Biochemical identification of citrobacteria in the clinical laboratory.

We biochemically identified 235 Citrobacter strains to the species level on the basis of the recently proposed taxonomic changes of Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993). Citrobacter isolates were initially identified as C. koseri or as members of the C. freundii complex or C. amalonaticus group on the basis of indole production, formation of H2S, malonate utilization, and acid production from D-arabitol and adonitol. On the basis of the results of these tests, 68% of the Citrobacter strains were identified as members of the C. freundii complex, 25% were C. koseri, and 8% were members of the C. amalonaticus group. By using a 15-test system recently proposed by Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993) to help identify new species in the C. freundii complex and C. amalonaticus group, 81% of the C. freundii complex strains and 100% of the C. amalonaticus strains could be definitively assigned to one of the previously established or recently designated species or hybridization groups of the genus Citrobacter. Within the C. freundii complex, C. freundii predominated overall (37%), followed by C. youngae (24%), C. braakii (13%), and C. werkmanii (6%). Only one strain each of C. sedlakii and Citrobacter DNA group 11 was identified in this study. Among C. amalonaticus complex members, all were identified as C. amalonaticus with the singular exception of one fecal isolate of C. farmeri. C. freundii and C. koseri were the two Citrobacter species most commonly (80 of 93 [86%]) isolated from extraintestinal sources (genitourinary tract, wounds, blood).     

Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats.

We have previously reported that vitamin A deficiency resulted in a reduced IgA antibody response to cholera toxin (CT) after per-oral immunization. In the present investigation we have studied the in vivo and in vitro immune response in vitamin A-deficient rats to two parenterally applied antigens, beta-lactoglobulin (beta-LG) and picrylsulphonic acid (TNP)-Ficoll. The serum IgG and IgM antibody responses to the T-cell dependent antigen beta-LG were significantly lower in the vitamin A-deficient rats than in the pair-fed control rats. No such differences were seen with the IgG and IgM responses to the T-cell independent antigen TNP-Ficoll. However, the biliary IgA and the serum IgE antibodies against both antigens were decreased in the vitamin A-deficient rats. In vitro lymphocyte stimulation with concanavalin A (Con A) or beta-LG gave higher T-cell proliferation rates in the vitamin A-deficient than in the control rats. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) levels in supernatants from Con A-stimulated mesenteric lymph node cells were also higher in the vitamin A-deficient rats, while IL-6 levels were decreased, which is consistent with an up-regulated Th1 activity. Proliferation studies on purified accessory cells and T cells from the deficient and the control rats, mixed in different combinations, showed that the T cells, but not the accessory cells, were disturbed in the vitamin A-deficient rats. Despite the increased T-cell activity in vitro the vitamin A-deficient rats had a lower delayed-type hypersensitivity (DTH) reaction than the pair-fed control rats. In conclusion, the increased IL-2 and IFN-gamma levels may reflect an up-regulation of Th1 cell function, while the decreased IgA, IgE and IL-6 levels indicate a suppression of Th2 cells. The disturbed T-lymphocyte function is manifested in vivo as a decreased DTH reaction and suppressed antibody production, the latter possibly due to a lack of B-cell switching and proliferation factors in vitamin A-deficient rats.     

Secretory IgA and IgM antibodies to E. coli O and poliovirus type I antigens occur in amniotic fluid,meconium and saliva from newborns. A neonatal immune response without antigenic exposure: a result of anti-idiotypic induction?

Antibodies to E. coli O-antigens and poliovirus type I antigen as well as total SIgA were analysed using enzyme-linked immunosorbent assay (ELISA), in amniotic fluid and in meconium, urine and saliva from neonates. Secretory IgA and IgM antibodies to E. coli and poliovirus antigens were found in saliva as well as in most meconium samples taken during the first day of life. SIgA could be quantified in all types of samples including amniotic fluid. The finding of secretory IgA and IgM antibodies to E. coli and poliovirus type I antigens in early samples from an infant of a hypogammaglobulinaemic mother, given regular intravenous (i.v.) immunoglobulin prophylaxis, but still lacking IgA and IgM antibodies, supports a fetal origin for at least part of the secretory antibodies detected in the different samples. Since it is unlikely that the fetus has been exposed to poliovirus, which is rare in Sweden, it is hypothesized that the stimulus inducing the SIgA and IgM antibodies found in the neonate could be anti-idiotypic antibodies from the mother.     

Human leukocyte antigen-DQ8 transgenic mice: a model to examine the toxicity of aerosolized staphylococcal enterotoxin B

Staphylococcal enterotoxins (SEs) belong to a large group of bacterial exotoxins that cause severe immunopathologies, especially when delivered as an aerosol. SEs elicit the release of lethal amounts of cytokines by binding to major histocompatibility complex (MHC) class II and cross-linking susceptible T-cell receptors. Efforts to develop effective therapeutic strategies to protect against SEs delivered as an aerosol have been hampered by the lack of small animal models that consistently emulate human responses to these toxins. Here, we report that human leukocyte antigen-DQ8 (HLA-DQ8) transgenic (Tg) mice, but not littermate controls, succumbed to lethal shock induced by SEB aerosols without potentiation. Substantial amounts of perivascular edema and inflammatory infiltrates were noted in the lungs of Tg mice, similar to the pathology observed in nonhuman primates exposed by aerosol to SEB. Furthermore, the observed pathologies and lethal shock correlated with an upsurge in proinflammatory cytokine mRNA gene expression in the lungs and spleens, as well as with marked increases in the levels of proinflammatory circulating cytokines in the Tg mice. Unlike the case for littermate controls, telemetric evaluation showed significant hypothermia in Tg mice exposed to lethal doses of SEB. Taken together, these results show that this murine model will allow for the examination of therapeutics and vaccines developed specifically against SEB aerosol exposure and possibly other bacterial superantigens in the context of human MHC class II receptors.     

