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New biomarkers are needed for monitoring the effectiveness of treatment for visceral leishmaniasis (VL). They might also improve the detection of the asymptomatic population in Leishmania- endemic areas. This paper examines the IL-2, IFN-γ, IFN-γ-induced protein 10 (IP-10), and monokine-induced-by-IFN-γ (MIG) levels in whole blood-stimulated in vitro with soluble Leishmania antigen (SLA)-taken from asymptomatic individuals and patients treated for VL living in a post-outbreak ( Leishmania infantum ) area in Spain, and in an endemic ( Leishmania donovani ) area of Bangladesh. IP-10 was found to be an accurate global marker of asymptomatic subjects with positive cellular/humoral tests, while MIG was found to be a better marker of contact with L. donovani than IL-2 but no for those with L. infantum . Determining IP-10, MIG, and IFN-γ levels proved useful in monitoring the cellular immune response following treatment for active disease caused by L. infantum .

Visceral leishmaniasis is a tropical disease caused by Leishmania donovani, an obligate intracellular parasite. Host cells of these parasites are macrophages. The conventional therapy of visceral leishmaniasis uses pentavalent antimony (Pentostam). To minimize the undesired side effects of antimony and to possibly reduce the therapeutical dose of antimony it was combined with interferon gamma (IFN gamma) and encapsulated both in multilamellar vesicles for treatment of murine visceral leishmaniasis. Using liposomes composed of one synthetic phospholipid and applying the freeze-thawing technique an encapsulation rate of about 30-70% of the offered antimony concentration was obtained. In the case of IFN gamma an entrapment of 20-30% was reached. Thus these liposomes were used to perform experiments in order to achieve pharmacological data about organ distribution of the encapsulated vs. free drug. For this purpose defined amounts of antimony were injected i.v. in B 10D2/n mice. At several time-points mice were sacrificed and lung, spleen, liver, kidneys and blood samples were assessed for antimony concentration. The results presented evidence that liposomal drugs were enriched in spleen and liver, organs mainly affected by visceral leishmaniasis. It has been shown previously that liposomes are phagocytozed by macrophages, the host cells of the parasites. Therefore treatment of L. donovani infected mice with liposomal antimony and IFN-gamma resulted in a nearly complete parasite reduction, while treatment with the free drug slightly reduced the parasite burden only in the liver. The aim of these studies was to minimize the undesired side effects of antimony and to possibly reduce the necessary dosage by 1 encapsulating it into multilamellar vesicles and 2, by combining the liposomal antimony with liposomal encapsulated IFN gamma, which is known to be a potent macrophage activating agent.

Active targeting of doxorubicin to macrophages was studied by incorporating it in mannose-coated liposomes by use of visceral leishmaniasis in BALB/c mice as the model macrophage disease. Mannosylated liposomal doxorubicin was more effective than liposomal doxorubicin or free doxorubicin. Because leishmaniasis is accompanied by immunosuppression, immunostimulation by interferon (IFN)-gamma was evaluated to act synergistically with mannosylated liposomal doxorubicin therapy. Combination chemotherapy with a suboptimal dose of IFN-gamma resulted in possibly complete elimination of spleen parasite burden. Analysis of mRNA levels of infected spleen cells suggested that targeted drug treatment together with IFN-gamma, in addition to greatly reducing parasite numbers, resulted in reduced levels of interleukin (IL)-4 but increased levels of IL-12 and inducible nitric oxide synthase. Such combination chemotherapy may provide a promising alternative for the cure of leishmaniasis, with a plausible conversion of antiparasitic T cell response from a Th2 to Th1 pattern indicative of long-term resistance.

Interferon (IFN)-gamma, in both natural endogenous form and administered as exogenous protein, induces control over visceral Leishmania donovani in experimentally infected BALB/c mice. To further characterize the therapeutic role of IFN-gamma in host defense against intracellular L. donovani, the efficacy of IFN-gamma delivered by gene transfer was tested. One week after infection, normal and IFN-gamma gene-disrupted (GKO) BALB/c mice were injected with an IFN-gamma gene-bearing mammalian expression plasmid (pIFN). Plasmid-specific IFN-gamma transcripts were detected in liver and spleen. Whereas liver parasite burdens more than doubled in untreated and mock-treated normal and GKO mice during the subsequent 2 weeks, animals injected with pIFN had controlled visceral infection and reduced parasite burden. These results indicate that, in infected tissues, IFN-gamma delivered by gene transfer enhances control over disseminated intracellular infection.

