Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells.

Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy.

HLA-B35 correlates with a favorable outcome following adjuvant administration of an HLA-matched allogeneic melanoma vaccine.

This work presents survival data of 42 melanoma patients at high risk for disease recurrence who received an allogeneic melanoma vaccine composed of three cell lines, each matching at least one allele of the recipient's human leukocyte antigen (HLA)-A and -B loci. The 5-year overall survival (OS) rate and disease-free survival (DFS) compared favorably with the standard interferon-α regimen. Interestingly, patients bearing HLA-B35 had significantly better OS and DFS (OS of 100% and DFS of 90% for HLA-B35 vs 56% and 23%, for the non-B35 patients). In contrast, patients expressing HLA-B07 did not fare well with the vaccine. Although the data include a relatively small cohort of patients, it strongly hints toward a correlation between HLA types and potential benefit from anticancer immunotherapy.

Production and purification of melanoma gp100 antigen and polyclonal antibodies.

gp100 is a melanoma-associated antigen found to carry immunogenic epitopes that can induce a CTL response against tumor cells. Production and purification of large quantities of this polypeptide may be important in the context of diagnosis and vaccinating against melanoma. To overcome the hydrophobic nature of gp100, we cloned and expressed only a part of the protein, and obtained a hydrophilic recombinant polypeptide (HR-gp100) that contained most of the immunogenic peptides. High yield was achieved in an Escherichia coli expression system. The protein was purified by AKTA Prime using anionic-columns. Polyclonal antibodies developed in chicken against HR-gp100 were efficient at detecting gp100 in melanoma cells, as determined by Western blot analysis and by immunohistochemistry. HR-gp100 can be used to develop a vaccine against melanoma. Antibodies to HR-gp100 may be used to detect tumors of melanocytic origin or to determine the level of gp100 expression in tumors prior to immunotherapy with the protein or one of its peptides.

Interleukin-2 improves tumour response to DNP-modified autologous vaccine for the treatment of metastatic malignant melanoma.

This paper is a report of response rate (RR) and survival of 34 metastatic melanoma patients who received a dinitrophenyl (DNP)-modified autologous melanoma cell vaccine. In all, 27 patients started the vaccine as a primary treatment for metastatic melanoma and seven started it as an adjuvant, with no evidence of disease at the time, but had developed new metastases. Interleukin-2 (IL-2) was administered in 24 out of the 34 patients: 19 who progressed on vaccine alone and five who had the combination from start. Interleukin-2 was administered in the intravenous, bolus high-dose regimen (seven patients) or as subcutaneous (s.c.) low-dose treatment (17). Overall response for the entire group was 35% (12 patients out of 34), 12% having a complete response (CR) and 23% a partial response (PR). However, only two patients had tumour responses while on the vaccine alone, whereas the other 10 demonstrated objective tumour regression following the combination with IL-2 (two CR, eight PR), lasting for a median duration of 6 months (range 3-50 months). Of the 12 responding patients, 11 attained strong skin reactivity to the s.c. injection of irradiated, unmodified autologous melanoma cells. None of the patients with a negative reactivity experienced any tumour response. Patients with positive skin reactions survived longer (median survival - 54 months). The results suggest enhanced RRs to the combination of IL-2 and autologous melanoma vaccine. Skin reactivity to unmodified autologous melanoma cells may be a predictor of response and improved survival, and therefore a criterion for further pursuing of immunotherapeutic strategies.

Relapse of cutaneous leishmaniasis in a patient with an infected subcutaneous rheumatoid nodule.

Cutaneous leishmaniasis is a protozoal infection generally considered to be limited to the skin. In Israel, the disease is common in geographically defined areas and is caused predominantly by Leishmania major. Sporotrichoid subcutaneous spread has been reported but is uncommon. We describe a patient with rheumatoid arthritis, treated with methotrexate and prednisone, in whom numerous rheumatoid nodules concomitant with cutaneous leishmaniasis were found, mimicking sporotrichoid spread of the disease. In a rheumatoid nodule that was examined by electron microscopy, Leishmania parasites were found at intracellular and extracellular locations. This observation supports the hypothesis that cutaneous leishmaniasis parasites persist after clinical cure of the disease and may re-emerge as a result of immunosuppression.

