Characterization of dendritic cell differentiation pathways from cord blood CD34(+)CD7(+)CD45RA(+) hematopoietic progenitor cells.

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD7(+)CD45RA(+) progenitor population found among cord blood CD34(+) cells. Unlike CD7(-)CD45RA(+) and CD7(+)CD45RA(-) progenitors, this population displayed high natural killer (NK) cell differentiation capacity when cultured with stem cell factor (SCF), interleukin (IL)-2, IL-7, and IL-15, attesting to its lymphoid potential. In cultures with SCF, Flt3 ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha (standard condition), CD7(+)CD45RA(+) progenitors expanded less (37- vs 155-fold) but yielded 2-fold higher CD1a(+) DC percentages than CD7(-)CD45RA(+) or CD7(+)CD45RA(-) progenitors. As reported for CD34(+)CD1a(-) thymocytes, cloning experiments demonstrated that CD7(+)CD45RA(+) cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7(-)CD45RA(+) and CD7(+)CD45RA(+) progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7(+)CD45RA(+) or thymic progenitors differentiated into Lag(+)S100(+) Langerhans cells in the absence of exogenous transforming growth factor (TGF)-beta 1. Analysis of the DC differentiation pathways showed that CD7(+)CD45RA(+) progenitors generated CD1a(+)CD14(-) precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a(-)CD14(+) precursors that readily differentiated into DCs under the standard condition. Accordingly, CD7(+)CD45RA(+) progenitor-derived mature DCs produced 2- to 4-fold more IL-6, IL-12, and TNF-alpha on CD40 ligation and elicited 3- to 6-fold higher allogeneic T-lymphocyte reactivity than CD7(-)CD45RA(+) progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD34(+)CD7(+)CD45RA(+) progenitors represent an original population for their developmental pathways and function. (Blood. 2000;96:3748-3756)

The susceptibility to X4 and R5 human immunodeficiency virus-1 strains of dendritic cells derived in vitro from CD34(+) hematopoietic progenitor cells is primarily determined by their maturation stage.

Dendritic cells (DC) were sorted on day 8 from cultures of CD34(+) cells with stem cell factor/Flt-3 ligand/ granulocyte-macrophage colony-stimulating factor (GM-CSF)/tumor necrosis factor-alpha (TNF-alpha)/interleukin-4 (IL-4). Exposing immature CCR5(+)CXCR4(lo/-) DC to CCR5-dependent human immunodeficiency virus (HIV)-1Ba-L led to productive and cytopathic infection, whereas only low virus production occurred in CXCR4-dependent HIV-1LAI-exposed DC. PCR analysis of the DC 48 hours postinfection showed efficient entry of HIV-1Ba-L but not of HIV-1LAI. CD40 ligand- or monocyte-conditioned medium-induced maturation of HIV-1Ba-L-infected DC reduced virus production by about 1 Log, while cells became CCR5(-). However, HIV-1Ba-L-exposed mature DC harbored 15-fold more viral DNA than their immature counterparts, ruling out inhibition of virus entry. Simultaneously, CXCR4 upregulation by mature DC coincided with highly efficient entry of HIV-1LAI which, nonetheless, replicated at the same low level in mature as in immature DC. In line with these findings, coculture of HIV-1Ba-L-infected immature DC with CD3 monoclonal antibody-activated autologous CD4(+) T lymphocytes in the presence of AZT decreased virus production by the DC. Finally, whether they originated from CD1a+CD14(-) or CD1a-CD14(+) precursors, DC did not differ as regards permissivity to HIV, although CD1a+CD14(-) precursor-derived immature DC could produce higher HIV-1Ba-L amounts than their CD1a-CD14(+) counterparts. Thus, both DC permissivity to, and capacity to support replication of, HIV is primarily determined by their maturation stage.

IL-4 and CD40 ligation affect differently the differentiation, maturation, and function of human CD34+ cell-derived CD1a+CD14- and CD1a-CD14+ dendritic cell precursors in vitro.

