitative ELISA assays were used. We collected 41 maternal sera within 2 months of delivery of a child with NLE (21 with congenital heart disease block (CHB) and 20 with dermatologic NLE) and 19 sera from anti-Ro and/or anti-La antibody-positive mothers with systemic lupus erythematosus (SLE) who delivered a child without NLE. All sera were tested for anti-r-La and anti-r-Ro antibodies by ELISA, and most sera were tested for antibodies directed against La polypeptides by immunoblot. We found significantly higher anti-r-La antibody levels in the sera from mothers of children with NLE compared with sera from mothers of unaffected children (0.67 +/- 0.43 versus 0.14 +/- 0.30; P < 0.0001). There was a statistically significant difference in the mean anti-r-La levels between the sera of mothers of children with CHB compared with dermatologic NLE (0.51 +/- 0.45 versus 0.83 +/- 0.37 respectively; P = 0.0091). When we examined antibodies directed against the recombinant 52-kD Ro protein, there was a statistically significant elevation of titres in the sera of mothers of NLE children (0.77 +/- 0.35) compared with non-NLE mothers (0.29 +/- 0.39; P < 0.0001). There was no difference in the r-Ro levels between mothers of children with dermatologic NLE compared with CHB (0.82 +/- 0.37 versus 0.71 +/- 0.74; P = 0.32). When we examined polypeptides derived from the recombinant La protein, the mean number of polypeptides recognized by sera from mothers of children with NLE was significantly higher than the mean number of polypeptides recognized by sera from mothers of unaffected children (5.1 +/- 0.54 versus 2.3 +/- 0.54 respectively; P < 0.001). More importantly, when we examined the individual polypeptides, we found that only sera from mothers of children with NLE and not from mothers of unaffected children recognized a polypeptide designated DD (30% versus 0%, respectively). These studies indicate that the autoantibody response to the Ro/La particle can differentiate sera from mothers of children with NLE and sera from mothers of unaffected children. Furthermore, there was a difference in the anti-La autoantibody response between mothers of children with CHB and dermatologic NLE.(ABSTRACT TRUNCATED AT 400 WORDS)