Failure of dideoxynucleosides to inhibit human immunodeficiency virus replication in cultured human macrophages.

Primary human monocyte-derived macrophages (MDM) were shown to have diminished deoxynucleoside kinase activities compared to T lymphoblasts, and a reduced ability to phosphorylate dideoxynucleosides with anti-human immunodeficiency virus (HIV) activity. These drugs, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyadenosine (ddA), which are potent anti-HIV agents in CD4 lymphocytes, did not inhibit HIV replication in MDM, even at concentrations of 100 microM. This drug concentration of AZT is approximately 100-fold higher than the levels attained in the serum of treated patients and the levels required to inhibit HIV replication in lymphocytes. These observations may explain the failure of AZT therapy to clear viremia, consistent with the presence of a drug-resistant reservoir of infected cells in vivo. New therapeutic approaches to inhibit the replication of HIV in MDM may be needed

Uniform high frequency expression of autoantibody-associated crossreactive idiotypes in the primary B cell follicles of human fetal spleen.

At 23 wk of gestation, the fetal spleen contains follicles of lymphocytes that coexpress B cell differentiation antigens, surface Ig, and the 67-kD pan-T lymphocyte antigen, CD5 (Leu-1). Such cells are thought to represent the normal equivalent cells of B chronic lymphocytic leukemia (CLL). This B cell leukemia is distinctive in that high proportions of patients have leukemic cells that express sIg bearing one or more crossreactive idiotypes (CRIs) that commonly are found on IgM autoantibodies. We performed immunohistochemical studies on fetal spleen at 23 wk of gestation using a panel of mAbs specific for autoantibody-associated CRIs. We find that high proportions (5-17%) of the lymphocytes within each follicle react with any one of the anti-CRI mAbs. Furthermore, there is little variation between primary follicles in the proportions of cells that express a particular CRI. Using a cocktail of four anti-CRI mAbs, we detect autoantibody-associated CRIs on approximately one-third of the lymphocytes within each of the primary B cell follicles. These data indicate that the many of the Igs produced during early B cell development may be structurally related to IgM autoantibodies and to Ig expressed in CLL and related CD5 B cell malignancies. Furthermore, these studies suggest that the repertoire of Ig V genes expressed in each primary B cell follicle may be representative of the total restricted Ig V gene repertoire expressed during early B cell ontogeny

Evidence for somatic selection of natural autoantibodies.

Natural autoantibodies are primarily immunoglobulin M (IgM) antibodies that bind to a variety of self-antigens, including self-IgG. Accounting for a large proportion of the early B cell repertoire, such polyspecific autoantibodies are speculated to contribute to the homeostasis and/or competence of the primary humoral immune system. Recent studies indicate that the leukemia cells from most patients with chronic lymphocytic leukemia (CLL) also express such IgM autoantibodies. Similarly, the leukemia cells from many CLL patients react with murine monoclonal antibodies (mAbs) specific for crossreactive idiotypes (CRIs) associated with human IgM autoantibodies. In particular, leukemic cells frequently react with G6, a mAb specific for an Ig heavy chain (H chain)-associated CRI, and/or with 17.109, a mAb that defines a kappa light chain (L chain)-associated CRI. Generated against IgM rheumatoid factor (RF) paraproteins, G6 and 17.109 each recognize a major CRI that is present in many IgM RF paraproteins. Furthermore, over 90% of the IgM paraproteins found to bear both H and L chain-associated CRIs also are found to have RF activity. Molecular characterization of these CRIs demonstrates that each is a serologic marker for expression of a highly conserved Ig V gene. As such, the frequent production of IgM polyspecific autoantibodies in CLL simply may reflect the frequent use of such highly conserved autoantibody-encoding Ig V genes with little or no somatic mutation. To test this hypothesis, we generated murine transfectomas to pair the 17.109-reactive kappa L chain of SMI, a 17.109/G6-reactive CLL population, with the Ig H chain of SMI or other G6-reactive leukemia cells or tonsillar lymphocytes. Cotransfection of vectors encoding the Ig H and L chains of SMI generated transfectomas that produce IgM kappa RF autoantibodies reactive with human IgG1 and IgG4. In contrast to G6/17.109-reactive IgM kappa RF Waldenstrom's paraproteins, the SMI IgM kappa also reacts with several other self-antigens, including myoglobin, actin, and ssDNA. However, cotransfection of the SMI L chain with a vector encoding any one of 10 different G6-reactive Ig H chains generated transfectomas that produce IgM kappa antibodies without detectable polyspecific autoantibody activity. These results indicate that polyspecific antiself-reactivity of G6/17.019-reactive Ig is dependent on the somatically generated Ig third complementarity determining region. Collectively, these studies imply that selection may be responsible for the frequent expression of polyspecific autoantibodies in CLL and early B cell ontogeny

