AIDS has existed in subsaharan Africa at least since 1980. However, the genesis of this condition and its emergence as a health problem remain obscured by lack of data and by antibody data that are now questionable. In Africa, as elsewhere, AIDS is associated with an immunodeficiency associated with the LAV/HTLV-III retrovirus. The clinical manifestations vary somewhat because of the different range of opportunistic pathogens in that environment. Although the "classical," more indolent, endemic form of African Kaposi sarcoma is not associated with this virus or with immunodeficiency, a new, aggressive variety of Kaposi sarcoma in Africa appears to be. The origin of LAV/HTLV-III remains unclear, but the clinical syndrome of AIDS has emerged in Africa only in the past decade. A pattern of geographic spread can be recognized, in which AIDS was seen earliest in Kinshasa, Zaïre, and then emerged in Zambia, Rwanda, and Uganda. Recently, reports indicate spread into Tanzania and Kenya. Transmission appears to be primarily heterosexual, but the factors enhancing heterosexual spread in Africa to a greater extent than in the U.S. and Europe need to be further studied.
Membranes were prepared from mammary tumors and fractionized using gel filtration and gradient polyacrylamide gel electrophoresis. To obtain monoclonal antibodies against tumor-associated antigens, mice were immunized against membrane fraction 2a. After hybridization, we obtained 746 hybridoma cell lines. We performed the immunodot assay for screening. Every supernatant was tested against 15 antigens. Eight antigens were purified fractions from tumor membrane preparation, and five were crude membrane preparations from benign and malignant breast cell lines. Additionally, the Thomsen-Friedenreich antigen of erythrocytes was tested. We selected 83 hybridomas for further characterization.
Tuftsin is the tetrapeptide Thr-Lys-Pro-Arg. It is spontaneously released from the Fc fragment of IgG by two specific enzymes. One 25-micrograms dose administered to mice in good immunologic status stimulated phagocytosis, macrophage killing of tumor cells, delayed hypersensitivity, cytolytic T-cell activity, antibody production, antibody-dependent cell-mediated cytotoxicity (ADCC), and natural killer (NK) cell activity. Administered for 6 months at the dose of 10 micrograms once a week to old, immunodepressed mice, tuftsin restored macrophage and T-cell cytotoxic activities. At this dosage, tuftsin prevented spontaneous tumor development. Tuftsin was also well tolerated in phase I studies in humans in increased polymorphonuclear leukocytes and OKT4-positive lymphocytes. Bestatin is extracted from Streptomyces olivoreticuli. One 100-micrograms dose of bestatin injected in young mice with normal immunologic status increased macrophage cytotoxicity, antibody production, ADCC, and NK cell activities. Long-term administration of bestatin (100 micrograms once a week) corrected macrophage and T-cell cytotoxicity and prevented age-related spontaneous tumors. Bestatin inhibited lymphocyte membrane aminopeptidase, which degrades tuftsin into a tripeptide that is an antagonist competing with it for receptors. Tuftsin and bestatin constitute a biopharmacologic system that can be developed as other aminopeptidase inhibitors are available for study.
Although human tumor xenografts have been extensively used for preclinical evaluation of antitumor agents, most of this work has utilized subcutaneous or subrenal capsule assays based on change in tumor size. To obtain experimental models more reflective of the human clinical situations, we have developed several metastatic models that are based on and complement a panel of cell strains used in large-scale in vitro drug screening. One melanoma and four lung tumors produced metastatic lesions in the lung within 60 days following subcutaneous, intraperitoneal, or intrasplenic inoculation of BALB/C athymic nude mice. Several tumors also produced liver lesions, and one lung tumor strain showed metastasis to the brain. The metastatic lesions histologically resembled the tumors that grew at the inoculation site. In vitro and in vivo cell strains were rederived from the metastatic lesions. These systems may provide practical models for experimental drug and immunotherapeutic trials.
Picibanil (OK432), a pharmaceutical preparation of a low virulent Su strain of Streptococcus pyogenes, possesses cell regulatory activity particularly in its ability to augment natural killer (NK) cell activity and to activate macrophages to exert a tumoricidal effect both in vitro and in vivo. It is effective in retarding and/or inhibiting the growth of three different tumors: MBL-2 lymphoma, M109 alveolar adenocarcinoma, and B16 melanoma. The antitumor effect is mediated through regulation of NK cells and macrophages, possibly by its ability to stimulate the production and secretion of interferon and interleukin 1 and 2. It is a very effective adjuvant for tumor cell vaccines that elicit cytotoxic T-cell responses. Following cytoreductive chemotherapy (Cytoxan) Picibanil treatment leads to an earlier reconstitution of both bone marrow cellularity and differentiation to granulocyte-macrophage colonies.
