Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis

We report the clinicopathologic characteristics of pulmonary lymphomatoid granulomatosis (LYG) in 11 patients (identified from a series of 330 consecutive patients who underwent autopsy between 1984 and 1995 at the University of Texas Medical Branch at Galveston, Texas) with a diagnosis of acquired immunodeficiency syndrome (AIDS). We used immunohistochemical stains, RNA in situ hybridization (ISH), and gene rearrangement studies to identify the immunophenotype and the presence or absence of Epstein-Barr virus (EBV) infection. All of the patients were men ranging in age from 27 to 65 years (mean age, 38.6 yr). Autopsy lungs of 21 age-matched controls were examined for EBV using ISH; these included 9 patients with AIDS who did not have pulmonary lesions and 12 HIV-negative individuals who died accidentally (mean age, 38.6 yr). All of the 11 pulmonary lesions showed the gross and microscopic characteristics of LYG, with zonal necrosis and prominent angioinvasion. The tumor nodules consisted of a mixture of atypical large lymphocytes, with vesicular nuclei and prominent nucleoli and with a background of small and intermediate-size lymphocytes, histiocytes, and plasma cells. The large lymphocytes were CD20 positive, consistent with a B-cell phenotype. Ten of the 11 cases demonstrated EBV1-encoded RNA and CD20 positivity in the large, atypical lymphocytes by double labeling. One patient showed EBV positivity in CD20-negative, CD45RO-positive large cells, but these cells were CD3 negative and showed a monoclonal heavy chain gene rearrangement by polymerase chain reaction, indicating that these were of B-cell origin. Aberrant CD43 coexpression was identified in four cases. EBV latent membrane protein was demonstrated in 9 of 11 cases by immunohistochemical stains. The lungs of all of the 21 control patients were negative for EBV by ISH. We conclude that, in our series, AIDS-associated LYG is a B-cell neoplasm and that it has a strong association with EBV infection.     

Epstein-Barr virus associated natural killer cell leukemia: report of an autopsy case

A 20-year-old female was admitted because of high fever, hepatosplenomegaly, severe hepatic dysfunction and coagulopathy. Peripheral blood showed pancytopenia and granular lymphocytes bearing the natural killer cell phenotype (CD2+CD3-CD16+CD56+CD57-TCR alpha beta-TCR gamma delta-) constituted 97% of leucocytes. Southern blot analysis of DNA obtained from peripheral blood mononuclear cells showed germ-line configuration of TCR beta, gamma and delta chain genes. EBV-DNA was detected in a single episomal form by using EBV-terminal repeat probe. Bone marrow findings were consistent with hemophagocytic syndrome and administration of VP-16 was effective transiently. After ten months she died from massive gastrointestinal bleeding. An in situ hybridization study identified EBV-RNA (EBER-1) in atypical lymphocytes infiltrating bone marrow, spleen and lymph nodes. Sections of liver showed steatosis and infiltration of T cells (CD3+ and EBER-1-negative) in the portal areas and few atypical lymphocytes in sinusoids. The patients developed an EBV-associated clonal proliferation of natural killer (NK) cells, but the clinical features were suggestive of chronic active EBV infection or virus-associated hemophagocytic syndrome (VAHS) rather than leukemia. Bone marrow transplantation for NK cell leukemia is an issue to be discussed.     

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage

The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement.     

Monoclonality in reactive lymphadenopathy: gene rearrangement and multiparameter analysis

Multiparameter analysis of lymph nodes with follicular, interfollicular, and/or atypical hyperplasia was undertaken to search for monoclonality. Twenty-three patients aged 7 to 75 years (mean 32 years) were studied. One patient had a history of lymphoma; two were HIV-positive. Nodes were removed for clinical suspicion of lymphoma. Light microscopy revealed increased and&or abnormal follicular proliferation and occasional progressive transformation of germinal centers. Immunostaining of frozen sections revealed CD4, CD8, kappa, and lambda positivity with more CD4+ than CD8+ cells. Flow cytometry showed a mixed population of T and B cells with no evidence of clonality. Hybridization studies with JH and JK probes showed rearranged bands in one case. No rearrangements were seen with CT beta and bcl-2 probes. Follow-up of 3 to 5 years showed no new occurrences of lymphoma. Although no evidence of monoclonality was seen with other parameters, DNA hybridization revealed heavy and light chain gene rearrangement in 4% (1 of 23).     

