Impact of MHC class I alleles on the M. tuberculosis antigen-specific CD8+ T-cell response in patients with pulmonary tuberculosis.

Challenged by scattered understanding of protective immunity to Mycobacterium tuberculosis (MTB), we have mapped peptide epitopes to human leukocyte antigen (HLA)-A*0101, A*0201, A*1101, A*2402, B*0702, B*0801 and B*1501 of the secreted mycobacterial antigen Ag85B, a vaccine candidate that may be associated with immune protection. Affinity (ED(50)) and half-life (t(1/2), off-rate) analysis for individual peptide species on HLA-A and HLA-B molecules revealed binding ranges between 10(-3) and 10(-7) M. After selection of the best matches, major histocompatibility complex class I/peptide tetramer complexes were constructed to measure the CD8+ T-cell responses directly ex vivo in peripheral blood mononuclear cells (PBMC) derived from 57 patients with acute pulmonary tuberculosis. Three patterns of (allele-) specific CD8+ recognition were identified: (a). Focus on one dominant epitope with additional recognition of several subdominant T-cell epitopes (HLA-A*0301, A*2402, B*0801 and B*1501); (b). Co-dominant recognition of two distinct groups of peptides presented by HLA-B*0702; and (c). Diverse and broad recognition of peptides presented by HLA-A*0201. Peptides that bound with slow off-rates to class I alleles, that is HLA-A*0201, were associated with low frequency of CD8+ T cells in PBMCs from patients with tuberculosis. HLA-B alleles showed fast off-rates in peptide binding and restricted high numbers (up to 6%) of antigen-specific CD8+ T cells in patients with pulmonary tuberculosis.

Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells.

Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching.

We have studied the pathogenic changes in Khaki Campbell ducks injected in mid embryogenesis with ALV subgroup C virus td daPR-C derived from a molecular clone. The employed duck flock was shown to be highly genetically homogeneous and was controlled for the absence of current infections. Clear symptoms of wasting disease, which appeared since one week post hatching, represented the early consequence of the virus infection. They were manifested by decreased body weight, including clear involution of thymic tissue and pronounced anaemia. Microscopically, thymuses of infected animals displayed lymphatic depletion, clearly visible in the lobular cortex. Similarly, in the bursa Fabricii follicles, a marked reduction of the cortical layer and a decrease in folicullar centres was revealed. A decrease in the antibody response correlated with bursa Fabricii atrophy. The clear signs of anaemia were confirmed by haematological measurements, red blood cell count, haematocrit value and haemoglobin included. On the basis of these and additional observations we propose that inoculation of duck embryos provides a suitable model for analysis of the wasting disease produced by ALV-C.

HIV-1 protease inhibitor ritonavir modulates susceptibility to apoptosis of uninfected T cells.

OBJECTIVE: Clinical experience with HIV-1 protease inhibitors (PIs) in the treatment of AIDS frequently has shown that increases in CD4+ T-cell counts can be independent of HIV-1 inhibition by these drugs. This disconnection between viral load and CD4 counts led us to investigate how the PI ritonavir directly affects leukocyte activation in vitro, using peripheral blood mononuclear cell (PBMC) fractions derived from normal donors. METHODS AND RESULTS: When uninfected PBMC cultures were treated for 72 hours with ritonavir at concentrations similar to or lower than that shown to be effective in vivo, an increase in cell viability was observed. The susceptibility of PBMCs to apoptosis was markedly decreased after ritonavir treatment and correlated with lower levels of caspase-1 expression, decreases in annexin V staining, and reduced caspase-3 activity. Induction in vitro of tumor necrosis factor (TNF) production by PBMCs and monocytes was inhibited by ritonavir in a time- and dose-dependent manner at nontoxic concentrations. CONCLUSION: Based on our data, we conclude that the HIV-1 PI ritonavir is an immune modulator that may affect leukocyte activation and apoptosis as an important part of its therapeutic benefit.

Recombinant IFN-alpha (2b) increases the expression of apoptosis receptor CD95 and chemokine receptors CCR1 and CCR3 in monocytoid cells.

