Chemical, biological, and immunochemical properties of the Chlamydia psittaci lipopolysaccharide.

The lipopolysaccharide (LPS) of Chlamydia psittaci was extracted from yolk sac-grown elementary bodies, purified, and characterized chemically, immunochemically, and biologically. The LPS contained D-galactosamine, D-glucosamine, phosphorus, long-chain fatty acids, and 3-deoxy-D-manno-2-octulosonic acid in the molar ratio of approximately 1:2:2:6:5. The antigenic properties of the isolated LPS were compared with those of the LPS from Chlamydia trachomatis and Salmonella minnesota Re by the passive hemolysis and passive hemolysis inhibition tests, absorption, hydrolysis kinetics, and Western blot analysis with rabbit polyclonal antisera against chlamydiae and with a mouse monoclonal antibody recognizing a genus-specific epitope of chlamydial LPS. Two antigenic determinants were identified, one of which was chlamydia specific and the other of which was cross-reactive with Re LPS. Both determinants were destroyed during acid hydrolysis, whereby a third antigen specificity was exposed which was indistinguishable from the lipid A antigenicity. In rabbit polyclonal antisera prepared against Formalin-killed elementary bodies or detergent-solubilized membranes, two antibody specificities were differentiated. One of these was chlamydia specific, and the other was cross-reactive with Re LPS. The LPS of C. psittaci was inactive within typical endotoxin parameters (lethal toxicity, pyrogenicity, local Shwartzman reactivity); it was, however, active in some in vitro assays, such as those testing for mouse B-cell mitogenicity and the induction of prostaglandin E2 in mouse peritoneal macrophages.     

Correlation between intracellular cAMP content, kinematic parameters and hyperactivation of human spermatozoa after incubation with pentoxifylline

Various compounds have been used in the attempt to improve sperm motility, including pentoxifylline (PF), a methylxanthine derivative. It has been postulated that PF, being a phosphodiesterase inhibitor, increases sperm kinematic parameters and the number of spermatozoa exhibiting hyperactivated motility by raising the intracellular content of cAMP, a molecule involved in the generation of sperm energy. However, it has not been clarified whether the biological effects of PF on sperm motility correlate with its ability to increase intracellular cAMP levels. To examine this relationship, the kinematic parameters, hyperactivation, and intracellular cAMP content were evaluated in motile spermatozoa, obtained by discontinuous Percoll gradient and swim- up from 21 normozoospermic semen samples, incubated without and with PF for 0, 1, 2, and 4 h. PF increased beat cross frequency after 1 and 2 h of incubation, curvilinear velocity and lateral head displacement (ALH) after 4 h, and hyperactivation after 1, 2, and 4 h, and decreased linearity (LIN) after 1 h of incubation. The intracellular cAMP content of spermatozoa incubated with PF increased at all time-points examined. Both intracellular cAMP content and increase in hyperactivation in response to PF decreased with the length of incubation. In the absence of PF, cAMP content was unchanged and was correlated significantly only with ALH and the percentage of spermatozoa with hyperactivated motility. Following incubation with PF, cAMP content correlated with hyperactivation and all sperm kinematic parameters, with the exception of LIN and straightness. These findings suggest that the beneficial effects of PF on sperm kinematic parameters and hyperactivation are related to its ability to increase intracellular cAMP content.      

Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta, are uncommon in healthy individuals and HIV patients, and are apparently without prognostic significance. In contrast to earlier reports, IL-2 antibodies were found only in HIV patients treated with rIL-2.     

GM-CSF counteracts hemorrhage-induced suppression of cytokine-producing capacity

Objective and design: To study whether a treatment with the hematopoietic growth factor GM-CSF restores the attenuated ex-vivo cytokine-producing capacity of macrophages after sublethal hemorrhagic shock.Subjects: Male Sprague-Dawley rats.Treatment: 20 g/animal of recombinant murine GM-CSF after shock via arterial line.Methods: Hemorrhagic shock was established by pressure-controlled bleeding to a mean arterial pressure of 50 mm Hg for 35–40 min and consecutive resuscitation. 24 h after hemorrhage, lipopolysaccharide (LPS)-induced cytokine production of isolated macrophages derived from different compartments was measured.Results: A significant reduction of LPS-induced TNF production was found in whole blood cultures (1.0 ± 0.7 ng/ml after sham vs. 0.23 ± 0.08 ng/ml after shock operation), macrophages derived from spleen (0.88 ± 0.23 ng/ml after sham vs. 0.03 ± 0.1 ng/ml after shock operation), peritoneum (2.2 ± 0.7 ng/ml after sham vs. 0.29 ± 0.4 ng/ml after shock operation) and bronchoalveolar fluid (0.65 ± 0.13 ng/ml after sham vs. 0.003 ± 0.027 ng/ml after shock operation, mean ± S.D.). In cells from animals treated with GM-CSF a significantly enhanced LPS-induced TNF production in splenic, alveolar and peritoneal macrophages was found after shock compared to the cells derived from untreated animals (peritoneum: 289 ± 366 ng/ml TNF after shock vs. 2066 ± 94 ng/ml TNF after shock and GM-CSF; lung: 9 ± 12 ng/ml TNF after shock vs. 64 ± 17 ng/ml TNF after shock and GM-CSF; spleen: 58 ± 96 ng/ml TNF after shock vs. 548 ± 47 ng/ml TNF after shock and GM-CSF). Blood cultures collected from rats after hemorrhagic shock did not show a significant increase of TNF-production after GM-CSF treatment.Conclusion: Hemorrhagic shock caused a depression of the TNFa response to LPS which was partly counteracted by treatment with GM-CSF. Therefore, GM-CSF represents a promising approach to normalise trauma- and shock-induced immune dysfunction.Received 4 April 2003; returned for revision 3 July 2003; accepted by A. Falus 25 August 2003     