Autoantibody-associated kappa light chain variable region gene expressed in chronic lymphocytic leukemia with little or no somatic mutation. Implications for etiology and immunotherapy.

Recently the minor B cell subpopulation that expresses the CD5 (Leu-1) antigen has been implicated as a source of IgM autoantibodies. Chronic lymphocytic leukemia (CLL), the most common leukemia in humans, represents a malignancy of small B lymphocytes that also express the CD5 antigen. However, little is known concerning the antibody variable region genes (V genes) that are used by these malignant CD5 B cells. We have found that a relatively high frequency of CLL patients have leukemic B cells with surface immunoglobulin (sIg) recognized by 17.109, a murine mAb specific for a kappa light chain associated crossreactive idiotype (CRI) associated with rheumatoid factor and other IgM autoantibodies. Flow cytometric analyses revealed that the relative expression of the 17.109-CRI by circulating leukemic B cells was directly proportional to the levels of sIg kappa light chain, indicating that there exists stable idiotype expression in the leukemic population. To examine this at the molecular level, the nucleic acid sequences encoding the Ig kappa light chains of two unrelated patients with CLL bearing sIg with the 17.109-CRI were determined. Analyses of multiple independent kappa light chain cDNA clones did not reveal any evidence for sequence heterogeneity in the CLL cell population. Furthermore, the nucleic acid sequences expressed by the leukemic cells of these two patients were identical or very homologous to a germline V kappa gene isolated from placental DNA, designated Humkv 325, or "V kappa RF" because of its association with IgM autoantibodies. This study suggests; (a) that the malignant CD5+ B lymphocytes in CLL use the same V kappa gene that has been highly associated with IgM autoantibodies and (b) that the expression of V genes is stable in CLL, in contrast to other B cell malignancies examined to date. We propose that many CLL cases represent malignancies of autoreactive CD5 B cells that use a restricted set of conserved V genes. This property may render CLL particularly amenable to immunotherapy with antiidiotypic antibodies

Isolation and characterization of a light chain variable region gene for human rheumatoid factors.

Previously, we isolated a Vk gene (Humkv325) from a human placenta that encodes RF light chains bearing the PSL2 and PSL3 CRI markers. Here we report the isolation and characterization of a second human Vk gene (Humkv328) that can be used for RF synthesis. This Vk gene probably encodes at least two 6B6.6 CRI+ RF light chains (Les and Pom) from unrelated subjects, and thus may be related to the light chain-associated 6B6.6 CRI

Function of B cells expressing a human immunoglobulin M rheumatoid factor autoantibody in transgenic mice.