IFN-alpha-2b, known as potent immune modulator, can either inhibit or enhance immune cell activity within the tightly regulated microenvironment of inflammation, depending upon the concentration of the cytokine and the activation stage of the cell. Chemokine receptors, which not only mediate chemotaxis of immune cells to the site of inflammation but also affect cellular activation by transferring corresponding signals, represent yet another level of immune regulation. Here we demonstrate that IFN-alpha increases the expression of CCR1 and CCR3 in primary mononuclear phagocytes, as well as in the monocytoid cell line U937. Enhanced receptor mRNA expression correlated with functional readouts such as increased intracellular calcium mobilization and cell migration in response to ligands. Expression of CCR2b, CCR4, CCR5, and CXCR4 was unchanged or decreased after IFN-alpha treatment. These observations indicate a differentially regulated cellular signaling relationship of IFN-alpha pathways and chemokine receptor expression. We also provide evidence that, under these conditions, IFN-alpha treatment increased the expression of CD95 (Fas, Apo1), resulting in enhanced susceptibility to apoptosis. Taken together, these data add important information for the rational application of IFN-alpha (2b) in immune and cancer therapies.

Human immunodeficiency virus type 1 protease inhibitor modulates activation of peripheral blood CD4(+) T cells and decreases their susceptibility to apoptosis in vitro and in vivo.

CD4(+) T cells from patients with human immunodeficiency virus (HIV) infection undergo apoptosis at an increased rate, which leads to their depletion during disease progression. Both the Fas-Receptor (Fas-R) and interleukin-1beta (IL-1beta)-converting enzyme (ICE; caspase 1) appear to play a role in the mechanism of apoptosis of CD4(+) lymphocytes. Although Fas-R is upregulated on both CD4(+) and CD8(+) cells in HIV-infected patients, results from our laboratory and others indicate that, in patients with advanced disease, CD4(+) cells preferentially express ICE. Protease inhibitors have successfully halted the progression of HIV disease and increased CD4(+) T counts. In this study, we examined the effect of protease inhibitors on Fas-R (CD95), ICE (caspase 1) expression, apoptosis, and cell death in CD4(+) T cells of (1) HIV-infected patients who were receiving protease inhibitors, and (2) normal and patient CD4(+) T cells cultured with a protease inhibitor in vitro. Fifteen patients with advanced HIV disease on treatment showed dramatically decreased CD4(+) T-cell ICE expression, diminished apoptosis, and increased numbers of CD4(+) cells within 6 weeks of institution of protease inhibitor therapy, and before down-modulation of Fas-R (CD95) expression was evident. To determine the role of HIV infection, we studied the effect of ritonavir, a protease inhibitor, on normal and patient cells in vitro. Stimulated and unstimulated normal CD4(+) T cells, cultured with protease inhibitor, demonstrated markedly decreased apoptosis and ICE expression (P =. 01). While Fas-R expression was not significantly altered during short-term culture by such treatment, Fas-Ligand (Fas-L) membrane expression of phytohemagglutinin (PHA)-stimulated blood lymphocytes was decreased by protease inhibitor. In the presence of ritonavir, CD4(+) T cells from HIV-infected patients showed similar changes in ICE intracellular levels without alteration of Fas expression. In conclusion, protease inhibitors appear to decrease CD4(+) T-cell ICE expression and apoptosis before they affect Fas-R expression in HIV-infected patients. This action was independent of HIV infection, as similar effects were seen in CD4(+) T cells from normal controls. Some of the benefit of protease inhibitors may be related to modification of programmed cell death, which increases CD4(+) T-cell number. Whether this is due to directly to the changes effected in the caspase system remains to be determined.

A novel sensitive assay to define immune status using short-term peripheral blood derived cell culture and dual-color flow cytometry.

In this study we describe a novel and highly sensitive in vitro system to determine the functionality of immune cells based on short term culture of peripheral blood derived mononuclear cells (PBMCs) and subsequent analysis of cellular proliferation and surface marker expression by automated dual-color flow cytometry. The standardized mild stimuli introduced into the culture system by supplemented medium (containing exogenous interleukin-2 (IL-2), and fetal bovine serum (FBS)) allow a more physiological interaction of the different cell subsets contained in PBMCs (including CD14+ accessory cells) than other methods that are based on potent and harsh cell activators, such as phytohemagglutinin (PHA) or anti-CD3 antibodies. Measurement of T-cell proliferation and cell surface marker (CD3, C25, CD26, CD71, HLA-DR) analysis revealed that activation response capacity in our assay depends on both the status of the obtained cells and their ability to interact in culture with CD14+ cells. This in vitro assay proved to be very sensitive in detecting changes in the status of T-cell activation and proliferation capacity, and avoid the use of radioactive reagents.