The spectrum of vitamin B12 deficiency

Low serum vitamin B12 levels are not uncommon in the elderly. Patients with vitamin B12 deficiency manifest a spectrum of clinical findings. Pernicious anemia and malabsorption syndrome are the usual causes of vitamin B12 deficiency. Pernicious anemia is confirmed by the presence of intrinsic factor blocking antibody or abnormal results on the Schilling test. Patients with neuropsychiatric symptoms of vitamin B12 deficiency may have a normal Schilling test and no evidence of macrocytic anemia. In such patients, vitamin B12 deficiency is confirmed by determining serum levels of homocysteine and methylmalonic acid.     

Loss of volunteer blood donors because of unconfirmed enzyme immunoassay screening results. Retrovirus Epidemiology Donor Study

BACKGROUND: Blood donors who test repeatably reactive on enzyme immunoassay (EIA) and are not confirmed as positive are a continuing problem for blood banks. Units are discarded and donors are deferred, in spite of multiple studies indicating that such donors are very rarely infected with the transmissible agents. Few data are available, however, with which to evaluate whether the discarded units are more likely to come from particular demographic subgroups. STUDY DESIGN AND METHODS: The Retrovirus Epidemiology Donor Study database of over 2 million allogeneic whole-blood donations collected in the years 1991 through 1993 was utilized. The prevalence of false-positive and indeterminate test results within demographic subgroups was computed for antibodies to human immunodeficiency virus, hepatitis C virus, and human T-lymphotropic virus (anti-HIV, anti-HCV, anti-HTLV, respectively) and hepatitis B surface antigen (false-positive only) as the proportion of donations that were repeatably reactive on EIA but negative or indeterminate on the confirmatory or supplemental test. RESULTS: Several demographic groups with increased prevalence of false- positive and indeterminate anti-HIV results were the same females, younger age groups, blacks, and first-time donors. Likewise, many of the demographic subgroups with increased prevalence of false-positive and indeterminate anti-HCV results were similar: older age groups, non- whites, lower education levels, first-time donors, donors making directed donations, and donors who had received transfusions. For anti- HTLV, by contrast, the oldest group had the highest prevalence of false- positive results but the lowest prevalence of indeterminate results: blacks had the lowest prevalence of false positive results but the highest prevalence of indeterminate results. CONCLUSION: If units that test repeatably reactive on EIA but that are not confirmed as positive are almost always from individuals not infected with the virus in question, then these results indicate that there may be sex-, race-, and/or age-linked proteins cross-reacting with the test kit materials. Elucidation of these antigenic determinates and their subsequent removal should be a priority.     

Frequency of human immunodeficiency virus (HIV) infection among contemporary anti-HIV-1 and anti-HIV-1/2 supplemental test- indeterminate blood donors. The Retrovirus Epidemiology Donor Study

Background: Follow-up studies from the mid-1980s showed that 1 to 5 percent of blood donors testing reactive in anti-human immunodeficiency virus type 1 (HIV-1) enzyme immunoassay (EIA) and testing indeterminate in Western blot were infected with HIV-1 and were in the process of seroconverting. The present study was conducted to establish the rate of HIV infection among contemporary anti-HIV-1/HIV type 2 (HIV-2) EIA- reactive, Western blot-indeterminate donors. Study Design and Methods: Donations (n = 607) with indeterminate HIV supplemental test results were identified by screening 3,021,342 donations given from November 1990 through August 1993 at five participating blood centers. Consenting donors were enrolled and samples taken 4 to 8 weeks after donation. Follow-up sera were tested by EIA and Western blot for anti- HIV-1 seroconversion and by type-specific peptide assays for antibodies to HIV-2 and HIV-1 subtype O. Peripheral blood mononuclear cells and/or plasma from the follow-up samples were tested for HIV-1 DNA and/or RNA by polymerase chain reaction. The rate of HIV-1 infection among Western blot-indeterminate donors was also estimated by multiplying the incidence rate of HIV-1 seroconversion in this donor population by the estimated duration of the EIA-reactive and Western blot-indeterminate window during seroconversion (8.5 days). Results: Supplemental test- indeterminate donors (n = 355) enrolled a median of 38 days after donation; 265 (75%) of these donors were identified as indeterminate after an anti-HIV-1/2 EIA-reactive donation. Enrolled and non-enrolled donors had similar distributions of demographic characteristics and band patterns. Follow-up samples from all 355 donors tested negative for HIV-1 in polymerase chain reaction. Follow-up sera tested Western blot-negative in 54 cases (15%) and Western blot-indeterminate in 299 (84%). Two follow-up sera (0.6%) were interpreted, according to manufacturer's package insert criteria, as Western blot positive with p24 and gp41 bands and/or gp120/160 bands; however, paired testing of index and follow-up sera from these two cases showed identical Western blot and EIA reactivity, and polymerase chain reaction was negative for HIV RNA and DNA, which ruled out HIV infection. The absence of HIV infection in 355 Western blot-indeterminate donors was consistent with our incidence-based model analysis, which yielded an estimate of one HIV-1 infection for every 215 Western blot-indeterminate donations (95% CI, 1/39-1/8333). Conclusion: Contemporary blood donors classified as indeterminate in supplemental HIV testing are infrequently infected with HIV. Donors whose follow-up samples test negative in anti-HIV-1/2 EIAs and negative or persistently indeterminate in Western blots should be considered eligible for reinstatement.