T lymphocyte roles during Eimeria acervulina and Eimeria tenella infections

This study evaluated the effects of selective depletion of T lymphocytes on Eimeria infections in chickens. Cell depletions were initiated in day- or week-old Hyline SC strain chickens using intra-peritoneal injections of monoclonal antibodies to CD4, CD8, or T cell receptor (TCR) alpha/beta. Control chickens received injections of irrelevant monoclonal antibody or phosphate buffered saline (PBS). Following the establishment of cell depletion, chickens were infected orally with E. acervulina or E. tenella, 1 x 10(4) oocysts for primary infections and 2 x 10(5) oocysts for secondary infections. Chickens treated with anti CD4 monoclonal antibody produced significantly more oocysts than controls following primary E. tenella but not E. acervulina infections. Development of resistance to challenge infection was unaffected. These results suggest that CD4+ lymphocytes are important in controlling primary infection with E. tenella. Chickens treated with anti-CD8 or anti-TCR alpha/beta monoclonal antibodies produced significantly fewer oocysts than controls following primary infection but significantly more oocysts than controls following secondary infection with both E. tenella and E. acervulina. Additionally, anti-CD8 treatment abrogated resistance to challenge infection. CD8-depleted chickens may exhibit decreased oocyst production following primary infection due to a lack of CD8+ lymphocytes to serve as transporting cells for sporozoites. The abrogation of resistance to secondary infection in CD8- and TCR alpha/beta-depleted chickens suggests that these cells are necessary for the development of protective immunity to coccidia.     

Migration resistant, blood-compatible plasticized polyvinyl chloride for medical and related applications

AIMS: To determine the morphology, immunophenotype and bcl-2 protein status of intraepithelial lymphocytes in HIV-positive lymphoepithelial lesions. METHODS AND RESULTS: Seventeen cases (from adults and children) of HIV-associated parotid and lung lymphoid lesions were examined. In addition, three lymphoepithelial cysts from HIV-negative patients were studied in parallel. Immunohistochemistry was performed on paraffin embedded tissue with the following antibodies: CD20, CD79a, CD3, CD4, CD8, bcl-2, CAM5.2, AE1/3, MIB1, kappa/lambda light chains and EBV-LMP-1. Heavy chain rearrangement was sought by polymerase chain reaction (PCR) in four of the cases. The lymphocytes participating in lymphoepithelial lesions of HIV-positive patients had the morphology of centrocyte-like cells with occasional cells resembling centroblasts. The majority of these cells were of B-cell lineage, but occasional intraepithelial T-cells (CD8 positive, CD4 negative) were also present. T-cells also formed a significant component of the infiltrative lymphoid cells outside the lymphoepithelial lesions. These were mainly CD8 positive, but very occasional CD4-positive T-cells were also noted. None of the cases showed light chain restriction and the four cases did not demonstrate heavy chain rearrangement by molecular biology. The interesting finding was the absence of bcl-2 expression by the intraepithelial lymphocytes. In contrast, the intraepithelial lymphocytes seen in the non-HIV setting were strongly bcl-2 positive. The majority of these were B-cells, and very occasional CD8 and CD4 positive T-cells formed the intraepithelial population. CONCLUSION: It is postulated that this finding is due to the HIV causing down-regulation of bcl-2 protein.     

Study of T-lymphocyte subsets of healthy and Mycobacterium avium subsp. paratuberculosis-infected cattle

