Anti-HIV-1 activity in vitro of ceftazidime degradation products.

Cephalosporins in aqueous solutions generate degradation products that inhibit in vitro HIV-1 replication in cell lines, as well as in primary cells (lymphocytes and macrophages). This effect is observed at concentrations that do not interfere with the normal functions of these cells. Upon chromatographic fractionation of an aqueous solution of hydrolysed ceftazidime, a high molecular weight fraction (MW 8000) with antiviral activity was isolated. The exact chemical nature of the active component responsible for the anti-HIV activity in vitro appears to be complex and is currently unknown. Inhibition of HIV-1 reverse transcriptase and RNase H activity was observed, however, higher concentrations than those needed to inhibit HIV replication were required. The inhibitory action of the hydrolysed ceftazidime was manifested during the early phase of the HIV-1 life-cycle. Despite a lack of a direct effect of the CD4/gp120 interaction, HIV-1 mediated cell fusion was inhibited by the hydrolysed ceftazidime, suggesting that the active principle acts in a very early stage of the viral life-cycle.

An analysis of the safety of the single dose, two drug regimens used in programmes to eliminate lymphatic filariasis.

This review of the safety of the co-administration regimens to be used in programmes to eliminate lymphatic filariasis (albendazole + ivermectin or albendazole + diethylcarbamazine [DEC]) is based on 17 studies conducted in Sri Lanka, India, Haiti, Ghana, Tanzania, Kenya, Ecuador, the Philippines, Gabon, Papua New Guinea, and Bangladesh. The total data set comprises 90,635 subject exposures and includes individuals of all ages and both genders. Results are presented for hospital-based studies, laboratory studies, active surveillance of microfilaria-positive and microfilaria-negative individuals, and passive monitoring in both community-based studies and mass treatment programmes of individuals treated with albendazole (n = 1538), ivermectin (9822), DEC (576), albendazole + ivermectin (7470), albendazole + DEC (69,020), or placebo (1144). The most rigorous monitoring, which includes haematological and biochemical laboratory parameters pre- and post-treatment, provides no evidence that consistent changes are induced by any treatment; the majority of abnormalities appear to be sporadic, and the addition of albendazole to either ivermectin or DEC does not increase the frequency of abnormalities. Both DEC and ivermectin show, as expected, an adverse event profile compatible with the destruction of microfilariae. The addition of albendazole to either single-drug treatment regimen does not appear to increase the frequency or intensity of events seen with these microfilaricidal drugs when used alone. Direct observations indicated that the level of adverse events, both frequency and intensity, was correlated with the level of microfilaraemia. In non microfilaraemic individuals, who form 80-90% of the 'at risk' populations to be treated in most national public health programmes to eliminate lymphatic filariasis (LF), the event profile with the compounds alone or in combination does not differ significantly from that of placebo. Data on the use of ivermectin + albendazole in areas either of double infection (onchocerciasis and LF), or of loiais (with or without concurrent LF) are still inadequate and further studies are needed. Additional data are also recommended for populations infected with Brugia malayi, since most data thus far derive from populations infected with Wuchereria bancrofti.

In vitro effect of alpha1-acid glycoprotein on the anti-human immunodeficiency virus (HIV) activity of the protease inhibitor CGP 61755: a comparative study with other relevant HIV protease inhibitors.

Protein binding can impair the potency of human immunodeficiency virus (HIV) protease inhibitors. Therefore, the activity of a novel compound, CGP 61755, was studied in the absence or presence of alpha1-acid glycoprotein (alpha1AGP). In MT-2 cells, the activity loss was 4-fold (EC90 without alpha1AGP, 29 nM vs. 122 nM with alpha1AGP). In primary lymphocytes, the loss was 8-fold (EC90, 45 nM vs. 364 nM). In identical experiments, the activity loss in MT-2 cells and lymphocytes was 2- and 3-fold, respectively, for indinavir, 11- and 10-fold for saquinavir, and 11- and 48-fold for ritonavir. For SC-52151, a 17-fold loss was seen in MT-2 cells, whereas no EC90 with alpha1AGP was reached in lymphocytes. This study demonstrates that the impact of alpha1AGP on in vitro activity varies greatly among different HIV protease inhibitors. The magnitude of such differences is greater in human lymphocytes than in a standard cell line.

Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

Profile of CGP 61755: a novel and potent HIV-1 protease inhibitor that shows enhanced anti-HIV activity when combined with other antiretroviral agents in vitro.