Interferon-gamma (IFN-gamma) enhances the therapeutic response to pentavalent antimony in patients with visceral leishmaniasis. To determine the effect of cytokine immunotherapy alone, 9 patients with kala-azar were treated with IFN-gamma before receiving antimony. After 20 days of IFN-gamma therapy, 4 patients showed no parasitologic response; in the remaining 5 patients, however, splenic aspirate parasite scores declined from 4.2 +/- 0.2 to 1.2 +/- 0.5 (mean +/- SE). These results indicate that treatment with IFN-gamma alone can induce visceral antileishmanial activity. However, the limited efficacy in this uncontrolled pilot trial suggests that the therapeutic role of IFN-gamma in kala-azar is that of an adjunct to conventional antimony treatment.

A 41-year-old AIDS patient with fever, nightly perspiration, diarrhoea, anaemia and leukopenia was diagnosed as having visceral leishmaniasis (VL). After 8 weeks of antimony treatment combined with gamma-interferon, given in 2 courses of 3 and 5 weeks, 12 weeks apart, the bone marrow revealed no parasites by microscopy and culture. Parasitic DNA could still be demonstrated by polymerase chain reaction. Weekly intravenous pentamidine maintenance therapy seemed to prevent relapses. Over time the patient was treated for disseminated M. avium infection, CMV retinitis, porphyria cutanea tarda and renal tubular acidosis. Ultimately he succumbed, 2.5 years after the diagnosis of VL and 4.5 years after the diagnosis of AIDS was established.

To determine if interferon (IFN)-gamma can enhance intracellular antimicrobial defense in a T cell-deficient host, nude BALB/c mice were infected with Leishmania donovani and treated with IFN-gamma. IFN-gamma induced killing of L. donovani in livers of euthymic mice but had no effect in nude mice despite activating peritoneal macrophages in vivo. Transfer of CD4+ or CD8+ T cells permitted nude mice to respond to IFN-gamma; treatment with T cell-regulated antileishmanial cytokines (interleukin-2, granulocyte-macrophage colony-stimulating factor, or tumor necrosis factor-alpha) could not substitute for T cells. NK cells played no apparent role. In reconstituted nude mice, the antileishmanial effect of IFN-gamma correlated with markedly enhanced mononuclear cell recruitment to infected liver foci. Thus, although IFN-gamma activates macrophages in the absence of host T cells, a T cell mechanism is required for antileishmanial activity in tissue. Provided one T cell subset is adequately preserved, IFN-gamma may prove useful in intracellular infections in the T cell-deficient host.

Visceral leishmaniasis (kala-azar) affecting HIV-infected patient is being reported in increasing frequency. A 40-year-old German bisexual patient with full-blown AIDS is described who presented with Kaposi's sarcoma, epigastric pain, diarrhea, and weight loss but without fever. Leishmania amastigotes were initially found in biopsies from stomach, duodenum, and a cutaneous Kaposi's sarcoma lesion but were later also recovered from bone marrow and lymph node. The patient received three courses of a combination of pentavalent antimony and interferon-gamma. In addition to the common side effects such as fever, thrombocytopenia, and elevated amylase and lipase, a vivid progression of the Kaposi's sarcoma was noted. Tumor progression was temporally closely associated with treatment with interferon-gamma. Because this phenomenon has also been observed in other patients, we advise caution when using interferon-gamma in patients with Kaposi's sarcoma.

