Comparison of elapegademase and pegademase in ADA-deficient patients and mice.

The absence of adenosine deaminase (ADA) causes severe combined immune deficiency (SCID), which has been treated with PEGylated bovine-extracted ADA (ADAGEN). ADAGEN was recently replaced by a PEGylated recombinant bovine ADA, expressed in Escherichia coli (elapegademase, ELA-ADA). Limited information on ELA-ADA is available.  ADA enzymatic activity of ELA-ADA and ADAGEN was assessed in vitro at diverse dilutions. ADA activity and immune reconstitution in an ADA-SCID patient treated with ELA-ADA were compared with age-matched patients previously treated with ADAGEN. ADA activity and thymus reconstitution were evaluated in ADA-deficient mice following ELA-ADA or ADAGEN administered from 7 days postpartum. In vitro, ADA activity of ELA-ADA and ADAGEN were similar at all dilutions. In an ADA-SCID patient, ELA-ADA treatment led to a marked increase in trough plasma ADA activity, which was 20% higher than in a patient previously treated with ADAGEN. A marked increase in T cell numbers and generation of naive T cells was evident following 3 months of ELA-ADA treatment, while T cell numbers increased following 4 months in 3 patients previously treated with ADAGEN. T cell proliferations stimulation normalized and thymus shadow became evident following ELA-ADA treatment. ADA activity was significantly increased in the blood of ADA-deficient mice following ELA-ADA compared to ADAGEN, while both treatments improved the mice weights, the weight, number of cells in their thymus and thymocyte subpopulations. ELA-ADA has similar in- vitro and possibly better in-vivo activity than ADAGEN. Future studies will determine whether ELA-ADA results in improved long-term immune reconstitution.

T lymphocytes possess the machinery for 5-HT synthesis, storage, degradation and release.

AIM: Although activated T lymphocytes express tryptophan hydroxylase 1 and produce 5-HT, the metabolic fate and cellular handling of this 5-HT is unclear. Here, we investigated key proteins in T cells linked to 5-HT metabolism and storage and compare differences in 5-HT synthesis and metabolism between T-cell subsets. METHODS: We cultured human Jurkat T cells and mouse splenic CD3(+) , CD4(+) and CD8(+) T cells with or without T-cell activators (phorbol ester/ionomycin, concavalin A or plate-bound anti-CD3 antibody). Subsequently, we measured mRNA and/or protein for monoamine oxidase A and B, vesicular monoamine transporter 1 and 2, N-acetyl transferase and tryptophan hydroxylase 1. In addition, we measured the release of exogenously loaded [(3) H]5-HT and endogenously synthesized 5-HT from CD4(+) and CD8(+) T-cell subsets. RESULTS: Human and mouse T cells selectively express monoamine oxidase A. Following T-cell activation, mRNA levels of MAO-A increase robustly in parallel with tryptophan hydroxylase 1. Concomitant with these changes, T cells increase the expression of the type 1 vesicular monoamine transporter. Raised intracellular [Ca(2+) ] rapidly releases preloaded [(3) H]5-HT from CD4(+) and CD8(+) T cells indicating that these cells have the capacity for the storage and regulated secretion of 5-HT. Notably, both the expression of tryptophan hydroxylase 1 and monoamine oxidase A, and 5-HT production are significantly greater in CD8(+) compared with CD4(+) T cells. CONCLUSION: These data reveal coordinated changes in 5-HT production, metabolism and storage that may optimize 5-HT secretion from the CD8(+) T cell subset in response to activation stimuli.

Attenuation of massive cytokine response to the staphylococcal enterotoxin B superantigen by the innate immunomodulatory protein lactoferrin.

Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome.

Analysis of HIV type 1 protease and reverse transcriptase sequences from Venezuela for drug resistance-associated mutations and subtype classification: a UNAIDS study.

We report the first study on prevalence of antiretroviral drug-associated resistance mutations in Venezuela. Protease and reverse transcriptase (RT) coding regions were analyzed in DNA samples obtained from 100 HIV-1-infected individuals. Primary resistance mutations to RT inhibitors were identified in 26% of patients treated with these drugs. Transmission of HIV-1-resistant strains was detected in a drug-naive patient (3%). Primary resistance mutations to protease inhibitors (PIs) were present in 9% of the 44 PI-treated patients and in 1 PI-naive individual. Phylogenetic analysis of these samples has resulted in the most extensive survey, to date, of HIV-1 genetic forms circulating in Venezuela. Ninety-nine samples clustered with subtype B, and 1 individual harbored the first B/F recombinant virus reported in Venezuela, with protease clustering with subtype F and RT with subtype B. In addition, this isolate had a new insertion (Glu-34 duplication) in the protease gene.