We examined the effect of interleukin (IL)-4 or CD40 ligation on the differentiation and maturation of CD1a+CD14- and CD1a-CD14+ dendritic cell (DC) precursors. Cord blood CD34+ cells were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha), to which stem cell factor and Flt-3 ligand were added for 5 days. Phenotypic analysis of DC precursors on culture day 7 showed that CD1a+CD14- cells expressed higher CD11c and CD80 levels and lower CD116/GM-CSFR and CCR-5 levels than their CD1a-CD14+ counterparts. Culturing CD1a+CD14- precursors with GM-CSF and TNF-alpha resulted in DC with heterogeneous CD1a, HLA;SMDR (DR), CD11b, and CD83 expression, 10% of which acquired CD14. IL-4 and CD40 ligation affected their differentiation in contrasting ways: IL-4 induced CD1ahiCD14-DRloCD11b+CD83-S100+ DC with reduced MLR-stimulating capacity, whereas CD40 ligation led to CD1alo/-CD14-CD40-DRhiCD11b-CD83+S100+/- DC with stronger MLR-stimulating capacity. Also, both IL-4 and CD40 ligation promoted ReIB expression and nuclear translocation. When CD1a-CD14+ precursors were maintained in only the presence of GM-CSF and TNF-alpha, this led to mixed populations of adherent macrophages and nonadherent CD1a-CD14+ monocytes, and of CD1a+CD14- and CD1a+CD14+ DC, which were DRloCD11b+CD83-S100-. IL-4 or CD40 ligation prevented their differentiation into macrophages and resulted in DC with phenotypes close to those issued from CD1a+CD14- precursors, with only a minority staying CD14+ but most being S100-; their MLR-stimulating capacity also increased but remained lower than that of DC differentiated from CD1a+CD14- precursors. Thus, IL-4 or CD40 ligation induced CD1a+CD14- and CD1a-CD14+ DC precursors to differentiate into phenotypically close but functionally different DC populations, suggesting that DC function is primarily determined by their origin. The heterogeneity of DC should then be related to different developmental pathways and to different stages of maturation/activation.

Special susceptibility to apoptosis of CD1a+ dendritic cell precursors differentiating from cord blood CD34+ progenitors.

We analyzed the effect of tumor necrosis factor (TNF)-alpha on the differentiation and viability of dendritic cells (DC) generated from cord blood CD34+ progenitors cultured for five days with GM-CSF, Flt-3 ligand (FL), and stem cell factor (SCF), and then with GM-CSF only [TNF(-) cultures]. Adding TNF-alpha from the start [TNF(+) cultures] potentiated progenitor cell proliferation and promoted early differentiation of CD1a+ DC precursors without affecting differentiation of CD14+ cells, which comprise bipotent precursors of DC and macrophages, nor of CD15+ granulocytic cells. Use of TNF-alpha was associated with increased cell mortality, which peaked on culture day 10 and mainly involved CD1a+ DC. Selective apoptosis of CD1a+ DC precursors was confirmed by showing that survival of day-7-sorted CD1a+CD14- cells from TNF(+) cultures was lower than that of CD1a-CD14+ cells. That similar findings were noted for sorted CD1a+CD14- cells of TNF(-) cultures, further cultured with GM-CSF without or with TNF-alpha, indicates that apoptosis of CD1a+ DC precursors was not induced by TNF-alpha. Apoptosis of CD1a+ DC precursors occurred after the cells had lost the capacity to incorporate bromodeoxyuridin. Finally, using higher GM-CSF concentrations or adding interleukin 3 (IL-3) improved viability of CD1a+ cells. Other cytokines, such as IL-4 and transforming growth factor (TGF)-beta1, were ineffective in this respect, though they promoted differentiation of CD1a+ DC. These results indicate that TNF-alpha promotes the differentiation of CD1a+ DC precursors, which display a high susceptibility to apoptosis that can be prevented by high concentrations of GM-CSF or use of IL-3, without affecting the differentiation of the CD14+ DC precursors.

Position-dependent variegation of a CD4 minigene with targeted expression to mature CD4+ T cells.

The CD4 gene follows a complex and highly regulated pattern of expression throughout T cell development. This expression is governed by different regulatory elements that have been partly identified, including a promoter, a proximal enhancer, and a silencer. Here we show that a CD4 minigene comprising a combination of these elements is specifically expressed in mature CD4+ T cells of transgenic mice, but not in CD4+CD8+ double positive thymocytes. The proportion of transgene-expressing CD4+ T cells was constant within a given transgenic line, but varied greatly from one line to another. We demonstrate that this pattern of expression is due to integration of the transgene within or in the vicinity of centromeric heterochromatin. This position-effect variegation demonstrated with a short CD4 transgene has not been observed with larger ones containing additional regulatory sequences, suggesting that the CD4 gene contains a locus control region. Such position-dependent effects must be taken into consideration when developing transgenic models or gene transfer vectors because they can result in the absence of transgene expression in a subpopulation of target cells. Finally, the combination of the CD4 gene silencer, proximal enhancer, and promoter provides an interesting tool to selectively express genes of interest in mature CD4+ T cells of transgenic mice and for the development of gene therapy vectors.

