Rapid expansion and IL-4 expression by Leishmania-specific naive helper T cells in vivo

CD4 T cells are pivotal for effective immunity, yet their initial differentiation into effector subsets after infection remains poorly defined. We examined CD4 T cells specific for the immunodominant Leishmania major LACK antigen using MHC/peptide tetramers and IL-4 reporter mice. Comprising approximately 15 cells/lymph node in naive mice, LACK-specific T cells expanded over 100-fold, and 70% acquired IL-4 expression by 96 hr. Despite their pathogenic role in susceptible mice, LACK-specific precursor frequency, expansion, and IL-4 expression were comparable between susceptible and resistant mice. When injected with unrelated antigen, Leishmania efficiently activated IL-4 expression from naive antigen-specific T cells. CD4 subset polarization in this highly characterized model occurs independently from IL-4 expression by naive T cells, which is activated indiscriminately after parasitism.     

Production of immune interferon is regulated by more than one T cell subset: Lyt-1,2,3 and Qat-5 phenotypes of murine T lymphocytes involved in IFN-γ production in primary and secondary mixed lymphocyte reaction

Lymphocytes responsible for the production of IFN-γ (immune interferon) in primary and secondary mixed lymphocyte reactions have been characterized with antisera specific for the Lyt-1,2,3 and Qat-5 alloantigens. A comparison was made between selected T cell subsets with respect to their ability to proliferate, generate cytolytic activity and produce IFN-γ in response to H-2 alloantigens. The data indicate that (a) in primary mixed lymphocyte reactions, IFN-γ is produced by Lyt-1⁺, Qat-5⁺ and by Lyt-123⁺, Qat-5⁺ T cells, (b) in secondary mixed lymphocyte reactions, an additional T cell subset, which is Lyt-23⁺, Qat-5^?, participates in the generation of IFN-γ and (c) the production of IFN-γ does not correlate with either proliferation or the generation of cytotoxic lymphocytes.     

Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge

This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.     

Viruses Infecting Marine Brown Algae

Viruses infecting algal hosts possess large double-stranded DNA as genomes. We have recently identified a family of viruses specific for filamentous brown algae. In contrast to the better known Chlorella viruses with their lytic infection cycle, marine brown algal viruses latently occur in their host cells and are induced to multiply in response to a variety of external stimuli such as change in light and temperature. Here, I summarize the known properties of this family of viruses and discuss their taxonomic classification.     

DRD4 exon 3 variants are not associated with symptomatology of major psychoses in a German population

We previously reported an association of DRD4 exon 3 long alleles with delusional symptomatology, independently from psychiatric diagnoses [Am. J. Med. Genet. 105 (2001) 283; Psychiatry Res. 80 (1998) 129]. The aim of this investigation was to replicate these results in an independent sample from Germany. We studied 394 subjects, affected by bipolar disorder (n = 32), schizoaffective disorder (n = 45), and schizophrenia (n = 317). All affected subjects were evaluated using the Operational Criteria for Psychotic Illness (OPCRIT) checklist. DRD4 variants were not associated with symptomatology of major psychosis. Our present results, obtained in an independent German sample, did not confirm the association between DRD4 variants and delusional symptomatology. However it should be considered that the original sample included a much higher rate of mood disorders and this could partially explain the discrepancy.     

C4d]FlowPRA screening--a specific assay for selective detection of complement-activating anti-HLA alloantibodies

On waiting lists, transplant candidates are routinely screened for potentially harmful complement-fixing alloantibodies using complement-dependent cytotoxicity (CDC) panel-reactive antibody (PRA) testing. We have recently developed a novel cell-independent assay for assessment of complement-activating panel reactivity ([C4d]FlowPRA), which is based on selective flow-cytometric detection of alloantibody-triggered C4 complement split product deposition to human leukocyte antigen (HLA)-coated FlowPRA beads. Serum specimens selected from 120 transplant candidates were evaluated by [C4d]FlowPRA (HLA class I vs II) in comparison with FlowPRA IgG alloantibody screening (HLA class I vs II), a method detecting both complement- and noncomplement-activating alloantibodies, and with CDC-PRA on separated T (T-CDC) or B cells (B-CDC, evaluation on platelet-absorbed sera). For each assay, >/=10% PRA reactivity was considered positive. Comparing complement-dependent PRA assays with standard FlowPRA, the specificity calculated for [C4d]FlowPRA (HLA class I: 92%; class II: 100%) was found to be superior to that of CDC testing (T-CDC-PRA: 79%; B-CDC-PRA: 86%). Because noncomplement-activating alloreactivities were not detected for both techniques, low sensitivities were calculated ([C4d]FlowPRA HLA class I: 61%; class II: 31%; T-CDC-PRA: 70%; B-CDC-PRA: 55%). Compared with CDC-PRA, [C4d]FlowPRA gave a high specificity (HLA class I compared with T-CDC: 89%, HLA class II compared with B-CDC: 95%) but, at least in part because of false-positive CDC results, a modest sensitivity (66% and 38%, respectively). For both HLA classes, we found a highly significant association between absolute [C4d]FlowPRA and CDC-PRA levels (p < 0.0001). Our results suggest that detection of C4 split product deposition to FlowPRA beads may represent an attractive HLA-specific and time-effective alternative to CDC-PRA screening.     

Emergence of translocation t(9;11)-positive leukemia during treatment of childhood acute lymphoblastic leukemia

Therapy-related acute myeloid leukemia (t-AML) characterized by the t(9;11)(p22;q23) translocation is one of the most frequent secondary malignancies. The timing of the initiation of translocation and of development of the malignant t(9;11) clone during chemotherapy is presently unknown. In the present study, we backtracked bone marrow samples from three children during treatment for acute lymphoblastic leukemia (ALL). Two patients developed a t(9;11)-positive t-AML 19 and 30 months after therapy start, whereas the third patient, diagnosed with a rare t(9;11)-positive ALL, suffered from an ALL relapse 23 months after initial diagnosis. The genomic MLL-MLLT3 (MLL-AF9) fusion site was amplified by a multiplex, nested long-range PCR and used as a clonal marker for quantification of the MLL-MLLT3-positive cells during chemotherapy. The t(9;11)-positive clone was detectable 13 and 18 months after therapy start in both t-AML cases, which was 6-12 months before clinical diagnosis of the secondary malignancy. In the t(9;11)-positive ALL patient, the identical leukemic clone reoccurred during maintenance therapy after a short molecular remission, 8 months before clinically overt ALL relapse. The time course and characteristics of the genomic breakpoints in the present t-AML cases support the hypothesis of translocation formation as a result of defective breakage repair after topoisomerase II cleavage.