Comparative performances of an HTLV-I/II EIA and other serologic and PCR assays on samples from persons at risk for HTLV-II infection.

BACKGROUND: HTLV-I and HTLV-II are related exogenous pathogenic human retroviruses. Until recently, ELISAs based on HTLV-I antigens have been used to screen for the presence of HTLV-I or -II antibodies. The HTLV-I-based assays have not been as sensitive in detecting antibodies to HTLV-II as in detecting antibodies to HTLV-I. The Abbott HTLV-I/HTLV-II ELISA uses a combination of HTLV-I and HTLV-II antigens to detect antibodies to the whole HTLV group. The performance of this ELISA was compared to that of several HTLV-I-based serologic assays and an HTLV-II PCR assay in cohorts of South American Indians and New York City IV drug users (IVDUs) in whom HTLV-II is endemic. STUDY DESIGN AND METHODS: Sera from 429 South American Indians and New York City IVDUs were evaluated for HTLV antibodies by the use of three ELISAs (rgp21-enhanced HTLV-I/II, Cambridge Biotech; Vironostika HTLV-I/II, Organon Teknika; and HTLV-I/HTLV-II, Abbott), and a Western blot (WB) assay. Peripheral blood leukocyte DNA from each person was analyzed for HTLV-I and HTLV-II pol DNA via PCR. The HTLV-II PCR-positive samples were further subtyped via cloning and sequencing and/or oligomer restriction. RESULTS: Two hundred four samples (48%) were positive for HTLV-II by serologic and/or PCR assays. All of the positive samples from the Indians and approximately one-third of the positive samples from the IVDUs were of the HTLV-IIB subtype. Comparative analyses indicate that the sensitivity and specificity of the various assays were: PCR, 98 and 100 percent; Abbott HTLV-I/HTLV-II, 78 and 95 percent; Cambridge Biotech HTLV-I/II, 76 and 96 percent; Vironostika HTLV-I/II, 71 and 98 percent; and WB, 73 and 100 percent, respectively. CONCLUSION: There were no significant differences among the sensitivities and specificities of the HTLV-I/II ELISAs (p values, 0.056-0.438). The WB and PCR assays were much more specific than the other serologic assays (p

The complete genomic sequence of an HTLV-II isolate from a Guahibo Indian from Venezuela.

A polyclonal CD3(+), CD8(+) T-cell line, G2, was derived from the peripheral blood of a seropositive, PCR-positive, HTLV-IIB infected Guahibo Indian from Venezuela. The cell line is productively infected with HTLV-IIB. The entire HTLV-II G2 proviral DNA was sequenced via PCR using overlapping HTLV-II primer pairs. Phylogenetic analyses indicate that HTLV-II G2 is the most divergent HTLV-IIB strain identified to date. Characterization of its deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

Endemic infection with HTLV-IIB in Venezuelan Indians: molecular characterization.

The peripheral blood of 41 Yaruro and Guahibo Indians from Venezuela was examined for HTLV antibodies and DNA. Twenty-five samples (61%) were found to be infected with HTLV-IIB. The sensitivities of the serologic and DNA polymerase chain reaction (PCR) analyses were 80% and 96%, respectively. Epidemiologic studies supported both sexual and perinatal transmission of the virus. Sequence analyses of the HTLV-IIB strains from these Indians indicate that they are unique relative to HTLV-II detected in other groups of humans. HTLV-IIB-G2 isolated from a Guahibo Indian is the most divergent HTLV-IIB strain relative to the prototype HTLV-II NRA.

Highly endemic human T-lymphotropic virus type II (HTLV-II) infection in a Venezuelan Guahibo Amerindian group.

Sera from 166 Guahibo Indians (55% of the population) living in southwest Venezuela were screened by enzyme-linked immunoassay for antibodies to human T-cell lymphotropic virus (HTLV) I and II. Positive samples were confirmed by immunofluorescence and Western blot. Forty-one Guahibos (24.8%) were found to be seropositive. Polymerase chain reaction (PCR) analysis of proviral DNA in mononuclear cell lysates revealed the virus to be HTLV-II. Prevalence increased with age, and sexual contact with HTLV-II-seropositive partners was identified as a risk factor for infection. PCR amplification of a region of the pol gene, utilizing the primer pair SK110/SK111, with subsequent digestion of the 140-base-pair amplification products with HinfI and MseI restriction enzymes, showed an HTLV-II subtype-b restriction pattern in all cases. These data suggest that the substrain infecting this Guahibo community belongs to the b subtype, the most frequent among Paleo-Amerindian populations.

