American Society for Microbiology

Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rRNA genes (rDNAs) were used to amplify the 16S-23S rDNA internal transcribed spacers (ITS) from eight species of pseudomonads which have been associated with human infections. Amplicons from reference strains of Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas mallei, Pseudomonas mendocina, Pseudomonas pickettii, Pseudomonas pseudomallei, and Xanthomonas maltophilia were cloned from each species, and sequence analysis revealed a total of 19 distinct ITS regions, each defining a unique sequevar with ITS sizes ranging from 394 (P. cepacia) to 641 (P. pseudomallei) bp. Five distinct ITS sequevars in P. cepacia, four in P. mendocina, three in P. aeruginosa, two each in P. gladioli and P. pseudomallei, and one each in P. mallei, P. pickettii, and X. maltophilia were identified. With the exception of one P. cepacia ITS, all ITS regions contained potential tRNA sequences for isoleucine and/or alanine. On the basis of these ITS sequence data, species-specific oligonucleotide primers were designed to differentiate P. aeruginosa, P. cepacia, and P. pickettii. The specificities of these primers were investigated by testing 220 clinical isolates, including 101 strains of P. aeruginosa, 103 strains of P. cepacia, and 16 strains of P. pickettii, in addition to 24 American Type Culture Collection (ATCC) Pseudomonas strains. The results showed that single primer pairs directed at particular ITSs were capable of specifically identifying the ATCC reference strains and all of the clinical isolates of P. aeruginosa and P. pickettii, but this was not the case with several ITS-based primer pairs tested for P. cepacia. This pathogen, on the other hand, could be specifically identified by primer pairs directed against the 23S rDNA.

While monoclonal antibodies show promise for use in the treatment of a variety of disease states, including cancer, autoimmune disease, and allograft rejection, generation of anti-antibody responses still remains a problem. For example, 50% of the patients who receive OKT3 produce blocking antibodies that interfere with its binding to T cells, thus decreasing the therapeutic effect (51). HAMA responses have also interfered with tumor imaging (39,40) and radioimmunotherapy (56). The generation of an anti-antibody response is dependent on many factors. These include the dose of antibody, the number of injections of antibody, the immunogenicity of the antibody, the form of the antibody, and the immunocompetence of the recipient. Predictably, both the number of injections of antibody and the dosage are influential in the generation of an anti-antibody response. It is apparent that human antibodies, chimeric antibodies, and mouse Fab fragments are much less likely to induce anti-antibody responses than intact mouse monoclonal antibodies or mouse F(ab')2 fragments when one injection is administered. Injections of human or chimeric antibodies appears to reduce immunogenicity, but the probability that anti-antibody responses can still be induced on multiple injections must be considered and appropriately evaluated. Several areas demand extensive investigation to enhance the clinical utility of monoclonal antibodies. First, results of thorough clinical trials with human or chimeric antibodies need to be evaluated for the induction of anti-antibodies after multiple injections of antibodies. Second, less immunogenic forms of antibodies (Fab, Fv) need to be studied for their clinical efficacies and for their abilities to induce anti-antibody responses.

The antibody response in patients with American cutaneous leishmaniasis was analyzed by immunoblotting with soluble and insoluble antigens ofLeishmania braziliensis. The recognition of the 27- and/or 30-kDa soluble antigens was considered relevant for the diagnosis of cutaneous leishmaniasis. Immunoblotting was found to be significantly more sensitive and specific than indirect immunofluorescence and enzyme-linked immunosorbent assay.

The retrospective analysis of 494 solid-organ transplant recipients revealed that during the follow-up period (mean duration, 3.2 years) 184 (88%) of 209 anti-human cytomegalovirus (HCMV) immunoglobulin A (IgA)-positive patients remained IgA positive, as did 128 (74.85%) of 171 anti-HCMV IgM-positive patients. We conclude that anti-HCMV IgA and IgM testing for management of clinically relevant HCMV infections in solid-organ transplant recipients is dispensable.

Antibody responses toAcinetobacter(five strains),Pseudomonas aeruginosa,Escherichia coli, myelin basic protein (MBP), and neurofilaments were measured in sera from 26 multiple sclerosis (MS) patients, 20 patients with cerebrovascular accidents (CVA), 10 patients with viral encephalitis, and 25 healthy blood donors. In MS patients, elevated levels of antibodies against all strains ofAcinetobactertested were present, as well as antibodies againstP. aeruginosa, MBP, and neurofilaments, but not antibodies toE. coli, compared to the CVA group and controls. The myelin-Acinetobacter-neurofilament antibody index appears to distinguish MS patients from patients with CVAs or healthy controls. The relevance of such antibodies to the neuropathology of MS requires further evaluation.

A neutralization enzyme immunoassay (N-EIA) was used to determine the neutralizing serum antibody titers to influenza A/Taiwan/1/86 (H1N1) and Beijing/353/89 (H3N2) viruses after vaccination of 51 human immunodeficiency virus (HIV) type 1-infected individuals and 10 healthy noninfected controls against influenza virus infection. Overall, the N-EIA titers correlated well with the hemagglutination-inhibition (HAI) titers that were observed in the same samples in a previous study (F. P. Kroon, J. T. van Dissel, J. C. de Jong, and R. van Furth, AIDS 8:469–476,1994). The N-EIA appeared to be more sensitive than the HAI test. Significantly more fourfold or higher rises in N-EIA titer and higher mean N-EIA titers occurred in HIV-infected individuals with ≥200 CD4 ⁺ cells per μl than in those with <200 CD4 ⁺ cells per μl.

