Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization.     

Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells

Autosomal-dominant polycystic kidney disease is a multiorgan disease and its vascular manifestations are common and life-threatening. Despite this, little is known about their pathogenesis. Somatic mutations to the normal PKD allele in cystic epithelia and cyst development associated with the unstable Pkd2(WS25) allele suggest a two-hit model of cystogenesis. However, it is unclear if this model can account for the cardiovascular pathology or if haploinsufficiency alone is disease-associated. In the present study, we found a decreased polycystin-2 (PC2, protein encoded by Pkd2 gene) expression in Pkd2( +/-) vessels, roughly half the wild-type level, and an enhanced level of intracranial vascular abnormalities in Pkd2 (+/-) mice when induced to develop hypertension. Consistent with these observations, freshly dissociated Pkd2 (+/-) vascular smooth muscle cells have significantly altered intracellular Ca(2+) homeostasis. The resting [Ca(2+)](i) is 17.1% lower in Pkd2 (+/-) compared with wild-type cells (P=0.0003) and the total sarcoplasmic reticulum Ca(2+) store (emptied by caffeine plus thapsigargin) is decreased (P<0.0001). The store operated Ca(2+) (SOC) channel activity is also decreased in Pkd2 (+/-) cells (P=0.008). These results indicate that inactivation of just one Pkd2 allele is sufficient to significantly alter intracellular Ca(2+) homeostasis, and that PC2 is necessary to maintain normal SOC activity and the SR Ca(2+) store in VSMCs. Based on these findings, and the fact that [Ca(2+)](i) signaling is essential to the regulation of contraction, production and secretion of extracellular matrix, cellular proliferation and apoptosis, we propose that the abnormal intracellular Ca(2+) regulation associated with Pkd2 haploinsufficiency is directly related to the vascular phenotype.     

Inflammatory Responses in Blood Samples of Human Immunodeficiency Virus-Infected Patients with Pulmonary Infections

We analyzed the characteristics of the inflammatory response occurring in blood during pulmonary infections in human immunodeficiency virus (HIV)-infected patients. A prospective study of consecutive hospital admissions of HIV-infected patients with new-onset radiologic pulmonary infiltrates was carried out in a tertiary university hospital from April 1998 to May 2001. Plasma cyclic AMP receptor protein (CRP), interleukin 1β (IL-1β), IL-6, IL-8, IL-10, and tumor necrosis factor alpha (TNF-α) levels were determined at the time of admission and 4, 5, and 6 days later. Patients were included in a protocol addressed to study etiology and outcome of disease. A total of 249 episodes of infection were included, with the main diagnoses being bacterial pneumonia (BP) (118 episodes), Pneumocystis carinii pneumonia (PCP) (41 episodes), and mycobacteriosis (36 episodes). For these three patient groups, at the time of admission the median CRP and cytokine levels were as follows: CRP, 10.2, 3.8 and 5 mg/dl, respectively (P = 0.0001); IL-8, 19, 3, and 2.9 pg/ml (P = 0.045); and TNF-α, 46.4, 44, and 75 pg/ml, respectively (P = 0.029). There were no significant differences in levels of IL-1β, IL-6, or IL-10 among the patient groups. A total of 23 patients died. At the time of admission, HIV-infected patients with BP had higher plasma CRP and IL-8 levels than did PCP and mycobacteriosis patients. TNF-α levels were higher in patients with mycobacteriosis. An elevated IL-8 level (>61 pg/ml) at the time of admission was an independent factor associated with higher mortality (odds ratio, 12; 95% confidence interval, 1.2 to 235.5).     

Correlation between in vitro and in vivo antifungal activities in experimental fluconazole-resistant oropharyngeal and esophageal candidiasis

Oropharyngeal and esophageal candidiasis (OPEC) is a frequent opportunistic mycosis in immunocompromised patients. Azole-resistant OPEC is a refractory form of this infection occurring particularly in human immunodeficiency virus (HIV)-infected patients. The procedures developed by the Antifungal Subcommittee of the National Committee for Clinical Laboratory Standards (NCCLS) are an important advance in standardization of in vitro antifungal susceptibility methodology. In order to further understand the relationship between NCCLS methodology and antifungal therapeutic response, we studied the potential correlation between in vitro susceptibility to fluconazole and in vivo response in a rabbit model of fluconazole-resistant OPEC. MICs of fluconazole were determined by NCCLS methods. Three fluconazole-susceptible (FS) (MIC, /=64 microgram/ml) isolates of Candida albicans from prospectively monitored HIV-infected children with OPEC were studied. FR isolates were recovered from children with severe OPEC refractory to fluconazole, and FS isolates were recovered from those with mucosal candidiasis responsive to fluconazole. Fluconazole at 2 mg/kg of body weight/day was administered to infected animals for 7 days. The concentrations of fluconazole in plasma were maintained above the MICs for FS isolates throughout the dosing interval. Fluconazole concentrations in the esophagus were greater than or equal to those in plasma. Rabbits infected with FS isolates and treated with fluconazole had significant reductions in oral mucosal quantitative cultures (P < 0.001) and tissue burden of C. albicans in tongue, soft palate, and esophagus (P < 0.001). In comparison, rabbits infected with FR isolates were unresponsive to fluconazole and had no reduction in oral mucosal quantitative cultures or tissue burden of C. albicans versus untreated controls. We conclude that there is a strong correlation between in vitro fluconazole susceptibility by NCCLS methods and in vivo response to fluconazole therapy of OPEC due to C. albicans.     