Interferon-gamma increases expression of chemokine receptors CCR1, CCR3, and CCR5, but not CXCR4 in monocytoid U937 cells.

Chemokine receptors (CR), which can mediate migration of immune cells to the site of inflammation, also function as coreceptors for human immunodeficiency virus (HIV) entry into CD4+ T lymphocytes and antigen-presenting cells. We demonstrate here that interferon-gamma (IFN-gamma) increases the expression of chemokine receptors CCR1, CCR3, and CCR5 in monocytoid U937 cells as detected by cell surface molecule labeling and mRNA expression, as well as by intracellular calcium mobilization and cell migration in response to specific ligands. The increased expression of these chemokine receptors also results in an enhanced HIV-1 entry into cells. Our data provide evidence for a relationship of cellular pathways that are induced by IFN-gamma with those that regulate chemokine receptor expression.

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures.

Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38-) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

The role of interleukin-converting enzyme in Fas-mediated apoptosis in HIV-1 infection.

Apoptosis of CD4+ lymphocytes is partially responsible for the depletion of these cells in HIV-infected individuals. CD4+ lymphocytes from HIV-1-infected patients express higher membrane levels of the Fas receptor, and are particularly susceptible to apoptosis after Fas triggering. IL-1beta- converting enzyme (ICE) is a key enzyme of the apoptotic machinery involved in Fas-mediated apoptosis of normal lymphocytes. The role of ICE in mediating the increased susceptibility of CD4+ lymphocytes from HIV-1-infected patients to apoptosis has not been examined. In this study, we found that ICE mRNA was present in T cells from both HIV-1-infected patients and controls. Active ICE proteins, p10 and p20, were demonstrated by immunoblot in lymphocytes from HIV-1-infected patients and in normal lymphocytes after treatment with Fas agonist, CH11 mAb. Cocultivation of lymphocytes from HIV-1-infected persons with Fas antagonist, antibody ZB4, resulted in decreased expression of ICE protein in lymphocytes from HIV-infected patients, and decreased apoptosis. Similar effects were obtained when cells were treated with synthetic ICE inhibitors, which blocked apoptosis in response to Fas triggering. When CD4+ and CD8+ cells were sorted by flow cytometry and analyzed by reverse transcriptase PCR, ICE mRNA was present in both CD8+ and CD4+ cells. However, flow cytometric analysis of lymphocytes with intracellular staining for ICE demonstrated ICE protein in the CD4+ but not the CD8+ cell fraction derived from blood of HIV-1-infected patients. These data suggest that activation of ICE occurs in vivo in CD4+ lymphocytes from HIV-1-infected individuals, and may account for the increased susceptibility of CD4+ cells to apoptosis.

Regulation of a graft-versus-leukemia effect by major histocompatibility complex class II molecules on leukemia cells: HLA-DR1 expression renders K562 cell tumors resistant to adoptively transferred lymphocytes in severe combined immunodeficiency mice/nonobese diabetic mice.

To understand the role of key molecules in determining the strength and nature of allogeneic T-cell response to leukemia, we transfected HLA-DR1 into the major histocompatibility complex (MHC)-deficient, natural killer (NK)-cell sensitive K562 leukemia cell line. Untransfected K562 cells stimulated NK proliferation in vitro and formed subcutaneous tumors in severe combined immunodeficiency/non-obese diabetic (SCID/NOD) mice. Tumor growth was inhibited by adoptive intravenous transfer of fresh unprimed peripheral blood mononuclear cells (PBMC). In contrast, HLA-DR1 transfected cells stimulated CD4(+) T cells, but not NK-cell proliferation in vitro and formed tumors resistant to fresh PBMC in SCID/NOD mice. Tumors not expressing MHC were infiltrated with CD16(+)CD56(+) lymphocytes whereas nonregressing HLA-DR1 expressing tumors showed only a scanty infiltration with both T-cell and NK-cell subsets. The results indicate that MHC class II expression by leukemia cells can determine the effector cell type that it engages. In vivo MHC class II expression rendered K562 cell tumors resistant to NK-cell mediated antitumor reactivity.