We have generated transgenic mice that express the immunoglobulin (Ig)M heavy chain and kappa light chain genes coding for a human IgM rheumatoid factor (RF), Les. Transgenic B cells expressing human IgM RF show striking similarities to their counterparts in normal humans. They comprise a significant proportion of the adult B cell population, but secrete only low levels of RF into the serum. The RF transgene-expressing B cells localize to primary B cell follicles and the mantle zone regions of secondary follicles in the spleen. Using these mice we have been able to show that one of the central functions of normal RF-expressing B cells may be to act as highly efficient antigen-presenting cells for low concentrations of immune-complexed antigen. High levels of secretion of IgM RF can not be induced under normal circumstances, although RF-expressing B cells proliferate well in vitro to both aggregated human IgG and anti-human IgM antibodies. However, these mice are not intrinsically secretion deficient. By crossing the RF transgenic mice with the autoimmune MRL/lpr background, we find a dramatic increase, > 200-fold, in levels of serum RF. The results strongly suggest that a major function of normal resting RF B cells is unrelated to antibody secretion. Rather, the RF B cells in the follicles may play a role in antigen presentation and regulation of immune responses to antibody-bound nonself-, and possibly self-antigens. This physiologic role of RF B cells may be disrupted in RF-associated autoimmune disease

Genetic analysis of self-associating immunoglobulin G rheumatoid factors from two rheumatoid synovia implicates an antigen-driven response.

Although much has been learned about the molecular basis of immunoglobulin M (IgM) rheumatoid factors (RFs) in healthy individuals and in patients with mixed cryoglobulinemia and rheumatoid arthritis, little is known about the genetic origins of the potentially pathogenic IgG RFs in the inflamed rheumatoid synovia of patients. Recently, we generated from unmanipulated synovium B cells several hybridomas that secreted self-associating IgG RFs. To delineate the genetic origins of such potentially pathogenic RFs, we adapted the anchored polymerase chain reaction to rapidly clone and characterize the expressed Ig V genes for the L1 and the D1 IgG RFs. Then, we identified the germline counterparts of the expressed L1 IgG RF V genes. The results showed that the L1 heavy chain was encoded by a Vh gene that is expressed preferentially during early ontogenic development, and that is probably located within 240 kb upstream of the Jh locus. The overlap between this RF Vh gene and the restricted fetal antibody repertoire is reminiscent of the natural antibody-associated Vh genes, and suggests that at least part of the "potential pathogenic" IgG RFs in rheumatoid synovium may derive from the "physiological" natural antibody repertoire in a normal immune system. Indeed, the corresponding germline Vh gene for L1 encodes the heavy chain of an IgM RF found in a 19-wk-old fetal spleen. Furthermore, the comparisons of the expressed RF V genes and their germline counterparts reveal that the L1 heavy and light chain variable regions had, respectively, 16 and 7 somatic mutations, which resulted in eight and four amino acid changes. Strikingly, all eight mutations in the complementarity determining regions of the V gene-encoded regions were replacement changes, while only 6 of 11 mutations in the framework regions caused amino acid changes. Combined with L1's high binding affinity toward the Fc fragment, these results suggest strongly that the L1 IgG RF must have been driven by the Fc antigen

Evaluation of rK39 rapid diagnostic tests for canine visceral leishmaniasis : longitudinal study and meta-analysis

Canine visceral leishmaniasis is a vector-borne disease caused by the intracellular parasite Leishmania infantum. It is an important veterinary disease, and dogs are also the main animal reservoir for human infection. The disease is widespread in the Mediterranean area, and parts of Asia and South and Central America, and is potentially fatal in both dogs and humans unless treated. Diagnosis of canine infections requires serological or molecular tests. Detection of infection in dogs is important prior to treatment, and in epidemiological studies and control programmes, and a sensitive and specific rapid diagnostic test would be very useful. Rapid diagnostic tests (RDTs) have been developed, but their diagnostic performance has been reported to be variable. We evaluated the sensitivity of a RDT based on serological detection of the rK39 antigen in a cohort of naturally infected Brazilian dogs. The sensitivity of the test to detect infection was relatively low, but increased with time since infection and the severity of infection. We then carried out a meta-analysis of published studies of rK39 RDTs, evaluating the sensitivity to detect disease and infection. The results suggest that rK39 RDTs may be useful in a veterinary clinical setting, but the sensitivity to detect infection is too low for operational control programmes