A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila

Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in rainbow trout. A gene encoding an elastolytic activity, ahyB, was cloned from Aeromonas hydrophila AG2 into pUC18 and expressed in Escherichia coli and in the nonproteolytic species Aeromonas salmonicida subsp. masoucida. Nucleotide sequence analysis of the ahyB gene revealed an open reading frame of 1,764 nucleotides with coding capacity for a 588-amino-acid protein with a molecular weight of 62,728. The first 13 N-terminal amino acids of the purified protease completely match those deduced from DNA sequence starting at AAG (Lys-184). This finding indicated that AhyB is synthesized as a preproprotein with a 19-amino-acid signal peptide, a 164-amino-acid N-terminal propeptide, and a 405-amino-acid intermediate which is further processed into a mature protease and a C-terminal propeptide. The protease hydrolyzed casein and elastin and showed a high sequence similarity to other metalloproteases, especially with the mature form of the Pseudomonas aeruginosa elastase (52% identity), Helicobacter pylori zinc metalloprotease (61% identity), or proteases from several species of Vibrio (52 to 53% identity). The gene ahyB was insertionally inactivated, and the construct was used to create an isogenic ahyB mutant of A. hydrophila. These first reports of a defined mutation in an extracellular protease of A. hydrophila demonstrate an important role in pathogenesis.     

Folate status and a new repletion cut-off value in a group of healthy Majorcan women

BACKGROUND AND AIMS: Folate status is complex, complicated to assess and we lack consensus for laboratory determination.Total plasma homocysteine (Hcy) is a sensitive marker of folate status. The aim of this study was choosing a plasma total homocysteine (tHcy) measurement method and folate repletion level; assess the mutation C677T frequency for methylenetetrahydrofolate reductase (MTHFR) and the prevalence of hyperhomocysteinemia in healthy Majorcan women. METHODS: The measurement methods were compared using 219 women. Folate status assessment was determined by plasma tHcy, serum and erythrocyte folate and C677T for MTHFR in 342 healthy women. The mutation frequency is established with 146 of them.RESULTS: The measurement method comparison is summarized by Y = 1.013 (IC 95% 0.959, 1.069) X +0.829 (IC 95% 0.485, 1.170). The folate repletion level was set at Hcy < or = 8.6 micromol/l. Women (67.1%) are heterozygotic and do not carry the mutation. Homozygotic frequency is 18.5%, significantly higher in women under 40 years (P = 0.033). Hyperhomocysteinemia prevalence is 19.3%, and 51.7% in the younger group. CONCLUSIONS: Hcy determination by polarized fluorescence immunoassay reflects folate status. Levels < or = 8.6 micromol/l suggest folate repletion, which is less frequent in homozygotes. Population frequency for homozygotic-TT alleles for C677T-MTHFR reductase and hyperhomocysteinemia is significantly higher in women under 40 years.     

Molecular Characterization of the Aeromonas hydrophila aroA Gene and Potential Use of an Auxotrophic aroA Mutant as a Live Attenuated Vaccine

The aroA gene of Aeromonas hydrophila SO2/2, encoding 5-enolpyruvylshikimate 3-phosphate synthase, was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined. The nucleotide sequence of the A. hydrophila aroA gene encoded a protein of 440 amino acids which showed a high degree of homology to other bacterial AroA proteins. To obtain an effective attenuated live vaccine against A. hydrophila infections in fish, the aroA gene was inactivated by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila AG2 by means of the suicide vector pSUP202. The A. hydrophila mutant AG2 aroA::Ka^r was highly attenuated when inoculated intraperitoneally into a rainbow trout, with a 50% lethal dose of >2 × 10⁸ CFU. The mutants were not recoverable from the internal organs after 48 h postinoculation. Immunohistochemical studies demonstrated that immunopositive materials, but not whole cells, reacting with a polyclonal antiserum against A. hydrophila were present in the kidney and spleen 9 days postinjection. Vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection against the wild-type strain of A. hydrophila.     

Modulation of murine lymphocyte functions by sulfated cholecystokinin octapeptide

The effects in vitro of the sulfated octapeptide form of cholecystokinin (CCK-8) at concentrations ranging from 10(-13) M to 10(-6) M on several functions of murine lymphocytes were studied, i.e. adherence to substrate, mobility (spontaneous and directed by chemical gradient or chemotaxis) and spontaneous and phytohemagglutinin (PHA)-mediated proliferation. Lymphocytes were obtained from peritoneal suspension as well as from axillary nodes, spleen and thymus of BALB/c mice. CCK-8, at concentrations from 10(-10) M to 10(-8) M, significantly inhibited the mobility capacity and the PHA-induced proliferation and increased the adherence and the spontaneous proliferation of lymphocytes. A dose-response relationship was observed, with a maximum effect on lymphocyte functions at 10(-10) M. In addition, CCK-8 induced a significant decrease in membrane and cytosol protein kinase C (PKC) activity in murine lymphocytes, as well as an increase of intracellular cyclic AMP levels. These results suggest that CCK-8 is a negative modulator of two important lymphocyte functions in the immune response, i.e. mobility and mitogen-induced proliferation, and that the PKC activity inhibition and cAMP increase could be the mechanisms through which CCK inhibits these lymphocyte activities.     

Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp

The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific.     

Application of the POSSUM System in Bariatric Surgery

Background: The POSSUM system has been devised for physiologic and operative scoring. The scoring system produced assessment for morbidity and mortality rates, which did not significantly differ from observed rates. The authors have applied this system to bariatric surgery. Patients and Methods: 20 patients were scored by the POSSUM system. All underwent elective bariatric surgery during 1997. All patients were scored at the time of surgery with the physiologic score (FIS) and at discharge with the operative severity score (IQ). The FIS score included age; cardiac signs; chest radiograph; respiratory history; blood pressure; pulse; Glasgow coma score; determinations of hemoglobin, leukocyte, urea, sodium, and potassium levels; and electrocardiogram. The IQ score included multiple procedures, total blood loss, peritoneal soiling, presence of malignancy, and mode of surgery. Results: The mean POSSUM score was 23.9. The mean FIS was 13.95 (12-22), and the mean IQ was 9.4 (7-16). The distribution of patients was performed for BMI. The group with BMI 35-45 (n = 4 patients) had a mean POSSUM score of 22.75, a mean FIS of 13.75, and a mean IQ of 9.0. The group with BMI >45 (n = 16 patients) had a mean POSSUM score of 24.18, a mean FIS of 14.62, and a mean IQ of 9.5. The morbidities were gastric fistula with peritonitis and deep venous thrombosis. The two complications had similar POSSUM scores with different BMIs. No mortality was observed. Conclusions: According to this experience, the POSSUM scoring system appears to provide an indicator of minor risk of morbidity and mortality in bariatric surgery with vertical banded gastroplasty.     

Significant Improvement in Extensive Lichen Sclerosus with Tacrolimus Ointment and PUVA

Lichen sclerosus is an uncommon, chronic inflammatory skin disorder of unknown origin. It is clinically characterized by sclerotic, whitish, atrophic-type lesions. The most frequent site of the lesions is generally the genital region, with about 15-20% having additional extragenital involvement. We present the case of a 62-year-old woman with a very extensive extragenital lichen sclerosus who showed great clinical and subjective improvement with 0.1% tacrolimus ointment and psoralen plus UVA (PUVA), without any topical or systemic adverse effects. Combined treatment of 0.1% tacrolimus ointment and PUVA may be a good option in extensive cases of lichen sclerosus or when other treatment options have failed, and has a good tolerability and safety profile.     

Beta2-microglobulin concentrations in cerebrospinal fluid correlate with neuroimaging findings in newborns with symptomatic congenital cytomegalovirus infection

Overview In newborns with symptomatic congenital cytomegalovirus (CMV) infection, neuroimaging is the best available predictor of neurodevelopmental outcome. Cerebrospinal fluid (CSF) findings in congenital CMV infection have seldom been described. Neonates with central nervous system infections present high CSF Beta₂-microglobulin (β₂-m) levels.Objectives The objectives of this study were: (1) to determine whether CSF β₂-m is increased in newborns with symptomatic congenital CMV infection, and (2) to examine its correlation with neuroimaging findings.Materials and methods Fourteen newborns with symptomatic congenital CMV infection admitted to La Paz Hospital from 1990 through 2004 underwent determination of CSF β₂-m. Ninety-three newborns, constituting the comparison group, underwent CSF β₂-m determination as part of a sepsis or meningo/encephalitis work-up, and at discharge had sterile cultures and normal neurological status. Neuroimaging findings were scored according to a semiquantitative system: (0) no abnormalities; (1) single punctate periventricular (PV) calcification and/or hyperechogenic areas in the thalamus and basal ganglia; (2) multiple discrete PV calcifications and/or ventriculomegaly; and (3) extensive PV calcifications and/or brain atrophy.Discussion and conclusion CSF β₂-m was increased in newborns with CMV infection (median 6.21 mg/L) compared with controls (1.68 mg/L) (P<.001). β₂-m showed a correlation with neuroimaging scores (r _s=0.753, P=.002). β₂-m was higher in patients who scored 2–3 (12.83 mg/L) than in patients who scored 0–1 (5.52 mg/L) (P=.028). CSF β₂-m is increased in newborns with symptomatic congenital CMV infection and correlates with neuroimaging abnormalities. β₂-m appears to be an indicator of the severity of brain involvement in congenital CMV infection.