We examined the effect of immune stimulation by a human immunodeficiency virus type 1 (HIV-1) immunogen (Remune) compared to a non-HIV vaccine (influenza) on HIV-1-specific immune responses in HIV-1-seropositive subjects. HIV-1 p24 antigen-stimulated lymphocyte proliferation was not augmented after immunization with the influenza vaccine. In contrast, subjects increased their lymphocyte proliferative responses to p24 antigen after one immunization with HIV-1 immunogen (Remune) (gp120-depleted inactivated HIV-1 in incomplete Freund’s adjuvant). Furthermore, p24 antigen-stimulated β-chemokine production (RANTES, MIP-1α, MIP-1β) was also augmented after immunization with the HIV-1 immunogen but not influenza vaccine. Taken together, these results suggest that in this cohort, HIV-specific immune responses to p24 antigen can be augmented after immunization with an HIV-1 immunogen. The ability to upregulate immune responses to the more conserved core proteins may have important implications in the development of immunotherapeutic interventions for HIV-1 infection.

Biopsy specimens of the antrum and corpus were obtained from four Helicobacter pylori -infected members of a family and from the same boy (son 1) in whom the infection reappeared after simultaneous successful eradication treatment of three family members, excluding the mother. A total of 18 to 60 H. pylori isolates were obtained from each specimen and subjected to rRNA gene restriction pattern analysis. The father's isolates and the initial isolates from son 1 showed the same Hin dIII type, which was divided into three Hae III subtypes. Isolates from the mother and a brother (son 2) and posttreatment isolates from son 1 showed a distinct Hin dIII type (with one minor subtype), which was divided into six Hae III subtypes. All subtypes of the initial isolates from son 1 were present in the father's isolates, and all subtypes of the posttreatment isolates from son 1 were present in the mother's isolates but not in son 2's. Electron microscopic analysis of the biopsy specimens demonstrated extremely high levels of H. pylori colonization in the father's gastric mucosa. H. pylori adherence with a ruffle formation was also demonstrated. The findings suggest that son 1 was infected initially with the H. pylori strain of the father and son 2 was infected with the H. pylori strain of the mother and that after eradication therapy son 1 was reinfected with the H. pylori strain of the mother, who did not undergo eradication therapy.

Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. We developed a semiautomated flow cytometric opsonophagocytosis assay using HL-60 granulocytes as effector cells and nonviable 5,6-carboxyfluorescein, succinimidyl ester-labeledStreptococcus pneumoniae(serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) as bacterial targets. The flow cytometric opsonophagocytosis assay was highly reproducible (for 87% of repetitive assays the titers were within 1 dilution of the median titer) and serotype specific, with ≥97% inhibition of opsonophagocytic titer by addition of homologous serotype-specific polysaccharide. In general, opsonophagocytic titers were not significantly inhibited by the presence of either heterologous pneumococcal polysaccharide or penicillin in the serum. The flow cytometric assay could reproducibly measure functional antibody activity in prevaccination (n= 28) and postvaccination (n= 36) serum specimens from healthy adult volunteers vaccinated with the 23-valent pneumococcal polysaccharide vaccine. When compared with a standardized manual viable opsonophagocytic assay, a high correlation (r= 0.89;P≤ 0.01) was found between the two assays for the seven serotypes tested. The flow cytometric assay is rapid (∼4 h) with high throughput (∼50 serum samples per day per technician) and provides a reproducible measurement of serotype-specific functional antibodies, making it a highly suitable assay for the evaluation of the immune responses elicited by pneumococcal vaccines.

CD69 is a lymphoid activation antigen whose rapid expression (< or = 2 h postactivation) makes it amenable for the early detection of T-cell activation and for subset activation analyses. In the present study we evaluated the utility of flow cytometric detection of CD69 expression by T cells activated with polyclonal stimuli (anti-CD3 and staphylococcal enterotoxin B [SEB]) and oligoclonal stimuli (tetanus toxoid and allogeneic cells) using flow cytometry. Following activation of T cells with anti-CD3 or SEB, CD69 is detectable at < or = 4 h following activation, with anti-CD3 peaks at 18 to 48 h. Dose titration experiments indicated that CD69 expression largely paralleled that in [3H]thymidine incorporation assays, although the former offered a more sensitive measure of T-cell activation at limiting doses of activator than [3H]thymidine incorporation when cells were activated with either anti-CD3 or SEB. However, activation of T cells with either tetanus toxoid or allogeneic stimulator cells failed to induce detectable CD69 expression at up to 7 days of culture. Subset analyses of anti-CD3- and SEB-activated T cells indicated that populations other than T cells can express CD69 following stimulation with T-cell-specific stimuli, indicating that CD69 can be induced indirectly in non-T cells present in the population. These findings indicate that CD69 is a useful marker for quantifying T-cell and T-cell subset activation in mixed populations but that its utility might be restricted to potent stimuli that are characterized by their ability to activate large numbers of cells with rapid kinetics.