Differentiation of human dendritic cells from monocytes in vitro.

Since either macrophages (Mphi) or dendritic cells (DC) differentiate from monocytes (MO) depending on culture conditions, we investigated the relationship of the DC and Mphi differentiation pathways. Culturing MO-enriched blood mononuclear cells with Mphi colony-stimulating factor (M-CSF) or with granulocyte/Mphi (GM)-CSF induced Mphi with a different morphology and CD14/CD1a expression. In contrast, in cultures with GM-CSF and interleukin (IL)-4, cells rapidly became nonadherent and acquired DC morphology, ultrastructure, CD1a expression, and most DC markers; they lost membrane CD14 and CD64 and capacity of phagocytosis, displayed less CD68 than Mphi, but retained nonspecific esterase activity. These DC directly developed from MO without proliferation inasmuch as only day 0 FACS-sorted MO, but not small CD14- cells, differentiated into DC when cultured with GM-CSF and IL-4, or to Mphi with M-CSF While overall cell numbers declined, DC numbers plateaued from culture day 2 onwards, indicating that most had differentiasted by then. This differentiation was radioresistant and occurred without [3H]thymidine incorporation. Commitment to differentiate into DC with GM-CSF and IL-4 was irreversible by day 2, since discontinuing IL-4 at this point did not revert cells to Mphi. Alternatively, cells rapidly converted to DC when IL-4 was added from day 2 to cultures initiated with GM-CSF only. If cultures were initiated with M-CSF and switched to GM-CSF and IL-4 after 2 or 5 days, about half of the cells still converted to DC. Thus, the capacity of MO and even of Mphi to differentiate into DC was conserved for at least this period. The increased capacity to stimulate the mixed leukocyte reaction correlated with the relative number of CD1a+ cells at any time and under each condition tested, a confirmation that these cells functionally qualify as DC. Thus, MO and even Mphi can be directed to differentiate into DC depending on the cytokine microenvironment.

The effect of in vitro human immunodeficiency virus infection on dendritic-cell differentiation and function.

CD1a+ dendritic cells (DC) differentiate from a major population of nonadherent CD13(hi)lin- cells that appear when human cord blood CD34+ hematopoietic progenitor cells are cultured with stem-cell factor, granulocyte/macrophage (MA) colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha) for 5 days. CD13hilin- cells, which also comprise MA and granulocyte precursors, are CD4+ and can thus be targets of human immunodeficiency virus (HIV). Low replication was noted when these day 5 cells were infected with lymphotropic HIV-1LA1 (p24: < or = 4 ng/mL on day 8 postinfection [PI]), while high virus production occurred with MA-tropic HIV-1Ba-L, HIV-1Ada, or HIV-1-m-n. (p24: 50 to > or = 1,000 ng/mL). Strong cytopathicity (CPE) was then observed in nonadherent cells as in adherent MA. However, FACS analysis on day 7 PI showed that HIV did not affect differentiation of DC that survived CPE: apart from CD4 downmodulation related to HIV production, overall expression of CD40, CD80, and CD86 costimulatory molecules, and of HLA-DR, was unchanged relative to controls. At that time, the capacity of DC from HIV-infected cultures to stimulate the mixed leukocyte reaction was only altered less than 10-fold. Immunocytochemistry on day 7 PI showed that most HIV-infected cells were included in syncytia that were stained by anti-CD1a, anti-S100, and anti-CD14 antibodies, indicating that syncytia consisted of DC and cells of the MA lineage. Polymerase chain reaction analysis of FACS-sorted CD1a+ cells confirmed that they harbored then HIV DNA. Viral DNA was also detected in CD1a+ DC from noninfected cultures that had been exposed to HIV only after sorting. Therefore, we examined whether in infected cultures DC precursors were infected at the onset or if virus spread later from other infected cells to differentiated DC. This was answered by showing that, 24 hours postexposure to HIV, viral DNA was preferentially detected in day 5 sorted CD13hilin- versus CD13hilin- cells, and that it was found in the CD1a+ progeny of CD13(hi)lin- cells 48 hours later. In addition, HIV replication did not affect myeloid clonogenic progenitors in day 0 to day 7 PI cultures, although viral DNA was detected in colony-forming unit-granulocyte/macrophage (CFU-GM)/CFU-M colonies derived from day 3 and 7 PI cultures. Thus, precursors of DC and their progeny are susceptible to HIV in vitro, but, apart from CPE, the effect of virus production on DC differentiation or function is limited.