First description of endemic HTLV-II infection among Venezuelan Amerindians.

We describe for the first time the presence of human T lymphotropic virus type II (HTLV-II) infection in Venezuela, among the Pume Amerindians living in the southern plains of the country. Antibodies to HTLV-II antigens were assessed by enzyme immunoassays (Elisa), Western blot, radioimmuno-precipitation, and immunofluorescence; titration studies against HTLV-I- and HTLV-II-infected cell lines were very useful in the differentiation of HTLV-I and HTLV-II antibodies. The HTLV-II general prevalence was 5%; however, there is a striking difference in prevalence between the truly isolated villages (0%) when compared to those living along the riverside and thus in contact with outsiders (9%). Preliminary evidence suggests sexual contact as the main source of transmission. These findings might suggest that HTLV-II in Venezuela originated through contact with outsiders rather than ancient infection related to the origins of the Pume.

Increased proportion of responders to a murine anti-CD3 monoclonal antibody of the IgG1 class in patients with systemic lupus erythematosus (SLE).

A group of Venezuelan patients with SLE showed an increased proportion of responders to Leu-4, an anti-CD3 MoAb of the IgG1 class, compared with ethnically matched non-SLE patients and healthy controls. The rate of proliferative responses or IL-2 production induced by MoAb Leu-4, and the helper effect of macrophages from Leu-4 responders on T cells from a third-party donor were comparable in patients and controls. No significant differences in the binding of murine IgG1 molecules by macrophages from SLE patients and controls were observed. The proportion of monocytes/macrophages expressing Fc gamma RI was significantly higher in SLE patients. However, the expression of FcRII, the type capable of supporting Leu-4-mediated responses, and of Fc gamma RIII was comparable in monocytes from SLE patients and controls. Our results suggest that Venezuelan patients with SLE may have a genetic predisposition for the expression of the phenotypic variant of Fc gamma RII capable of binding murine IgG1 molecules.

Bone marrow and peripheral blood natural killer cell activity in lymphomas. Its response to IL-2.

Natural killer (NK) cytotoxic activity was simultaneously investigated in bone marrow mononuclear cells (BMMC) and peripheral blood lymphocytes (PBL) from nine Hodgkin's disease (HD) and 15 non-Hodgkin lymphoma (NHL) untreated patients. Twenty-five PBL samples and seven bone marrow specimens from healthy individuals were also included as control group (C). NK cell activity was evaluated in basal condition and post-stimulation with human recombinant IL-2 (rIL-2). Data were expressed in K values (number of BMMC or PBL needed to lyse 50% of the target cells). In basal condition, both HD and NHL patients showed a NK cell activity comparable to the C group, both in BMMC (HD, K = 2.48 +/- 1.3; NHL, K = 3.8 +/- 2.0; C, K = 3.2 +/- 0.7) and PBL (HD, K = 2.0 +/- 1.0; NHL, K = 2.3 +/- 1.0; C, K = 2.2 +/- 0.2). Stimulation with rIL-2 induced a significant and comparable enhancement of the NK activity in PBL from HD, NHL and C while the response to rIL-2 of the BMMC in most of the HD and NHL patients was significantly greater than the C group. Responder cells were characterized by negative selection with specific MoAb plus complement as a CD3-, CD16+, CD56+ cytotoxic cell and further confirmed by flow cytometry. We postulate that IL-2 activation of bone marrow NK cell precursors, in addition to enhancing the activity of circulating NK, may be of value for the therapeutic rationale of IL-2 in patients with lymphoma.

Enhanced CD3-mediated T lymphocyte proliferation in patients with systemic lupus erythematosus.

Nonfractionated peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE) showed enhanced proliferative responses when stimulated via the CD3 pathway. In contrast, proliferative responses induced by phytohemagglutinin were diminished in SLE patients. Levels of CD3-induced interleukin-2 production and interleukin-2 receptor expression were comparable with normal levels. Highly purified T cells also showed augmented CD3 responses, but only in the presence of phorbol myristate acetate or a combination of phorbol myristate acetate plus calcium ionophore A23187, and not with calcium ionophore alone. The data suggest integrity of the T cell receptor/CD3 pathway for T cell activation in patients with SLE, as examined in cultures stimulated with specific anti-CD3 monoclonal antibodies rather than with multivalent lectins. An increased response via the CD3 complex could contribute to the autoimmune activity in human SLE.