Permanent impairment of embryo development by hydrosalpinges

Recent reports suggest a deleterious effect of hydrosalpinges on pregnancy outcome for in-vitro fertilization (IVF) and improvement following surgical treatment. We compared the effect of hydrosalpinx on pregnancy outcome in 286 patients having 348 IVF cycles and followed the development of untransferred embryos for 7 days to determine if hydrosalpinges affect oocyte quality or embryo development. The delivery rate per retrieval was significantly lower for patients with hydrosalpinx, but was restored by surgical treatment to that of patients without hydrosalpinx. However, the implantation rate per embryo transferred and normal blastulation of untransferred embryos, which were significantly decreased in patients with hydrosalpinx, and growth arrest and degeneration of untransferred embryos, which were significantly increased compared to patients without hydrosalpinx, were not restored by surgical treatment of hydrosalpinges. We conclude that surgical treatment of hydrosalpinges decreases early pregnancy loss and improves pregnancy outcome, possibly by diminishing reversible deleterious effects exerted on the endometrium. As we have seen in our laboratory, hydrosalpinges may have a permanent negative influence on ovarian function, follicular development and oocyte quality since implantation of transferred embryos and normal blastulation of untransferred embryos remain low, and in-vitro growth arrest and degeneration remain high despite surgical treatment of hydrosalpinges.      

Impaired mucosal antibody response to cholera toxin in vitamin A-deficient rats immunized with oral cholera vaccine.

To investigate the importance of vitamin A in the ability to respond to oral antigen administration, rats were fed a vitamin A-free diet. The animals were immunized perorally three times with a mixture of cholera toxin (CT) and a commercial cholera vaccine. The total immunoglobulin A (IgA) concentration as well as the specific IgA anti-CT antibody levels in serum and bile was significantly lower in the vitamin A-deficient animals than in the paired fed controls (animals that were fed a normal commercial diet in an amount equal to the amount the deficient animals consumed), while the levels of total and specific anti-CT IgG were not affected to the same extent by the vitamin A deficiency. The number of IgA anti-CT antibody-producing cells in the mesenteric lymph nodes after immunization was also significantly lower in the vitamin A-deficient rats than in the control rats. Supplementation of the diet with retinyl palmitate restored the ability to mount an IgA antibody response to the antigen, since the level of specific IgA anti-CT antibodies in relation to the total IgA concentration was as high in the vitamin A-supplemented group as in the paired fed control group. Restricted diet intake by itself did not affect the ability to respond adequately to the antigen since there was no difference in IgA anti-CT antibody level between paired fed rats and those being fed ad libitum. Assessment of transforming growth factor beta in cell cultures revealed no difference between vitamin A-deficient and paired fed animals. In summary, vitamin A deficiency resulted in a decreased number of IgA-producing cells, decreased IgA production, and a reduced ability to respond with IgA antibodies to the oral cholera vaccine.     

Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture

There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.     

Quantification of HIV-1 p24 by a highly improved ELISA: an alternative to HIV-1 RNA based treatment monitoring in patients from Abidjan, C?te d'Ivoire.

BACKGROUND: Quantification of HIV-1 RNA remains difficult to implement in Africa. Simple and inexpensive tests for antiretroviral treatment (ART) monitoring are needed. OBJECTIVE: To evaluate an HIV-1 p24 ELISA, which combines efficient virus disruption, heat-denaturation and signal amplification, in a West African setting. STUDY DESIGN: Eighty-six HIV-1 infected patients from Abidjan, C?te d'Ivoire, were tested for p24, HIV-1 RNA, and CD4+ count at baseline, and twice within 8 months after ART initiation. RESULTS: All patients responded to ART with a minimal HIV-1 RNA drop of 0.5 log(10) at first follow-up. Forty-one (47.7%) then rebounded >0.5 log(10) or persisted above 1000 copies/mL by week 24. The predicted baseline concentration of p24 corresponding to 100,000 copies/mL of HIV-1 RNA, above which ART is recommended, was 4546 fg/mL (95% confidence interval 3148-6566). A prediction model of virologic failure, occurring after an initial response to ART, correctly classified 84% of patients using baseline p24, p24 change on therapy, and achievement of undetectable p24 as explanatory variables. The model and further bootstrap evaluation suggested a good ability to discriminate between sustained or failing virologic response to ART. CONCLUSION: HIV-1 p24 and RNA based-ART monitoring in a low-resource country dominated by HIV-1 CRF02 AG appeared comparable.     

Antibody-dependent cell-mediated cytotoxicity against Tamm-Horsfall protein in acute pyelonephritis

The cytotoxic activity of leukocytes from humans and rats with pyelonephritis were examined in an antibody-dependent cell-mediated cytotoxicity assay (ADCC) with CrCl3-treated erythrocytes coated with Tamm-Horsfall (TH) as target cells. The specificity of the ADCC was confirmed by absorption with TH urinary glycoprotein and inhibition of the ADCC activity seen with polyclonal rabbit anti-TH antisera by monoclonal mouse antibodies. The ADCC activity detected in children with acute pyelonephritis was low in the initial phase of the disease, but increased significantly 9 days after the start of antibacterial treatment. In rats with experimental pyelonephritis, ADCC activity decreased significantly with increased duration of infection. Depletion of cells adhering to carbonyl iron led to higher ADCC activity. During the course of the infection the difference in ADCC activity between effector cell preparations depleted using carbonyl iron and those not depleted decreased. The decreased ADCC activity demonstrated during acute pyelonephritis may point to mechanisms operating to diminish the risk of tissue damage.