HIV-1 envelope glycoprotein gp120 does not bind to galactosylceramide-expressing rat oligodendrocytes.

It may be postulated that the encephalopathy induced by the human immunodeficiency virus HIV-1, in particular, the characteristic "myelin pallor," may result from binding of the envelope glycoprotein gp120 to galactosylceramide and/or its metabolite sulfatide in the plasma membrane of oligodendrocytes, the myelin forming cells in the central nervous system. (1) gp120 has been reported to have a high affinity for these molecules in vitro. (2) The binding of antibodies to these molecules increases intracellular free calcium levels, which may be cytotoxic. (3) The binding of gp120 to the CD4 receptor in the immune system has the same effect. We have investigated the binding of gp120 to rat oligodendrocytes in vitro by indirect immunofluorescence and have monitored changes in intracellular free calcium with the calcium-sensitive dye INDO-1, in individual oligodendrocytes exposed to the glycoprotein. Antibodies against galatosylceramide and sulfatide bound to the cell membrane, but gp120 did not. The antibodies also increased intracellular free calcium levels in the oligodendrocytes, whereas gp120 did not. It, therefore, seems highly improbable that the demyelination observed during HIV encephalopathy is a direct cytotoxic effect of gp120 on oligodendrocytes.

Extensive C-terminal deletion in human immunodeficiency virus type 1 Env glycoprotein arising after long-term culture of chronically infected cells.

Human immunodeficiency virus type 1 (HIV-1) chronically infected (CI) cell lines were established from HIV-1HIB/LAI-infected MT-4 cells that survived acute infection. The HIV env gene expressed in the two long-term cultured cell lines differed from that of the lines cultured for shorter periods, by coding for a glycoprotein gp 160 that had the C terminus deleted. One long-term cultured cell line, CI-17, was studied in detail. An insertion of a premature stop codon in the env gene caused about 90% of gp160 molecules to be truncated (gp160x), lacking both cytoplasmic and transmembrane domains; these species were secreted into the cell medium, and could form oligomers with other truncated gp160 molecules as well as with their normal counterparts. CI-17 cells constantly yielded high levels of viral protein and relatively low quantities of infectious virus, without cytopathicity. However, acute infection of fresh MT-4 cells with CI-17-derived virus led to cytopathicity, the rate of which as well as the Env glycoprotein pattern depended on multiplicity: (i) using an infection dose of 10(-4) ID50/cell, cells died 7 to 8 days post-infection with normal gp160 synthesis predominating; (ii) with 10(-2) ID50, gp160x was produced as early as 48 h post-infection and cell death was delayed. Predominant gp160x formation occurred again when new CI cell lines were obtained with CI-17-derived virus. Thus, two human immunodeficiency virus variants, a normal and a defective one, are persistently expressed in CI-17 cells. The other long-term cultured CI cell line also expressed gp160 with a similar (albeit slightly longer) deletion of a C-terminal region in most molecules, but the cell lines that were cultured for shorter periods did not. These results suggest that the emergence of HIV variants with a C-terminal deletion in the Env glycoprotein, which coexist with normal virus, may play a role in maintaining the long-term growth capacity and viability of CI cells.

Primary in vitro sensitization of human T-helper lymphocytes by peptides derived from the V3 loop of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein.

To generate CD4+ T-helper cell lines, lymphocytes from HIV-seronegative subjects were primed in vitro with peptides derived from the V3 loop of HIV-1 gp120. Antigen-specific reactivity was inhibited by an anti-DR monoclonal antibody, indicating HLA-class II dependency, but peptides could be recognized in different HLA-class II contexts. Three sites on V3LAI and two on V3MN were identified as targets of the respective V3LAI- and V3MN-specific lines. Recognition of V3 peptides was isolate-specific. The lines did not react against whole gp160, which suggests that V3 may be differently presented when used as such rather than as part of the entire glycoprotein. Similar results were obtained in chimpanzees immunized in vivo against V3LAI.

Antibody-dependent cellular cytotoxicity against HIV-1 in sera of immunized chimpanzees.