Fifteen Indian patients with relapsing or drug-refractory visceral leishmaniasis were retreated for 30 days with antimony plus interferon-gamma (IFN-gamma). All 15 had failure of an initial course of antimony and at least one additional course of antimony or pentamidine; 7 patients had failure of three or four prior courses of therapy. During the study, treatment was discontinued in 2 patients because of anemia and congestive heart failure in 1 and intractable vomiting in the other; both subsequently died. In the remaining 13 patients, IFN-gamma plus antimony treatment was associated with daily fever but no other adverse reactions. After 30 days of therapy, 9 (69%) of the 13 patients were apparently cured. Six months after treatment, all 9 were healthy, had parasite-free bone marrow aspirate smears, and were considered cured. None have relapsed during a mean follow-up of 15.9 +/- 1.7 months. These results support the use of antimony plus IFN-gamma as an immunochemotherapeutic alternative for kala-azar patients who have repeated failures of conventional treatment.

The optimal therapy for visceral leishmaniasis in patients with AIDS is unknown. We describe herein three patients with advanced AIDS and disseminated leishmaniasis who were treated with meglumine antimoniate plus interferon-gamma. The conditions of the three patients rapidly improved, and the therapeutic combination drastically reduced the parasite burden; after administration of antiprotozoal therapy for 4 weeks, cultures of bone marrow were negative for two of the three patients. Interferon-gamma was well tolerated. Because the patients died of other AIDS-related diseases in the months following therapy, the efficacy of the combination of meglumine antimoniate and interferon-gamma for preventing relapses remains unknown.

Traditionally, the mainstay of visceral and diffuse cutaneous leishmaniasis therapy has been pentavalent antimony, with pentamidine and amphotericin B reserved for refractory cases. Interferon-gamma (IFN-gamma) in combination with pentavalent antimony has been used successfully in treating patients refractory to pentavalent antimony and in patients with previously untreated visceral leishmaniasis (39 total). In addition, 6 patients with diffuse cutaneous leishmaniasis have been treated with combination IFN-gamma and antimony therapy. Preliminary experience with these patients indicates that IFN-gamma is a useful adjunct therapy for severe or refractory cases of visceral leishmaniasis; however, the potential of IFN-gamma in treating diffuse cutaneous leishmaniasis cannot be assessed as current clinical experience is too limited.

The clinical, parasitological, hematological, and serological evolution of visceral leishmaniasis in Brazilian patients was assessed during treatment with human recombinant interferon-gamma (rIFN-gamma; 0.1 mg/m2 i.m. days 1-14) followed by pentavalent antimony (Sbv; 10 mg/kg days 22-28). At day 30, 6 patients had improved, 2 had slightly improved, and 1 patient had deteriorated. IFN-gamma was well tolerated in the dose tested and may be very effective as an adjunct to conventional therapy with antimony.

The efficiency of immunotherapy with murine recombinant interferon-gamma (rIFN-gamma) in mouse visceral leishmaniasis caused by Leishmania donovani was examined. To avoid the side effects encountered after the in vivo administration of high dosages of free IFN-gamma, this lymphokine and muramyltripeptide (MTP-PE) were encapsulated into multilamellar liposomes. We established that a combination of 5 X 10(3) U of IFN-gamma and 6 micrograms of MTP-PE, encapsulated in liposomes and given i.v. in C56BL/6 and BALB/c mice activates macrophages from spleen and liver in vivo to kill L. donovani in vitro. Neither empty liposomes nor the same concentration of free IFN-gamma and/or MTP-PE injected i.v. resulted in a leishmanicidal activity of these macrophage populations. For verification of these results in an in vivo infection model, susceptible mice were infected with L. donovani and were treated with IFN-gamma and MTP-PE encapsulated in multilamellar vesicles. Treatment consisted of multiple i.v. injections beginning 4 and 2 days before infection (prophylactic), either simultaneously with the infection or at various times of the exacerbation and remission phases of visceral leishmaniasis. These mouse strains treated with IFN-gamma and MTP-PE in liposomes had significantly fewer splenic parasites than untreated mice or control animals treated with free drugs or empty liposomes. The targetting of multilamellar vesicles to liver and spleen make them particularly suited for the delivery of macrophage-activating substances used for treatment of visceral L. donovani infection.