The relative contributions of T-lymphocyte subsets to host defense in cattle infected with Mycobacterium avium subsp. paratuberculosis is reported. The subsets were purified with appropriate monoclonal antibodies and a magnetic bead column separation system, and their purity was verified by flow cytometry. Biological activity of each subset, expressed as lymphoproliferation and gamma interferon (IFN-gamma) production, was measured in response to phytohemagglutinin (PHA) and an M. avium antigen preparation (A-PPD). IFN-gamma was measured by antibody capture enzyme-linked immunosorbent assay. The results showed a correlation between proliferation and IFN-gamma production in response to A-PPD but not to PHA. In response to PHA, CD4+ lymphocytes were the most prolific producers of IFN-gamma. CD8+ lymphocytes produced IFN-gamma to a lesser extent, whereas gammadelta+ T lymphocytes produced little or no IFN-gamma. Differences observed between the amount of IFN-gamma produced by CD4+ versus CD8+ cells and CD4+ versus gammadelta+ cells were significant (P < 0.01), but those between peripheral blood mononuclear cells (PBMC) and CD4+ T cells were not. Similar responses to A-PPD were observed except that PBMC produced higher levels of IFN-gamma than did CD4+ T cells. These data for cattle are similar to observations made for other animal species, where CD4+ cells are the major type of T lymphocytes producing IFN-gamma. They further suggest that whatever the role gammadelta+ T cells may play in paratuberculosis, it is not likely to be mediated by IFN-gamma production.     

Tumour radiosensitization by high-oxygen-content gases: influence of the carbon dioxide content of the inspired gas on PO2, microcirculatory function and radiosensitivity

The rat testis is considered to be an immunologically privileged site because of its reduced capacity to support antigen-specific immune responses. To understand this phenomenon, it is essential to characterize both the lymphocyte subpopulations normally present in the testis and their regulation by testicular cytokines. Peripheral blood was obtained from adult male Dark Agouti or Sprague-Dawley rats, and testicular interstitial tissue was collected after perfusion of the testes to remove blood. Blood and testis lymphocytes were isolated using discontinuous Percoll density gradients, and the testicular lymphocytes were further purified by selective adherence to remove mononuclear phagocytes. The isolated lymphocytes were analyzed by flow cytometry using specific monoclonal antibodies and fluorescein labeling and were enumerated as total T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells. In contrast to peripheral blood, in which the CD4+ T-cell subset was the major lymphocyte subset, rat testis T cells were predominantly of the CD8+ subset, and a large population of NK cells also were present. Subsequently, peripheral blood lymphocytes were stimulated with the polyclonal T-cell activator, phytohemagglutinin, and cultured in the presence of activin, inhibin, or transforming growth factor beta (TGFbeta) prior to flow cytometric analysis. Activin and TGFbeta suppressed T-cell proliferation without any selective effect on either T-cell subset, and inhibin had no effect. The predominance of CD8+ T cells and NK cells, and the relatively minor proportion of CD4+ T cells, are consistent with both increased cellular immune surveillance and a reduced capacity for initiating antigen-specific immune responses in the adult rat testis.     

Expression of the CD8alpha beta-heterodimer on CD8(+) T lymphocytes in peripheral blood lymphocytes of human immunodeficiency virus- and human immunodeficiency virus+ individuals

CD8(+) T lymphocytes play a pivotal role in controlling human immunodeficiency virus (HIV)-1 replication in vivo. We have performed four-color flow cytometric analysis of CD8(+) peripheral blood lymphocytes (PBL) from 21 HIV-1 seronegative and 103 seropositive individuals to explore the phenotypic heterogeneity of CD8beta-chain expression on CD8(+) T lymphocytes and to clarify how its expression on CD8(+) T lymphocytes may relate to acquired immunodeficiency syndrome (AIDS) clinical progression. We showed that the single monoclonal antibody (MoAb) 2ST8-5H7, directed against the CD8alpha beta-heterodimer, identifies CD8(+) T lymphocytes as effectively as the conventional combination of anti-CD3 and anti-CD8alpha antibodies. However, we detected a significantly lower mean fluorescence (MF) of anti-CD8alpha beta staining on PBL from HIV-1 seropositive donors as compared with seronegative donors. In fact, CD8(+) T lymphocytes from HIV-1-infected individuals with the lowest CD4 counts showed the lowest levels of CD8alpha beta MF. To explore further this change in CD8alpha beta expression, we assessed the expression of 14 different cell surface molecules on CD8alpha beta+ T lymphocytes of PBL from 11 HIV-1 seronegative and 22 HIV-1 seropositive individuals. The MF of anti-CD8alpha beta staining was significantly reduced on CD8(+) T lymphocyte subsets that showed immunophenotypic evidence of activation. The subset of lymphocytes expressing low levels of CD8alpha beta expressed higher levels of activation, adhesion, and cytotoxic-associated molecules and was predominantly CD45RO+ and CD28(-). Finally, we monitored the expression of the CD8alpha beta-heterodimer on PBL of eight HIV-1-infected individuals over a 16-week period after the initiation of highly active antiretroviral therapy (HAART), including zidovudine (ZDV), lamivudine (3TC), and indinavir (IDV), and found a significant increase in the expression of the CD8alpha beta-heterodimer. These results suggest that antibodies recognizing the CD8alpha beta-heterodimer are useful tools to specifically identify CD8(+) T lymphocytes. Moreover, the quantitative monitoring of CD8alpha beta expression allows the detection of discrete CD8(+) T lymphocyte subsets and may be useful for assessing the immune status of individuals infected with HIV-1.     