After immunization of chimpanzees against HIV antigens, antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) were evaluated and compared with anti-HIV-antibody levels detected by enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody titers. Adult chimpanzees were immunized with different HIV-1 (LAV-BRU) antigen preparations: recombinant vaccinia virus (rVV) expressing gp160, p25 or p27nef; formalin- and beta-propiolactone-inactivated whole virus (inHIV); soluble recombinant gp160 either associated or not associated with other HIV proteins; a 25-mer peptide from the V3 region of gp120 coupled with KLH (V3-KLH). Immunization with the various rVV mixtures induced no or borderline ADCC increase above preimmune serum levels. Stronger and more sustained reactivity was elicited by inHIV. Purified HIV antigens elicited ADCC activity when the chimpanzees were naive; ADCC increased or remained at the same level when the animals had been preimmunized with rVV and/or inHIV. This type of reactivity apparently did not depend on whether gp160 alone or mixed with other proteins was used for immunization. The injection of V3-KLH resulted in only little, if any, recall ADCC response. ELISA antibody titers significantly correlated with ADCC and neutralizing antibody titers, but serum ADCC was independent of neutralizing antibody titers, an indication that the two latter serum activities are mediated by independent antibodies. Therefore, ADCC is elicited in the same manner as other antibody activities by the immunization of chimpanzees with inHIV or with purified recombinant HIV antigen preparations. The results obtained from the three chimpanzees of this series, which were subsequently challenged with infectious virus through the intravenous route, suggest that serum ADCC may be considered for vaccination purposes.

Immunogenicity of the human immunodeficiency virus (HIV) recombinant nef gene product. Mapping of T-cell and B-cell epitopes in immunized chimpanzees.

The nonstructural nef gene product of human immunodeficiency virus (HIV), p27, is a regulatory "early phase" protein produced by HIV-infected cells. As a possible negative regulator of transcription, it has been suggested that p27 may be involved in the control of HIV proviral latency. Immune reactivity to p27 may result in early destruction of HIV-replicating cells before viral assembly or of latently infected cells. It appeared, thus, of interest to investigate the immunogenicity of the molecule in chimpanzees immunized against HIV antigens. Two of the six chimpanzees that were injected with soluble recombinant p27 in association with other HIV proteins, displayed significant and sustained T-helper lymphocyte proliferative responses to p27 and to the other antigens. Using a set of synthetic peptides spanning the entire p27 sequence, two T-cell epitopes could be located: one within the last 20 amino-acids of the C terminus of the molecule, the other around the region of residues 118-122. Sera from the same animals also reacted to p27 in a radioimmunoassay as well as to some of the peptides in enzyme-linked immunosorbent assay. Sequential B-cell epitopes could thus be determined as being located in the regions of amino acids: 17-35, 52-66, and 185-205. The results obtained with peptides spanning the region between amino acid residues 65 and 172 indicate that at least two additional B-cell epitopes were present in the region comprised between amino acid 65 and 146. Interestingly, the extreme C terminus of the molecule encompasses both immunodominant T- and B-cell epitopes. Taken together, these observations should prove useful for the rational design of a HIV vaccine.

Characterization of sodium and pyruvate interactions of the two carrier systems specific of mono- and di- or tricarboxylic acids by renal brush-border membrane vesicles.

The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zero trans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and delta psi = 0. Under zero trans condition and delta psi = 0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (Kt = 88 microM) and a low affinity for sodium (Kt = 57.7 mM) (site I), the second one with a low affinity for pyruvate (Kt = 6.1 mM) and a high affinity for sodium (Kt = 23.9 mM) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25 mM and 8 mM pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously. Under chemical equilibrium (delta psi congruent to 0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation was n = 1.7. The direct method of measuring Na+/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives a n = 1.67. Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).

Cell-mediated immune proliferative responses to HIV-1 of chimpanzees vaccinated with different vaccinia recombinant viruses.