Human IFN-gamma up-regulates IL-2 receptors in mitogen-activated T lymphocytes.

This study examined the role of human recombinant interferon-gamma (rIFN-gamma) in the expression of interleukin-2 receptors (IL-2R) by human T lymphocytes. rIFN-gamma enhanced total numbers of IL-2R in mitogen-activated but not resting T cells. Scatchard plot analysis indicated that rIFN-gamma increased both high- and low-affinity receptors, with a predominant effect on the latter. Phytohaemagglutinin (PHA)-activated T cells treated with IFN-gamma showed higher IL-2 binding and greater IL-2 internalization and degradation than cells treated with PHA alone. There was a corresponding increase of mitogen-driven proliferative responses, indicating an increase of functional receptors in IFN-treated cultures. IFN-gamma may influence T-cell activation and proliferation by enhancing expression of IL-2R and promoting IL-2 uptake by mitogen-activated lymphocytes.

Actividad NK en pacientes con linfoma: capacidad de respuedsta AIL-2 / NK activity in patients with lymphoma capacity response AIL-2

En el presente estudio se evaluó la SP y MO de 21 pacientes, no tratados con diagnóstico de linfoma y provenientes de la Unidad de Linfomas del Hospital Universitario de Caracas. El protocolo de investigación incluyó parámetros clínicos e histológicos. En los grupos estudiados se evaluó el porcentaje de células killer naturales (NK) mediante el uso de anticuerpos monoclonales, así como la actividad NK basal postestimulación in vitro con 500/ml de IL 2. Los resultados muestran que los porcentajes de células en SP y MO son similares en pacientes con EH y LNH en relación al grupo de control

Down-regulation of immunoglobulin and IgM-rheumatoid factor synthesis by oral treatment of rheumatoid arthritis patients with a nonsteroidal antiinflammatory drug.

We examined the effect of treatment with piroxicam, a nonsteroidal antiinflammatory drug (NSAID), on immunoglobulin (Ig) and IgM-rheumatoid factor (IgM-RF) synthesis in vitro by lymphocytes of patients with rheumatoid arthritis (RA). Oral treatment with piroxicam induced a progressive decrease of spontaneous IgM-RF production by unstimulated lymphocyte cultures during 12 weeks of observation. Also, pokeweed mitogen (PWM)-driven Ig synthesis was significantly diminished and the effect on total IgM production was sustained until the end of the study. This modulation of humoral responses is consistent with the drop in RF sera level. In addition, we also showed that treatment with NSAIDs can decrease RF levels in the synovial space. NSAIDs may have a long-term beneficial effect in patients with RA due to their modulatory role of lymphocyte responses.

Helper activity by human large granular lymphocytes in in vitro immunoglobulin synthesis.

In the present study we have examined the effect of human large granular lymphocytes (LGL) from healthy donors on Ig synthesis by autologous B lymphocytes. The results showed that this cell population has a consistent helper activity in pokeweed mitogen-activated cultures even when added at very low numbers. LGL can mediate their effect by secreting soluble helper factors capable of modulating B-cell responses as evidenced by the enhancement of IgG and IgM production by supernatants obtained from LGL cultures. Preincubation with interferon gamma further potentiated the helper activity by LGL.

Abnormal immunoglobulin and rheumatoid factor synthesis by blood lymphocytes in patients with primary Sjögren's syndrome.

Peripheral blood B lymphocytes from patients with primary Sjögren's syndrome showed significantly higher spontaneous synthesis of IgG, IgM, and IgM rheumatoid factor in vitro, compared with B lymphocytes from healthy controls. Lymphocytes from patients also showed higher IgM rheumatoid factor production after mitogen stimulation. Patients had competent suppressor activity for IgG, but not for IgM synthesis. Pre-irradiation of T cells, but not depletion of OKT8+ cells, markedly enhanced IgG synthesis in cocultures with autologous B cells; therefore, the T lymphocyte responsible for this effect is radiosensitive and is not identified by OKT8. OKT8+ lymphocytes from patients did not suppress Ig synthesis by autologous B plus T cell cocultures. However, OKT8+ cells from normal controls down-regulated Ig synthesis by B plus T cells from patients. The abnormal proportion of helper and suppressor cells suggests that there is altered redistribution of regulatory subpopulations in peripheral blood from Sjögren's syndrome patients.