Controlled administration of penicillin to patients with a positive history but negative skin and specific serum IgE tests

BACKGROUND: Although subjects with a positive history of immediate allergy to penicillin and negative skin test are traditionally considered to tolerate penicillin, current evidence indicates that they may develop an immediate reaction despite negative skin and serum specific IgE tests. It is thought that these patients require additional tests to confirm the diagnosis. OBJECTIVE: To assess in a large group of patients with a history of immediate allergy to penicillins but with both skin test and CAP-FEIA-negative to classical and side chain penicillin determinants, the role of controlled administration of betalactams as a diagnostic test. METHODS: A group of 330 patients with a history of immediate allergic reactions to penicillins was studied by two evaluators from the same allergy unit using the following protocol: skin tests with major and minor determinants of benzylpenicillin (benzylpenicilloyl-poly l-lysine and minor determinant mixture), amoxicillin and ampicillin, and determination of specific IgE antibodies to penicillins, by CAP-FEIA, in serum. If both tests proved negative, a controlled administration of the drug was then carried out. RESULTS: A total of 89 (27%) patients were skin test and CAP-FEIA-negative and therefore required controlled administration of the drug. Of these, 49 developed an immediate response and were therefore considered allergic, and the remainder had good tolerance after administration of both benzylpenicillin and amoxicillin. The clinical characteristics of this group were similar to the other allergic patients who were skin test or CAP-FEIA-positive, except that they were younger (P < 0.01). Twenty-two (45%) developed a response to benzylpenicillin and 27 (55%) had a selective response to amoxicillin. Although all reactions appeared within 1 h, a positive correlation was found between the dose inducing the response and the time elapsed from drug administration, for both benzylpenicillin and amoxicillin (P < 0.001). CONCLUSION: These data indicate that an important number of subjects are not correctly identified if only skin tests and/or CAP-FEIA are used and that this is particularly relevant for side chain-specific reactions and younger subjects. This suggests that new diagnostic tests are required so as to limit the use of controlled administration.     

TonB is required for intracellular growth and virulence of Shigella dysenteriae

To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.     

Clinical evaluation of a commercial ligase-based gene amplification method for detection ofMycobacterium tuberculosis

The purpose of this study was to evaluate the clinical usefulness of a commercial ligase-based gene amplification method (LCxMycobacterium tuberculosis test; Abbott Laboratories, USA) for detection ofMycobacterium tuberculosis. The tuberculosis infection rate among clinical samples was 10.6%. The sensitivity, specificity, and positive and negative predictive values were 23.5%, 100%, 100%, and 91.7%, respectively, with the fluorochrome auramine stain; 32.4%, 100%, 100%, and 92.6%, respectively, with culture; and 76.5%, 95.8%, 68.4% and 97.2%, respectively, with the gene amplification method. When only samples from patients without current or previous treatment were studied, the sensitivity was 36.4% with the auramine stain, 63.6% with culture, and 100% with the gene amplification assay. The mean treatment time for culture-negative and assay-negative samples was greater than that of culture-negative and assay-positive samples. The LCxMycobacterium tuberculosis test is a sensitive method for detection and identification ofMycobacterium tuberculosis. It produces few false-positive results. However, as it can remain positive after the culture becomes negative, it is not recommended for evaluation of treatment efficiency.     

Bioactivity of prolactin isoforms: lactation and recovery of menses in nursing women

To assess whether plasma prolactin (PRL) characteristics relate to lactogenesis and absence or presence of menstrual cycles, we measured bioactive PRL (BIO-PRL) using the Nb2 assay, immunoreactive PRL (IR-PRL) by radio-immunoassay, calculated equations describing the BIO-PRL-IR-PRL relationship and separated charged PRL isoforms (by chromatofocusing) in five amenorrhoeic and five cycling nursing women at 6 months postpartum and in 10 cycling non-nursing women. Plasma samples were drawn before and 30 min after a suckling episode at 0800, 1600 and 2400 h in nursing women and at the same hours in non-nursing women. BIO-PRL and IR-PRL concentrations were highest in amenorrhoeic nursing women, intermediate in cycling nursing women and lowest in cycling non-nursing women. The BIO-PRL-IR-PRL relationship shows that a given amount of IR-PRL corresponds to equivalent amounts of BIO-PRL in cycling nursing and cycling non-nursing women, and to a larger extent in amenorrhoeic nursing women. IR-PRL was present in plasma as several charge isoforms. Bioactive isoforms eluting at pH 6.0-5.1 were found in amenorrhoeic and cycling nursing women, reaching similar concentrations after suckling. Bioactive isoforms eluting at pH 7.0-6.1 were found only in amenorrhoeic nursing women. We speculate that isoforms eluting at pH 6.0-5.1 may play a role in lactation and isoforms eluting at pH 7.0-6.1, in lactational amenorrhoea.