CD8 dimer usage on alpha beta and gama delta T lymphocytes from equine lymphoid tissues

Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.     

Development of autologous, oligoclonal, poorly functioning T lymphocytes in a patient with autosomal recessive severe combined immunodeficiency caused by defects of the Jak3 tyrosine kinase

Defects of the common gamma chain subunit of the cytokine receptors (gamma c) or of Jak3, a tyrosine kinase required for gamma c signal transduction, result in T-B+ severe combined immunodeficiency (SCID). However, atypical cases, characterized by progressive development of T lymphocytes, have been also reported. We describe a child with SCID caused by Jak3 gene defects, which strongly but not completely affect Jak3 protein expression and function, who developed a substantial number (> 3,000/microL) of autologous CD3+CD4+ T cells. These cells showed a primed/activated phenotype (CD45R0+ Fas+ HLA-DR+ CD62L(lo)), defective secretion of T-helper 1 and T-helper 2 cytokines, reduced proliferation to mitogens, and a high in vitro susceptibility to spontaneous (caused by downregulation of bcl-2 expression) as well as activation-induced cell death. A restricted T-cell receptor repertoire was observed, with oligoclonal expansion within each of the dominant segments. These features resemble those observed in gamma c-/y and in Jak3-/- mice, in which a population of activated, anergic T cells (predominantly CD4+) also develops with age. These results suggest that residual Jak3 expression and function or other Jak3-independent signals may also permit the generation of CD4+ T cells that undergo in vivo clonal expansion in humans; however, these mechanisms do not allow development of CD8+ T cells, nor do they fully restore the functional properties of CD4+ T lymphocytes.     

Bispecific monoclonal antibodies for intravenous treatment of carcinoma patients: immunobiologic aspects

Immunobiologic parameters measured during a phase I trial of intravenously (i.v.) administered bispecific monoclonal antibodies (BsmAb) in renal cell carcinoma (RCC) patients are described. The BsmAb used, BIS-1, is reactive with a pancarcinoma-associated 38 kDa transmembrane glycoprotein, EGP-2, as well with the CD3 complex. Patients received during a 2 h i.v. infusion F(ab')2 fragments of BIS-1 at doses of 1, 3, or 5 micrograms/kg body weight during concomitantly applied subcutaneous (s.c.) IL-2 treatment. Acute but transient BIS-1 F(ab')2-related toxicity was observed at the 3 and 5 micrograms/kg dose level, and the maximum tolerated dose (MTD) was set at 5 micrograms/kg. A dose-dependent binding of BIS-1 F(ab')2 to circulating T lymphocytes was found. The in vivo occupancy of CD3 molecules on T lymphocytes was highest at teh end of the infusion period and then rapidly decreased, as shown by flow cytometry. A much slower decrease of BIS-1 F(ab')2 binding was observed in vitro, suggesting migration of BIS-1 F(ab')2-loaded T lymphocytes from the circulation. A strong but transitory leukopenia was observed, in which LFA-1 alpha bright, CD3/CD8 double positive T cells left the circulation preferentially. This phenomenon was most likely induced by elevated TNF-alpha and IFN-gamma plasma levels, which were at a maximum shortly after the end of the infusion. Isolated peripheral blood mononuclear cells obtained from patients directly after treatment with BIS-1 F(ab')2 at the 3 and 5 micrograms/kg dose level showed increased EGP-2-directed antitumor activity.     