Secondary colony formation after long-term bone marrow culture using peripheral blood and bone marrow of HIV-infected patients.

OBJECTIVE: To determine if defective bone marrow function in HIV infection is associated with decreased numbers of progenitor cells or defective stromal cell function. DESIGN: Defective bone marrow function may in part be responsible for the cytopenias so frequently seen in patients with AIDS. Although a number of investigators have reported impaired growth of committed hematopoietic progenitor cells in HIV-1-infected patients, few studies have examined the most primitive hematopoietic stem cells. Our study was designed to determine the function and quality of the most immature hematopoietic progenitor and stem cells in the peripheral blood and bone marrow of HIV-1-infected patients and to assess stromal cell function. METHODS: For quantification of these cells we used a modified long-term culture-initiating cell (LTCIC) assay in which the number of secondary colony-forming cells after 5 weeks of stromal culture served as a measure of LTCIC. Stromal cells from normal controls and HIV-1-infected patients were used for cross-matching experiments. Normal CD34+ cells or those derived from HIV-infected patients were plated and colony growth assessed. RESULTS: We found that HIV-1-infected patients had a mean of 0.8 secondary colony-forming cells/10(5) peripheral blood mononuclear cells (PBMC), whereas normal controls showed 1.2 secondary colony-forming cells/10(5) PBMC (P = not significant) after long-term culture. Asymptomatic patients showed well preserved numbers of secondary colony-forming cells after long-term culture of PBMC, but a significant reduction was seen in patients with a history of opportunistic infections (P < 0.01), low CD4+ cell count (< 200 x 10(6)/l; P < 0.05), or leukopenia (P < 0.05). Decreased numbers of secondary colony-forming cells have also been found in bone marrow of HIV-1-infected patients with advanced disease. When normal CD34+ cells were cultured on stromal layers from bone marrow of HIV-1-infected patients or normal controls, no differences in the numbers of surviving progenitor cells were found. CONCLUSIONS: Our data indicate that the hematopoietic defect in HIV-1 infection involves the most immature hematopoietic cells and becomes evident in advanced disease. Stromal function of HIV-infected patients appears normal.

Paroxysmal nocturnal hemoglobinuria: new insights from murine Pig-a-deficient hematopoiesis.

A large fraction of the hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) are deficient in membrane expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Current evidence suggests that this deficiency is sufficient to account for the hemolytic and thrombotic manifestations of this disease but not for its frequent association with aplastic anemia, an autoimmune disorder in which the patients' own hematopoietic progenitor cells are the target. Mutations in X-linked gene PIG-A, encoding one of several enzymes required for the biosynthesis of the glycophosphatidylinositol anchor, have been found in all PNH patients studied to date. Recent experiments with murine Pig-a knock-out embryonic stem cells show that although embryogenesis is critically dependent on normal GPI-AP expression, Pig-a-deficient cells can undergo apparently normal hematopoietic differentiation if they develop in a GPI-AP-replete environment. Thus, in an in vitro mouse model of PNH, Pig-a mutations confer no gross proliferative or differentiative advantage or disadvantage, suggesting an unidentified process selecting for these mutations in the bone marrow of patients with the PNH-aplastic anemia syndrome. The rescue of hematopoiesis observed in chimeric cultures of knock-out and normal cells was accompanied by intercellular transfer of GPI-AP, suggesting exciting new possibilities for future therapeutic manipulations in PNH patients.

Role of Fas ligand and receptor in the mechanism of T-cell depletion in acquired immunodeficiency syndrome: effect on CD4+ lymphocyte depletion and human immunodeficiency virus replication.