Clinical data have shown that neoplastic diseases and/or related therapies frequently result in protein depletion of tumor-bearing patients. Depressions of acquired and specific immunity caused by protein depletion are well known. In an experimental model protein depletion was induced by lack of nutritional protein in otherwise isocaloric conditions in BALB/c and C57BL/6 mice over various time periods (max. 35 days). The results show that natural immune effector cells, natural killer cells, and monocyte/macrophages also during treatment with biological response modifiers (BRM) are depressed in their cytotoxic potentials in vitro and in vivo. Substantial and critical reductions of bone marrow cellularity (bone marrow nucleated cells) were also observed. In contrast, preliminary results show that if, following protein depletion, mice were treated parenterally with amino acids (Neo-aminomel, Boehringer-Ma. Co., FRG) complete restoration of immune parameters takes place. Adequate protein status is shown to be a crucial factor for natural immunity and therapy with BRM.
We studied the effects of exogenous T cell growth factor (TCGF) (= interleukin-2) and indomethacine on the lymphocyte transformation response in vitro to allogeneic cells, mitogens, and antigens in AIDS patients, those with AIDS-related complex (ARC), and in healthy controls. While low amounts of TCGF reduced the response of peripheral blood mononuclear cells (PBMC) to allogeneic cells in both healthy controls and AIDS patients, large amounts of TCGF augmented the response in both groups, although the response of the patients' cells were still subnormal. By depleting the PBMC for either CD4-positive or CD8-positive cells, the effect of TCGF on suboptimally mitogen-stimulated PBMC from controls was shown to be due to an increased response in both the CD4-positive and the CD8-positive cells. In contrast, with patient cells, TCGF only increased the response of the CD4-positive cells, while that of the CD8-positive cells was largely unchanged. Thus, the lack of normalization of the mitogen response of patient cells upon addition of TCGF may be largely due to unresponsiveness of CD8-positive cells to TCGF. This observation further supports the idea that CD8-positive cells are abnormal. To investigate the role of the inhibitor of TCGF production, PGE2, in AIDS, indomethacine was added to cultures of mitogen-stimulated PBMC from controls and patients. No differences were found between the three groups: the responses to PHA were slightly increased and those to Con A were unchanged.
Modulation of H-2Kb antigens on cells of a subline of the murine Lewis lung carcinoma was induced in vitro by a monoclonal antibody with specificity for H-2Kb antigens. High antibody concentrations resulted in a more spherical morphology of the cells, in a discrete loss of all antibody-antigen complexes within 4-6 hr, and in a corresponding maximal decrease in the total cellular antigen content 6-8 hr after antibody exposure. Restoration was complete within 2 hr after removal of the complexed antigen and occurred without any visible cap formation.
Biopsy specimens of lymphoid tissues were analysed by two-colour flow cytometry to determine the proportions and phenotypes of natural killer-like cells present in the lesions. No significant difference was found between the proportions of Leu 7+ cells in reactive and malignant nodes. Low numbers of Leu 11+ cells were found in both benign and malignant nodes. The most common phenotype among the tumour-infiltrating Leu 7+ cells in the malignant nodes was Leu 7+OKT3+OKM1-. Only low numbers of Leu 7+ cells in malignant nodes coexpressed OKM1. Isolated Leu 7+ cells from four out of five malignant nodes were unable to lyse autologous B lymphoma cells in vitro. However, in one of five malignant nodes tested, autologous B lymphoma cells were lysed by isolated tumour-infiltrating Leu 7+ cells but not by Leu 7- cells. These observations indicate that tumour-infiltrating Leu 7+ cells are infrequently capable of lysing autologous lymphoma cells.