Effects of lovastatin on natural killer cell function and other immunological parameters in man

Suppression of cholesterol synthesis by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, such as lovastatin, has been shown to inhibit mitogen stimulated proliferation of natural killer (NK) cells and other lymphocytes in vitro. This effect is only partially overcome by provision of exogenous free or lipoprotein cholesterol but is reversed by mevalonate, suggesting that proliferating lymphocytes have a specific requirement for a nonsterol isoprenoid product of mevalonate. The effect of lovastatin (20 mg bid) on a range of immune function parameters was determined in a randomized, placebo-controlled, double-blind ex vivo study in 52 patients with primary hypercholesterolemia. No significant differences (P < 0.05) were found between lovastatin and placebo groups for basal NK or interleukin-2 (IL-2)-induced cell-mediated cytotoxicity, PHA-stimulated lymphocyte proliferation, or relative numbers of T lymphocytes (CD3+), B lymphocytes (CD19+), total NK cells (CD3-, CD16+, CD56+) and CD57+ NK cells or in immunoglobulin levels after 4 or 8 weeks of treatment. In contrast to previous in vitro data, no statistically or clinically significant changes were observed in any parameter of lymphocyte function in patients treated with lovastatin.     

Chronic lymphocytic leukaemia with binucleated lymphocytes

Chronic lymphocytic leukaemia (CLL) is a monoclonal proliferation usually involving B cells and composed of mature lymphoid cells. Distinct morphologic subtypes have been recognized according to lymphocyte size, nuclear:cytoplastic ratio and nucleolus. However the presence of characteristically binucleated lymphocytes in patients fulfilling criteria for CLL diagnosis has never been described. We here report immunological and cytogenetic studies of four patients with CLL but with binucleated lymphocytes. Moreover, trisomy 12, known to be associated with atypical morphology in CLL, was detected in two of these four patients. We suggest that this be considered as a possible new entity.     

Molecular genetic analysis of the pullulanase B gene of Bacillus acidopullulyticus

In this study we describe immunotoxins prepared with different CD2 monoclonal antibodies (mAbs) and a ribosome-inactivating protein, saporin. The CD2 immunotoxins were tested on different models. Anti-CD2-saporin conjugates inhibited protein synthesis by a neoplastic CD2+ cell line (SKW-3) and by an interleukin 2 dependent polyclonal CD2+ lymphoid cell culture (T lymphoblasts), with IC50s ranging from 10(-13) M to 10(-11) M (as saporin). Similar results were obtained with proliferation inhibition tests (3H-thymidine incorporation) on phytohaemagglutinin (PHA) driven lymphoid cultures and on mixed lymphocyte culture activated lymphocytes. Moreover a CD2-ricin A chain conjugate was less effective than an analogous immunotoxin containing the same CD2 mAb and saporin in inhibiting lymphocyte proliferation induced by PHA (IC50 approximately 10(-9) M as ricin A chain versus 10(-12) M as saporin). The conjugates were not toxic on bone marrow stem cells. These results suggest that CD2-saporin immunotoxins could represent an effective tool for CD2+ lymphomas or leukaemias, and for T-dependent immune disorders, such as transplanted organ rejection and graft-versus-host disease.     

Production of monoclonal antibody to CD4 antigen and development of reagent for CD4+ lymphocyte enumeration

A hybridoma secreting monoclonal antibody (mAb) specific to CD4 protein was generated. This monoclonal antibody, named MT4, was proved to be specific to CD4 protein as it reacted with CD4-DNA transfected COS cells, CD4+ cell lines and CD4+ lymphocytes. Furthermore, MT4 mAb inhibited the binding of standard CD4 monoclonal antibodies to CD4 proteins on CD4+ cells. To develop a home made reagent for CD4+ lymphocyte determination by flow cytometry, fluorescein isothiocyanate (FITC) was conjugated to MT4 mAb. To evaluate the developed reagent, 30 HIV infected and 30 healthy individuals were determined for CD4+ lymphocytes by using both a commercial Simultest reagent kit and home made FITC labeled MT4 mAb simultaneously. The study has shown that both percentages and absolute CD4+ lymphocyte counts obtained from both reagents were equivalent. The correlation coefficient for regression analysis was 0.995 and 0.996 for percentages and absolute CD4+ lymphocyte counts, respectively. The results suggest that home made FITC labeled MT4 reagent is an acceptable alternative reagent for monitoring CD4+ lymphocytes in blood samples by flow cytometry.