Direct killing of CD4+ lymphocytes by human immunodeficiency virus-1 (HIV-1) probably cannot account for the magnitude of the loss of these cells during the course of HIV-1 infection. Experimental evidence supports a pathophysiologic role of the apoptotic process in depletion of CD4 cells in acquired immunodeficiency syndrome (AIDS). The Fas-receptor/Fas-ligand (Fas-R/Fas-L) system mediates signals for apoptosis of susceptible lymphocytes and lympoblastoid cell lines. A number of investigators have recently reported increased expression of the Fas receptor in individuals with HIV infection, along with increased sensitivity of their lymphocytes to anti-Fas antibody mimicking Fas ligand. We attempted to determine the role of Fas-mediated apoptosis in disease progression and viral replication. Increased Fas-receptor (CD95) expression on CD4+ and CD8+ lymphocytes was found in a large group of HIV-1-infected patients compared with normal controls; individuals with a diagnosis of AIDS and a history of opportunistic infection had significantly more Fas receptor expression than did asymptomatic HIV-infected persons and normal blood donor controls (P < .01). Triggering of the Fas-R by agonistic anti-Fas monoclonal antibody, CH11, was preferentially associated with apoptosis in the CD4+ cells; this effect was more pronounced in lymphocytes derived from HIV+ individuals. Soluble and membrane-bound forms of Fas-L were produced in greater amounts in peripheral blood mononuclear cells (PBMC) cultures and in plasma obtained from HIV-1-infected persons than from normal controls. Furthermore, triggering of lymphocytes from HIV-infected persons by CH11 increased levels of interleukin-1beta converting enzyme (ICE), a protein associated with apoptosis. When PBMC were cultured in the presence of CH11, p24 production per number of viable cells was decreased as compared with the same PBMC without CH11 (P < .01). These findings suggest that multiple mechanisms, including increased production of Fas-L by infected PBMC, increased Fas-R expression, and induction of a protease of ICE family, may play roles in the apoptotic depletion of CD4+ cells in HIV infection.

A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(-) hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins.

We created a "knockout" embryonic stem cell via targeted disruption of the phosphatidylinositol glycan class A (Pig-a) gene, resulting in loss of expression of cell surface glycosyl phosphatidylinositol-anchored proteins and reproducing the mutant phenotype of the human disease paroxysmal nocturnal hemoglobinuria. Morphogenesis of Pig-a- embryoid bodies (EB) in vitro was grossly aberrant and, unlike EB derived from normal embryonic stem cells, Pig-A EB produced no secondary hematopoietic colonies. Chimeric EB composed of control plus Pig-A- cells, however, appeared normal, and hematopoiesis from knock-out cells was reconstituted. Transfer in situ of glycosyl phosphatidylinositol-anchored proteins from normal to knock-out cells was demonstrated by two-color fluorescent analysis, suggesting a possible mechanism for these functional effects. Hematopoietic cells with mutated PIG-A genes in humans with paroxysmal nocturnal hemoglobinuria may be subject to comparable pathophysiologic processes and amenable to similar therapeutic protein transfer.

Antisense phosphorothioate oligodeoxynucleotides alter HIV type 1 replication in cultured human macrophages and peripheral blood mononuclear cells.

The use of antisense oligodeoxynucleotides as antiviral drugs to combat HIV-1 infection may offer an alternative to traditional pharmacological therapies. We compared the effects of two 28-mer antisense phosphorothioate oligodeoxynucleotides [PS-oligo(dN)] with non-sequence-specific controls on HIV-1 replication in long-term human monocyte/macrophage and PBMC cultures. The anti-rev PS-oligo(dN) was complementary to the messenger RNA (mRNA) sequences derived from the overlapping region of the HIV-1 regulatory genes tat and rev, while anti-gag targeted the translational initiation site of the gag mRNA. In vitro cytotoxicity of the PS-oligo(dN) was evaluated at concentrations ranging from 0.1 to 10.0 microM for a period of 20 days. Cell survival was 100% at 0.1 microM, but decreased to 5% at 10.0 microM in relation to the untreated control cultures. Our data demonstrate that replication of both the T cell-tropic and macrophage-tropic HIV-1 strains in primary cells can be inhibited by PS-oligo(dN) in a sequence-specific and dose-dependent manner at concentrations achievable in vivo. However, the sequence-dependent antiviral activity of the utilized PS-oligo(dN) was limited to a window of specificity at concentrations between 0.25 and 1.0 microM.

Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro.

We have tested a novel strategy of intracellular immunization to block human immunodeficiency virus (HIV) infection. The expression of a specific antibody within a cell was achieved by transduction of genes that encode for immunoglobulin chains with specificity to viral reverse transcriptase. We demonstrated that inhibition of this enzyme makes cells resistant to HIV infection by blocking an early stage of viral replication. If high efficiency transduction with a stable vector into lymphohaematopoietic stem cells or mature lymphocytes can be achieved, gene transfer-mediated intracellular immunization might be a feasible treatment strategy in AIDS.