CGP 61755 is a novel hydroxyethylene derivative produced by a high yield 10 step chemical synthesis. It is highly specific for HIV-1 protease with an IC50 of 1 nM. The ED90 in MT-2, PBLs and macrophages is infected with laboratory strains of HIV-1 or clinical isolates is 30-100 nM. In chronically infected macrophages the ED90 is 1000 nM (1000 nM for saquinavir and 10 microM for indinavir). When the antiviral activity of CGP 61755 on HIV-1 infected lymphocytes was examined using serum free medium an ED99 of 60 nM was determined, while in the presence of 10% human serum the same activity was achieved with 120 nM. When examined in combination with RT inhibitors or protease inhibitors, either in a co-culture of CEM-SS and chronically infected H9IIIB cells or in a free virus lymphocyte infection, cooperativity of the antiviral activities was observed. Dog pharmacokinetic studies comparing p.o. and i.v. data indicate that CGP 61755 has a bioavailability between 50 and 80%. Following oral administration the area under the concentration curve (AUC) values increased in a dose proportional manner. The plasma levels of the drug at 6 hours after oral administration were above the ED90. Based on these properties we believe that CGP 61755 has an attractive profile that justifies further preclinical evaluation of the drug.

The replicative restriction of lymphocytotropic isolates of HIV-1 in macrophages is overcome by TGF-beta.

In vitro exposure of human blood monocyte-derived macrophages to T-cell tropic human immunodeficiency virus (HIV) isolates fails to establish a productive viral infection. Several studies have shown that such preferential HIV-1 replication in T cells or in mononuclear phagocytes (HIV tropism) may be determined by distinct viral characteristics. In the present study it was demonstrated that transforming growth factor-beta (TGF-beta), a factor known to be produced by platelets, macrophages, and other cells present at a wound site, can act as a mediator in overcoming the lymphocytotropic restriction of several well-characterized viral isolates of HIV-1 (i.e., LAV, Z84, pLAI, NY5). Macrophages infected with these isolates show cytopathic changes comparable to those seen upon infection with the monocytotropic isolate ADA. To achieve this effect with TGF-beta, the factor must be present after the infection period. The emerging virus retains its original cellular tropism. Based on these observations the authors propose a role for TGF-beta in the establishment and progression of HIV infection and disease.

In vitro effect of transforming growth factor-beta on progression of HIV-1 infection in primary mononuclear phagocytes.

In vitro-differentiated monocytes can be infected with the monocytotropic isolate of HIV-1/ADA. The infection is characterized by formation of giant cells and production of virus that can be found in cell supernatants or cell-associated. In this study, we demonstrate that the above described parameters of infection can be enhanced by a factor present in acidified M phi supernatants, suggesting that it might be transforming growth factor beta-1 (TGF-beta 1). When recombinant or purified TGF-beta were examined, similar activities were detected. This effect apparently is not because of changes in the cellular phenotype that could favor infection. The effect of TGF-beta is exerted on cells once infection is established or on cells with active virus production. The activity can be also demonstrated using U-937 cells.

TGF-beta: upregulator of HIV replication in macrophages.

TGF-beta at physiological concentrations, when added to monocyte-derived macrophages following HIV1 infection, has an enhancing effect upon the rate of virus production. This effect is observed with the monocytotropic isolate ADA, as well as with HIV1 IIIB, which poorly replicates in macrophages.

Activation of human monocyte-derived macrophages to kill schistosomula of Schistosoma mansoni in vitro.

Human peripheral blood monocytes from normal donors were isolated by elutriation and differentiated by culture in the presence or absence of various immunomodulators. Cells were harvested between 0 and 24 days and tested for their ability to kill schistosomula of Schistosoma mansoni in vitro as a measure of activation. Freshly isolated monocytes showed no significant cytotoxic activity in the presence or absence of IFN-gamma or LPS. As the cells matured in vitro, there was a slight increase in their inherent toxicity against the parasite, which was greatly enhanced by pretreatment with either IFN-gamma or CSF-1. Optimal antibody-independent larvicidal activity occurred after stimulation with both IFN-gamma and CSF-1, using cells that had matured for at least 7 days in vitro. Under these conditions, killing of up to 70% of the larvae was observed. Although enhanced larvicidal activity was not found to strictly correlate with production of any of several proposed effector molecules examined, activated monocyte-derived macrophages were capable of producing significant amounts of H2O2 and TNF-alpha. These observations indicate that cytokine-activated human monocyte-derived macrophages are able to kill schistosome larvae by an antibody-independent mechanism, as has been observed using murine peritoneal macrophages. Stimulation with multiple differentiation and activation signals, as would occur in vivo, may be required for development of optimal larvicidal activity.