The present study was performed to evaluate the ability of lymphocytes from 18 children living in an endemic area of visceral leishmaniasis (VL) to produce gamma-interferon. These children had no previous history of VL and were considered to be infected with Leishmania chagasi based on leishmanial seroconversion. The gamma IFN levels were determined by radioimmunoassay on supernatants of lymphocyte cultures (3 x 10(6)/ml) stimulated with PHA (final dilution 1:10) and Leishmania chagasi antigen (10 micrograms/ml). The gamma-IFN production by lymphocytes from seroconverting children stimulated with PHA (178 +/- 151 U/ml) and Leishmania chagasi (47 +/- 77 U/ml) was significantly higher than that observed in visceral leishmaniasis. For clinical follow-up, these 18 seroconverting children were divided into three groups: asymptomatic infection (N = 4); self-healing subclinical illness (N = 9), and subclinical infection progressing to VL (N = 5). Gamma IFN levels in children with either asymptomatic or subclinical infection (65 +/- 85 U/ml) were significantly higher (P less than 0.003) than those observed in children progressing to VL (9 +/- 6 U/ml). The data demonstrate that there is an association between gamma IFN levels and the clinical course of Leishmania infection.

In experimental visceral leishmaniasis, intermittently administered interferon-gamma (IFN-gamma) induces antileishmanial activity, which is primarily microbistatic. To determine if the efficacy of IFN-gamma immunotherapy could be enhanced by continuous delivery, Leishmania donovani-infected mice were treated using a subcutaneous osmotic pump. Once-daily intraperitoneal injections of 10(5) or 10(6) units of IFN-gamma inhibited the replication of L. donovani within liver macrophages but overall did not reduce liver parasite burdens. In contrast, a comparable dose of IFN-gamma (2.4 x 10(5) units/day) administered continuously induced an enhanced effect and reduced liver burdens by almost 50%. Although pump delivery did not similarly increase the efficacy of antimony chemotherapy in infected mice, continuous treatment with IFN-gamma plus antimony produced an additive antileishmanial effect. These results suggest that continuous infusions of macrophage-activating lymphokines such as IFN-gamma (used alone or in combination with chemotherapy) may be required to optimize in vivo antimicrobial effects.

We have found that the crude lymphokines, which prime the human monocyte-derived macrophage to generate H2O2 and exert microbicidal activity against intracellular Leishmania donovani, are rich in interferon (IFN)-gamma (600-3,000 U/ml). To determine the role of this specific lymphocyte product in macrophage activation, lymphokines were pretreated with a monoclonal antibody that neutralizes human IFN-gamma. Antibody exposure completely abolished the capacity of both mitogen- and antigen-stimulated lymphokines to either enhance macrophage H2O2 release or induce leishmanicidal activity. In addition, partially purified and pure recombinant human IFN-gamma were as effective as crude lymphokines in activating macrophages, and 3 d of treatment with 300 U/ml resulted in a seven- to eightfold increase in H2O2 generation and the intracellular killing of both L. donovani promastigotes and amastigotes. The ability of crude lymphokines to induce monocytes and macrophages from a patient with chronic granulomatous disease to kill L. donovani promastigotes was similarly abrogated by anti-IFN-gamma antibody, and could also be achieved by IFN-gamma alone. These results suggest that IFN-gamma is the key macrophage-activating molecule present within human lymphokines, and indicate that IFN-gamma can enhance both the oxygen-dependent and -independent antiprotozoal mechanisms of human mononuclear phagocytes.

To determine if the macrophage-activating T cell lymphokine gamma interferon (IFN-gamma) can enhance the effect of conventional chemotherapy against intracellular Leishmania donovani, we treated human macrophages in vitro with both recombinant (r) IFN-gamma and sodium stibogluconate (Pentostam). After pretreatment with a nonactivating dose of rIFN-gamma (10 U/mL), ineffective concentrations of Pentostam (1 and 5 micrograms/mL) were converted to leishmanistatic concentrations, and a leishmanistatic dose (10 micrograms/mL) was converted to uptake of Pentostam. In a model of visceral leishmaniasis, infected mice were treated with ineffective concentrations of rIFN-gamma (10(4) U) plus suboptimal doses of Pentostam (10 or 50 mg/kg). With combination therapy, the doses of Pentostam required to achieve 50% inhibition or killing of visceral L. donovani were reduced by 10-fold and fourfold, respectively. These results suggest that IFN-gamma therapy may be a useful adjunct in visceral leishmaniasis and illustrate one potential role for IFN-gamma in the treatment of systemic intracellular infections.