The only animal that can be reproducibly infected with HIV, and that thus provides an experimental system for testing the effectiveness of prototype vaccines, is the chimpanzee. We compared proliferative responses to HIV and to vaccinia virus (VV) antigens of lymphocytes taken at various times from chimpanzees vaccinated with recombinant VV expressing different HIV genes. Animals were immunized with the original VV strain, as control, or with constructs expressing gp160 (VV160) given exclusively or in combination with one or two other constructs producing p25 (VV25), F/3'-orf (VVF), or the human interleukin-2 (IL-2) gene, which was included in an attempt to amplify immune responses. Irrespective of the HIV gene utilized, lymphocyte proliferation to HIV was usually weak and rapidly decreased after each inoculation, contrasting with strong and sustained responses to VV. Lack of adequate recall reactivity after challenge with fixed autologous lymphocytes expressing VV-produced HIV antigens indicated that vaccination resulted only in low levels of HIV-specific memory cell priming. The use of IL-2-producing VV did not lead to increased responsiveness. Reactivity to soluble purified gp160, but not to p25, could be detected in PBL from animals that had received both VV160 and VV25, while immunization with VVF resulted in a significant response to this protein in one of two animals. The transient nature of T cell reactivity to HIV might explain why, in similar studies, chimpanzees were not protected from infection with live HIV.

A molecular mechanism of inhibition of HIV-1 binding to CD4+ cells by monoclonal antibodies to gp110.

We have investigated the possible involvement in the interaction between HIV gp110 and its CD4 receptor of epitopes different from the currently known binding site(s) of the molecule. Four monoclonal antibodies (MAbs) to gp110 were used (Genetic Systems Corporation, Seattle, Washington, USA): one (110-1) recognized a peptide corresponding to the C-terminal part of gp110 (494-517); the other three (110-3, 110-4, 110-5) recognized the same peptide located at position 308-328. HIV or purified gp110 obtained from a vaccinia recombinant (Transgene S.A., Strasbourg, France) were pre-incubated with the MAb prior to addition to CD4+ cells. Specific binding of viral particles or of the soluble molecule was then determined by flow cytometer analysis, compared with that of control preparations where the MAb was added after HIV or gp 110 had been allowed to bind CD4+ cells. Significant inhibition of HIV binding was noted with the three MAbs to peptide (308-328), but not with 110-1. At the molecular level, these same MAbs decreased the affinity of interaction between CD4 and soluble gp110, although they could still label the latter molecule after it had bound to CD4+ cells. Therefore, steric hindrance may account for neutralization of HIV binding by antibodies that are actually directed to epitopes topographically distinct from the site of binding of gp110 to CD4.

RU-41740 (K. pneumoniae glycoprotein) enhances resistance to experimental candidiasis and stimulates phagocytic functions.

RU-41740, a purified glycoprotein extract from Klebsiella pneumoniae, (which is an efficient non-specific immune activator in a broad spectrum of in vitro and in vivo reactions) was administered either orally or parenterally in the mouse. It enhanced the resistance of mice to candidiasis, both in terms of survival rate and a decrease in viable yeast cell recovery in kidneys. The drug administered at 0.1 mg or 1 mg/kg augmented 4-fold the mean survival time (MST) of animals infected with 1 to 2 X 10(6) Candida albicans, both by the intraperitoneal and the intravenous route. The effect of the orally administered drug was less striking but nonetheless present. At 10 mg/kg, the MST of infected animals increased about 2-fold. In vitro, in the presence or absence of zymosan, the drug at 10 or 100 micrograms/ml was able to stimulate the phagocytic process of elicited mouse peritoneal cells (65% polymorphonuclear cells, 35% macrophages) and human peripheral blood cells (95% polymorphonuclear cells, 5% monocytes) in terms of activated oxygen species production. The involvement of polymorphonuclear cells in the mechanisms of natural resistance to C. albicans infection led us to discuss the role of these cells as targets for the drug.

Clinical trials with C 1740, an immunomodulator compound proposed for prevention of acute infectious exacerbations in chronic bronchitis.

C 1740 is an immunmodulating agent of biological origin proposed for the prevention of infectious exacerbations in chronic bronchopathy. The first placebo-controlled double-blind randomized clinical trials have led to opposite conclusions regarding the utility of C 1740. The rate of infectious exacerbations in the placebo group and a large Type II error could explain the "negative clinical trials". However, two out of four "positive clinical trials" were associated with high risk of falsely positive results. The activity of C 1740 is discussed here.

Tumor-promoting phorbol diesters inhibit in vitro antibody synthesis.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate prevents the synthesis of antibodies directed against sheep red blood cells in an in vitro system. The inhibitory effect of 3 phorbol diesters on this immune response was positively correlated with their tumor-promoting activity. The effect did not appear to be mediated through the inhibition of cell proliferation. Results suggest that the tumor promoter may alter the differentiation of the precursor cells to antibody-producing cells.