The lipophilic muramyl peptide MTP-PE is a potent inhibitor of HIV replication in macrophages.

Cultured monocyte-derived macrophages were productively infected with human immunodeficiency virus in vitro. Treatment of these cells shortly after infection and several times thereafter with the free form of MTP-PE had an inhibitory effect on virus production. When the liposomal formulation of MTP-PE was used, higher levels of protection were achieved. The drug was not only effective when added to cells immediately after infection, but it also reduced virus production by cells with an established infection. When the liposomal formulation of MTP-PE was used only one treatment was required to achieve maximal effects. During these studies it was noted that the placebo liposomes had some effect in reducing the reverse transcriptase levels found in the supernatants of infected cells. This reduction could not be explained by direct cytotoxic effect. Both free and liposomal MTP-PE lipid significantly prevented formation of giant cells during the course of infection as well as reduced the cell associated viral antigen.

In vitro effect of thymosin alpha 1 on the expression of peanut agglutinin binding by murine thymocytes.

Thymosin alpha 1 induces the loss of PNA binding ability by subpopulation of thymic cells. This loss is probably due to an endocytic process. Nevertheless this disappearance is not a permanent one, suggesting a recycling of the PNA binding molecule. The cells that modulate their PNA binding sites after exposure to Thymosin alpha 1 are a small proportion of the total PNA+ thymocytes, indicating that not all thymocytes are susceptible to the thymic hormone Thymosin alpha 1. Conversely the exposure of thymocytes to Thymosin alpha 1 induces the disappearance of the binding sites for this ligand without further recycling, behavior expected for the receptor of a regulatory ligand. These results also indicate that the Thymosin alpha 1 and the PNA binding sites are on different molecules on the surface of the PNA+ thymocytes.

Macrophages as effector cells of protective immunity in murine schistosomiasis. VI. T cell-dependent, lymphokine-mediated, activation of macrophages in response to Schistosoma mansoni antigens.

Macrophages from Schistosoma mansoni-infected mice kill significant numbers of skin stage schistosomula and murine fibrosarcoma cells in vitro. In order to determine whether the macrophage tumoricidal and larvicidal activation observed in mice as a result of S. mansoni infection are mediated through T cell-dependent (lymphokine) or B cell-dependent (antibody or immune complex) mechanisms, the development of macrophage populations with cytotoxic activity against schistosome larvae or tumor cells was monitored in S. mansoni-infected nude or mu-suppressed mice. Whereas peritoneal cells from S. mansoni-infected congenitally athymic mice had no activity in either assay, cells from mu-suppressed S. mansoni-infected mice showed cytotoxic activity equivalent to that of cells from untreated S. mansoni-infected counterparts. Cells from mu-suppressed uninfected mice were not activated. The mu-suppressed animals had no detectable nonspecific IgM or specific antischistosome IgM, IgG, or IgE antibodies and showed a 90% reduction in numbers of splenic IgM+ cells upon fluorescence activated cell sorter analysis. These results indicate that antibody is not required for in vivo activation of macrophages during S. mansoni infection. Further experiments showed that lymphoid cells from S. mansoni infected mice respond in culture with various specific antigens (such as living or dead whole schistosomula or soluble adult worm antigens) by production of factors capable of activating macrophages from uninfected control mice to kill schistosomula or tumor cells in vitro. Macrophage-activating factors were produced by T cell-enriched, but not T cell-depleted or B cell-enriched, populations from spleens of schistosome-infected mice in response to schistosome antigen. Similar lymphokines may be responsible for the macrophage activation observed during chronic murine schistosomiasis. These observations emphasize the potential contribution of T cell-mediated immune mechanisms in resistance to S. mansoni infection.

Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages.

Maximal rates of superoxide (O-2) release, and the cytochemical locales of peroxide staining in resident, elicited, and activated macrophages have been determined. Macrophages elicited into the peritoneum with either casein (1.2% w/v) or proteose-peptone (10.0% w/v) release about twice as much O-2 as macrophages activated by infection of the animals with either Listeria monocytogenes, or Bacille Calmette-Guerin (BCG) followed by immune boosting with Purified Protein Derivative (PPD) (i.e., about 35 vs. 14-18 nmol O-2/min/10(7) cells). Macrophages elicited with thioglycollate (3.0% w/v) and resident macrophages produce negligible amounts of O-2 upon stimulation with PMA. These data are compared with those reported by other investigators who used different procedures. A cytochemical procedure for localizing peroxide has been modified for use with murine macrophages. No production of H2O2 by macrophages is detected cytochemically in the absence of stimulation. Upon exposure to PMA, resident macrophages are still largely unresponsive. Approximately 20% of the casein elicited macrophages and BCG-PPD activated macrophages exhibit H2O2 staining, which is largely restricted to the cytoplasmic vesicles and channels induced by PMA in these cells. The only exception to this staining pattern is a small population (about 2%) of activated macrophages which exhibits H2O2 staining in the cytoplasmic vesicles and channels and on the plasmalemma as well.