Studies in hemophiliacs receiving factor concentrates demonstrated T-cell defects in vitro. Recently, B-cell dysfunctions were described in AIDS and pre-AIDS and in some hemophiliacs. To investigate the B-cell function in hemophiliacs in relation to factor substitution, we examined five patients with mild (substitution less than 20,000 U/year) and seven with severe (greater than 100,000 U/year) hemophilia A and compared the data with normal control individuals. The B-cell proliferative response (3H-thymidine uptake) to Staphylococcus aureus Cowan I and the differentiation response (Ig secretion into culture supernatants) to T-cell-dependent or -independent polyclonal B-cell activators (PBAs) were studied in vitro. In contrast to T-cell dysfunctions, which correlate with the amount of clotting factor concentrates, the B-cell proliferative response was not affected. Stimulation with PBAs however failed to increase elevated spontaneous IgG levels and showed a diminished increase in IgM levels in severe, but not in mild, hemophilia. Our data give evidence of a T-cell-independent B-cell dysfunction in asymptomatic hemophiliacs that correlates with factor substitution.
The specificities of six monoclonal antibodies produced against plasminogen activator of the human Bowes melanoma cell line are described. They have been used to detect membrane-bound plasminogen activator on cultured human lymphoid cell lines and in neoplastic human lymphocytic and myeloid cells of leukemic patients. These studies indicate that only certain phenotypic subsets of the T-cell lineage derived from patients with chronic lymphocytic leukemia or with Szezary syndrome express plasminogen activator on their surface membrane.
Pregnancy is a natural allograft and the mechanisms for its non-rejection are obscure. Depression of maternal cellular immunity was suggested as a possible explanation. Interleukin-2(IL-2) is a lymphokine release from OKT4+ lymphocyte. This factor has a crucial role in the proliferation and differentiation of T cell subsets, and controls functions associated with immune rejection mechanisms. We therefore examined the ability of lymphocytes from women in the 3 trimesters of pregnancy to produce IL-2 in culture. Mononuclear cells were cultured with PHA for 48 h. The IL-2-containing supernatant was added to and supported the proliferation of an IL-2 dependent T cell line. Proliferation of this line indicated the IL-2 content of the added supernatant. Using this assay, IL-2 production in all 3 trimesters of pregnancy was adequate and comparable to that of lymphocytes from non-pregnant women. These results suggest that the proposed defect in cellular immunity during pregnancy is not mediated by an inability of the lymphocytes to produce IL-2.
Patients with certain malignant diseases excrete in their urine elevated levels of modified nucleosides originating predominantly from the breakdown of transfer RNA (tRNA). Acquired immune deficiency syndrome (AIDS), often associated with rapidly progressing Kaposi's sarcoma (KS), is currently occurring in many countries. Male homosexuals are considered to be at highest risk of developing these disorders. We have previously reported that patients with AIDS excrete elevated levels of modified nucleosides. In this communication, we report on modified nucleoside levels measured in 77 male homosexuals without clinical manifestations of AIDS at the time of examination. A high frequency of abnormal nucleoside levels was found in this high-risk group. There was a trend towards higher levels in individuals with lymphadenomegaly, considered a prodrome of AIDS. Statistically significant correlations were found between some of the nucleosides (pseudouridine and dimethylguanosine) and degree of lymphadenomegaly. Pseudouridine, 1-methyl-adenosine and dimethylguanosine were inversely related to percentages of total T-lymphocytes (T11), suppressor T-lymphocytes (T8), and number of natural killer cells (Leu-7). These findings suggest that determination of urinary nucleoside levels may help identify individuals at high risk of developing AIDS.
T-cell colonies were generated from the peripheral blood and bone marrow of 61 patients with acquired immunodeficiency syndrome (AIDS), 54 patients with persistent lymphadenopathy syndrome (LAS), 14 clinically normal male homosexuals, and 17 healthy heterosexuals. Mononuclear cells were cultured in methylcellulose in the presence of IL2-containing conditioned medium. The number of T-cell forming cells (T-CFC) from healthy male homosexuals and AIDS and LAS patients was significantly (P less than 0.01) reduced compared to T-CFC from healthy heterosexuals. In AIDS patients, the low colony growth capacity of T-CFC was independent of the presence of either opportunistic infections or Kaposi sarcoma. Twelve LAS patients who subsequently developed AIDS showed the lowest capacity of peripheral blood and bone marrow T-CFC to proliferate. Pooled induced colonies from AIDS and LAS patients and normal homosexuals were composed of immature cells bearing the T3+, T4+, T6+, and T8+ surface phenotype, unlike colonies from normal heterosexuals, which displayed mature cells bearing the T3+, T4+, T6-, and T3+, T8+, T6- surface phenotype. Moreover, most T-CFC from primary colonies had lost their self-renewal capacity. In some AIDS and LAS patients but not healthy homosexuals peripheral blood and bone marrow T-CFC were capable of generating colonies with recombinant IL2 (rIL2) without any other mitogenic stimulation. The rIL2-induced colony growth was abrogated by a monoclonal antibody against the IL2 receptor. These results suggest that early impairment of T-CFC plays a predominant role in the pathogenesis of AIDS.