Although directly microbicidal, pentavalent antimony has failed as treatment for visceral leishmaniasis in patients who also have AIDS or are receiving immunosuppressive therapy. To define the role of T cells in the successful host response to chemotherapy, we examined the efficacy of pentavalent antimony (sodium stibogluconate, Pentostam) in normal and T cell-deficient BALB/c mice infected with Leishmania donovani. In euthymic (nu/+) mice, single injections of 250 and 500 mg/kg of Pentostam induced the killing of 67% and 89% of intracellular liver amastigotes, respectively. In contrast, in athymic nude (nu/nu) mice, up to three injections of 500 mg/kg achieved no L. donovani killing and did not retard visceral parasite replication. Once nude mice were reconstituted with nu/+ spleen cells, however, Pentostam exerted strong leishmanicidal activity, an effect that appeared to be transferred by either L3T4+ or Lyt-2+ cells. Responsiveness to chemotherapy could also be induced by providing nude mice with either interferon-gamma or interleukin 2 alone. The absence of this T cell- and probably lymphokine-dependent mechanism is a likely explanation for treatment failures in immunocompromised patients infected with L. donovani and perhaps other systemic intracellular pathogens as well.

Recombinant mouse interferon-gamma (IFN-gamma) was encapsulated into multilamellar vesicles and the proportion of encapsulated IFN-gamma determined by biological activity was 19%. The distribution of 125I-labeled IFN-gamma liposomes in C57BL/6 mice was analyzed. After an initial enrichment of liposomes in lung, more than 60% of total 125I-labeled IFN-gamma was accumulated in spleen and liver. Furthermore, it was observed if the encapsulation of IFN-gamma in liposomes prevented the rapid decay of IFN-gamma in serum of C57BL/6 mice after intravenous injection. We compared the serum decay curve of liposomal and free IFN-gamma, and showed that IFN-gamma encapsulated in liposomes has an elongated availability in the serum. In addition, we established that a combination of 10(2) U/ml IFN-gamma and 1 microgram/ml MTP-PE, encapsulated in liposomes, activates splenic and starch-elicited peritoneal macrophages in vitro synergistically to kill Leishmania donovani promastigotes. After intravenous injection of liposomal IFN-gamma (5 X 10(3) U) and muramyltripeptide (MTP-PE) (6 micrograms) in C57BL/6 mice, splenic and liver macrophages were activated in vivo to kill Leishmania species in vitro. Neither an injection of the same amount of free substances nor injection of empty liposomes resulted in an increased leishmanicidal activity.

During the first 2 to 4 weeks of progressive visceral infection with the intracellular protozoan, Leishmania donovani, spleen cells from BALB/c mice failed in response to leishmanial antigen to produce either of the activating T cell-derived lymphokines, interleukin 2 (IL 2) or gamma-interferon (IFN-gamma). Four weeks after infection, however, antigen-induced IL 2 and IFN-gamma secretion emerged and coincided with the onset of control over parasite replication and the subsequent killing of greater than 80% of intrahepatic L. donovani. The development of this immunosecretory activity correlated with the hepatic tissue response at the site of parasitized Kupffer cells. This response progressed from Kupffer cell fusion (week 1) to fusion plus a mononuclear cell infiltrate (week 2) to well-organized granuloma formation (weeks 4 to 8). In contrast, T cell-deficient nude BALB/c mice exerted no control over L. donovani, their spleen cells failed to generate antigen-induced IFN-gamma, and at 4 weeks, their livers were devoid of any tissue reaction. Since spleen cells from 2-week infected normal mice did not produce antigen-stimulated IL 2 or IFN-gamma, these mice were treated with recombinant (r) lymphokines. Various protocols using both high and low dose human rIL 2 had no antileishmanial effect. Hepatic parasite replication was completely halted, however, by macrophage-activating doses of murine rIFN-gamma. These results reemphasize that an intact T cell-dependent response is required for successful defense against L. donovani, indicate that this immune response can be measured at both the cellular (secretory) and tissue levels, and confirm that IFN-gamma can exert an antileishmanial effect in vivo.