HIV-1 suppression of hematopoiesis in vitro mediated by envelope glycoprotein and TNF-alpha.

Dysmorphic marrow morphology and bone marrow failure are common in AIDS patients, but the mechanism of HIV-1 effects on blood cell production is unclear. Experiments to test the susceptibility of hematopoietic progenitor cells to HIV-1 infection have led to conflicting results. We found that hematopoietic colony formation by burst-forming units-erythroid and CFU-GM was equivalently inhibited by both active and heat-inactivated, noninfectious virus. Inhibition was dependent on the presence of macrophages and was not observed in cultures derived from highly enriched CD34+ cells. We hypothesized that TNF-alpha, produced by mononuclear phagocytes after contact with HIV-1 or gp120 and itself a potent suppressor of hematopoiesis, might mediate this effect. The addition of anti-TNF-alpha neutralizing Abs to marrow cultures abrogated inhibition by gp120 or virus. In contrast, neutralizing Abs to Il-4, IFN-alpha, and TGF-beta failed to improve colony formation. TNF-alpha was released from blood monocytes and marrow mononuclear cells stimulated by gp120. TNF-alpha is increased in the blood of patients with late stage AIDS and may mediate many of the symptoms of the disease. Our data do not support a requirement of direct infection of hematopoietic progenitor cells by HIV-1 for the inhibition of hematopoiesis in vitro. We propose instead an indirect mechanism of viral suppression of hematopoiesis as a result of TNF-alpha induction by virus or viral envelope glycoprotein. The importance of local TNF-alpha production in patients' marrow is amenable to clinical testing.

Antisense oligodeoxynucleotide phosphorothioate complementary to Gag mRNA blocks replication of human immunodeficiency virus type 1 in human peripheral blood cells.

Gene-expression modulator 91 (GEM91) is a 25-nt antisense oligodeoxynucleotide phosphorothioate complementary to the Gag mRNA of human immunodeficiency virus type 1 (HIV-1). Cellular uptake and intracellular distribution of GEM91 within cells suggest that this oligomer is readily available for antisense activity. GEM91 inhibited HIV-1 replication in a dose-dependent and sequence-specific manner. In a comparative study, 2 microM GEM91 was as effective as 5 microM 3'-azido-3'-deoxythymidine in blocking virus replication during the 28-day treatment of an HIV-1-infected T-cell line. GEM91 also completely inhibited (> 99%) the growth of three different HIV-1 isolates in primary lymphocytes and prevented the cytopathic effect of the virus in primary CD4+ T cells. Similarly, treatment with GEM91 for 3 weeks of HIV-1/BaL-infected primary macrophages blocked virus replication. Based on GEM91 anti-HIV-activity, safety, and pharmacokinetic profile in animals, a clinical trial was started using this compound as an antisense oligonucleotide drug for the treatment of the acquired immunodeficiency syndrome.

Activation of human CD4+ and CD8+ cells by Sarcocystis gigantea lectin.

As recently reported, Sarcocystis gigantea lectin (SGL) is a powerful mitogen and a polyclonal activator (Syn. S. ovifelis) of human peripheral B-cells. In the present study we investigated the reactivity of human T-helper (CD4) and T-suppressor (CD8) cells to SGL. Mononuclear cells (MNCs) from five newborns and six adults were examined cytofluorometrically for the expression of cell-surface differentiation and activation antigens using a set of seven monoclonal antibodies. In all, 96% of cord-blood and 81% of adult CD4 cells expressed receptors for interleukin-2 (Tac+) after 64 and 164 h microgram/ml). The percentages of neonatal and adult Tac+ CD8 cells amounted to 67% and 59%, respectively. Major histocompatibility complex (MHC) class II antigens identified using the monoclonal anti-human leukocyte antigen (HLA)-DR antibody L243 were expressed on 30% (CD4) and 44% (CD8) of adult T-cells. Neonatal HLA-DR+ T-lymphocytes were not detectable. In parallel, functional tests were performed to examine cell proliferation and MNC antibody production.