Urinary neopterin levels are raised with a high incidence in all risk groups for AIDS. Neopterin elevations reflect activated cellular immunity in risk group members, in some cases independently of LAV/HTLV-III infection. Moreover, we are able to show that in patients receiving multiple blood transfusions at least a transient challenge of cell-mediated immunity occurs, which is indicated in part by increasing neopterin levels. We conclude that neopterin levels are a reliable index for assessment of susceptibility for AIDS when infection with LAV/HTLV-III occurs. Activated status of cell-mediated immunity might predispose infected persons to an overwhelming infection and secondary spreading of LAV/HTLV-III, thus leading to the development of full-blown AIDS or ARC. As a consequence of these observations, T-cell-stimulatory actions and agents should intentionally be avoided. Treatment of AIDS patients with immunosuppressants should be examined. The success of therapeutic regimens should be monitored by measurement of neopterin levels.
In addition to the well known T-cell dysfunctions in AIDS, hypergammaglobulinaemia and autoimmune phenomena indicate an involvement of the B cell as well. Reports of HTLV-III/LAV-infected B cells suggest T-cell-independent B-cell abnormalities. To look for early B-cell dysfunctions, we examined a high-risk group of AIDS consisting of six homosexuals with PGL and HTLV-III/LAV antibodies, comparing these data to those of patients with AIDS/ARC and a normal control. In vitro studies included the B-cell proliferation response (3H-thymidine uptake) to Staphylococcus aureus Cowan I and the differentiation response (Ig secretion into culture supernatants) to T-cell-dependent/-independent polyclonal B-cell activators (PBAs). Profound alterations were found in both the proliferation and the differentiation responses. The weak response even to T-cell-independent PBAs indicates a B-cell dysfunction that is not due only to a T-cell defect in patients with PGL, similar to that observed in AIDS.
Several biological response modifiers (BRMs) were demonstrated to increase myelopoiesis and effector cell responses (M phi and natural killer cell activity) in vivo. The increased myelopoiesis was reflected by an increase in bone marrow cellularity and granulocyte-M phi colony-forming cells (GM-CFU-C). The increase in myelopoiesis appeared to be related to a concomitant increase in colony-stimulated factor (CSF) production and secretion by M phi and bone marrow cells. CSF induction by BRMs increased myelopoiesis and counteracted the myelosuppressive and immunosuppressive effects of cyclophosphamide. CSF induced in vivo by BRMs attained high titers and were maintained over a longer period than exogenously injected CSF, which was rapidly cleared from serum.
Experimental studies of the survival curves of different species and of in vitro cell culture survival point to normal aging as a largely preprogrammed process. Reciprocal skin grafting among syngeneic young and old animals further demonstrates autonomous aging of this organ independent of the remainder of the body. This compartmentalization of change with age is also suggested when immune functions of various lymphoid organs are compared at different ages. Experimental studies also show that for some organs their susceptibility to certain carcinogens is diphasic, high early in life and high in senescence. A major question now is if preprogrammed age changes at the molecular level have steps in common with carcinogenetic processes.
An increased incidence of lymphoid neoplasias is associated with the states of immune deficiency both congenital and acquired. Thirty-five cases of lymphoma in males at high risk for AIDS were diagnosed in one community hospital in New York City within the past 2 years. The mean age of these patients was 39.6 years; 34 were homosexual, and one was an intravenous drug abuser. There were four Hodgkin and 31 non-Hodgkin lymphomas of various histologic types but almost all of high-grade categories. The proportion of extranodal lymphomas, the involvement of the gastrointestinal tract, central nervous system, bone marrow, and myocardium were significantly higher than in the lymphomas of the general population. The phenotypes were B-cell and non-B-non-T-cell types without any T-cell lymphomas. All patients had reversed helper-suppressor T-cell ratios, and all those tested had circulating HTLV-III and antilymphocyte antibodies. Nine patients have had previous lymph node biopsies showing the lesions of AIDS-related lymphadenopathies that were often directly associated with lymphoma. A variety of severe opportunistic infections and Kaposi sarcoma affected these patients. All lymphomas associated with immune deficiency were highly aggressive, involved multiple organs, and responded poorly to treatment resulting in early deaths.