Macrophages as effector cells of protective immunity in murine schistosomiasis. II. Killing of newly transformed schistosomula in vitro by macrophages activated as a consequence of Schistosoma mansoni infection.

Plastic adherent peritoneal cells from Schistosoma mansoni-infected mice have previously been shown to exhibit nonspecific tumoricidal activity in vitro. In this report we show that these same cell populations kill significant numbers of skin-stage schistosomula in vitro in the absence of added antibody. Larval killing by these activated cells could be enhanced by the use of suspension rather than monolayer cultures and by addition of heat-inactivated immune mouse serum to the cultures. Adherence of cells to schistosomula was also enhanced under the same conditions, suggesting that cell binding to the larvae might be critical in the development or expression of microbicidal activity. In support of this hypothesis, the same level of enhancement of cell binding and larval damage was observed upon substitution of concanavalin A for immune mouse serum. Killing of schistosomula appeared to be mediated solely by activated macrophages in the peritoneal cell suspensions from S. mansoni-infected mice, because partially purified preparations of eosinophils were virtually inactive in these assays. Likewise, inflammatory macrophages from uninfected mice were unable to kill schistosomula under the same conditions, emphasizing the importance of activation in the development of killing capability. The finding that macrophages activated as a consequence of S. mansoni infection are able to kill larval schistosomes in vitro suggests that these cells may play a role in concomitant immunity to schistosomiasis in vivo.

Macrophages as effector cells of protective immunity in murine schistosomiasis. III. Loss of susceptibility to macrophage-mediated killing during maturation of S. mansoni schistosomula from the skin to the lung stage.

Newly transformed skin-stage and lung-stage schistosomula were compared in terms of their susceptibility to killing mediated by activated mouse macrophages in vitro. Although skin-stage schistosomula were readily killed by macrophages activated as a consequence of either BCG or Schistosoma mansoni infection and used either as cell monolayers or in suspension, lung-stage larvae appeared to be totally resistant to this effector mechanism and survived normally when reinjected into mice. Resistance of schistosomula to in vitro damage by macrophages was evident as early as 18 hr after host infection and was complete in worms recovered at 42 hr. The insusceptibility of lung-stage larvae is apparently not due to a defect in effector cell-target contact, because the induction of extensive macrophage adherence to the worms by the addition of anti-mouse red blood cell antisera to the cultures had no effect on parasite viability. These findings provide additional support for the concept that schistosomula during their development to the lung stage undergo a generalized change affecting their susceptibility to a variety of different immunologic effector mechanisms.

Comparative aspects of oxidative metabolism of neutrophils from human blood and guinea pig peritonea: magnitude of the respiratory burst, dependence upon stimulating agents, and localization of the oxidases.

We have compared the subcellular sites of H2O2 and presumably also superoxide-(O2-) production, and certain aspects of metabolic responses (O2 consumption, O2- production) of stimulated neutrophils from human blood and those elicited into guinea pig peritonea. Stimulation was accomplished with either opsonized zymosan or phorbol-12-myristate-13-acetate (PMA). Striking quantitative differences were observed between these cell types with regard to the increased respiration and O2- production observed during stimulation. These differences were most apparent when opsonized zymosan served as the stimulating agent. They were minimized when the soluble stimulating agent, PMA, was used. With either stimulus, the subcellular sites of H2O2 production were the same for both types of neutrophils, i.e., the plasmalemma and phagosomal membranes. No H2O2 production could be detected cytochemically in the absence of stimulation. Treatment of both unstimulated human blood and elicited guinea pig peritoneal neutrophils with the nonpenetrating, covalently linking reagent, p-diazobenzenesulfonic acid, failed to diminish O2- production upon subsequent stimulation, in contrast to a previous report. These data are discussed in terms of the possible cytological arrangements of the respiratory enzyme(s), and the different modes of stimulation of neutrophil metabolism by various agents. Ancillary data on elicited mouse peritoneal neutrophils are presented.