Efficacious treatment of experimental leishmaniasis with amphotericin B-arabinogalactan water-soluble derivatives.

In this study, we tested the efficacy of amphotericin B (AmB)-arabinogalactan (AmB-AG) conjugates for the treatment of experimental leishmaniasis. Chemical conjugation of AmB to a water-soluble, biodegradable, and biocompatible polymer could present many advantages over presently available AmB formulations. Two conjugates were tested, a reduced (rAmB-AG) form and an unreduced (uAmB-AG) form. In vitro, the drug concentrations which lower the values of parasites (for promastigotes) or infected macrophages (for amastigotes) to 50% of the untreated values (ED(50)s) of uAmB-AG and rAmB-AG were 0.19 and 0.34 microg/ml, respectively, for Leishmania major promastigotes and 0.17 and 0.31 microg/ml, respectively, for amastigotes. The effect on Leishmania infantum-infected macrophages was more marked, with ED(50)s of 0.035 microg/ml for rAmB-AG and 0.027 microg/ml for uAmB-AG. In in vivo experiments, BALB/c mice injected with L. major were treated from day 2 onwards on alternate days for 2 weeks. Both conjugates, as well as liposomal AmB (all at 6 mg/kg of body weight) and Fungizone (1 mg/kg), significantly delayed the appearance of lesions compared to that in untreated mice. In addition, both conjugates, but not liposomal AmB, were significantly more effective than Fungizone. Subcutaneous injection of the conjugates (6 mg/kg) was significantly more effective than liposomal AmB in delaying the appearance of lesions. Higher AmB concentrations of up to 12 mg/kg could be administered by this route. When an established infection was treated, uAmB-AG was somewhat more effective than liposomal AmB. In summary, water-soluble polymeric AmB derivatives were found effective and safe for the treatment of leishmanial infections. The conjugates, which are stable and can be produced relatively cheaply (compared to lipid formulations), can be used in the future for the treatment of leishmaniasis infections.

Efficacious topical treatment for murine cutaneous leishmaniasis with ethanolic formulations of amphotericin B.

The goal of the present study was to evaluate the antileishmanial effects of topically applied lipid-based formulations containing amphotericin B (AmB) in CBA mice as a model for human cutaneous leishmaniasis. Such treatment, if efficacious, is expected to be superior to systemic treatments since, by acting in a localized manner, it will require lower, and therefore less toxic, drug dosages. Three preparations of AmB complexed to polar lipids were tested: Fungizone (mixed micelles composed of AmB and deoxycholate), Amphocil (AmB and cholesteryl sulfate complex), and ABPLC (AmB and phospholipid complex). All these formulations killed parasites in vitro with similar efficacies but were ineffective when they were applied topically. However, Amphocil and ABPLC, but not Fungizone, when dispersed in an aqueous solution containing 5 to 25% ethanol, induced a statistically significant improvement in lesion size from week 2 or 3 onward (a total of 15 mg of AmB per kg of body weight was applied over 3 weeks). AmB biodistribution measurements following topical application of Amphocil, determined by high-pressure liquid chromatography, showed that AmB was detectable in the skin but not in the internal organs. Application of at least 10 times more drug was necessary to obtain detectable levels of AmB in the internal organs. After application of therapeutic doses of ABPLC, very low levels of AmB were detected in the internal organs. These experiments show for the first time that AmB administered topically as a complex either with cholesteryl sulfate or with phospholipids and in the presence of ethanol can penetrate the skin and kill sensitive organisms in a localized manner by using very low total drug concentrations.

Vitamin A inhibits cytokines produced by type 1 lymphocytes in vitro.

The effect of vitamin A (retinol) on cell-mediated immune responses was studied. As an experimental model, Leishmania major infection in mice was used. In this model, resistant mouse strains develop a type 1 response, while susceptible strains develop a type 2 response. Using lymph node cells and T-cell lines developed from infected susceptible and resistant mice, it was found that vitamin A inhibited lymphocyte proliferation in a dose-dependent manner. By separately incubating antigen-presenting cells and T cells with vitamin A, it was shown that the inhibitory effect was on the T cells. Type 1 cytokine (IFN-gamma, GM-CSF, IL-2) secretion in vitro in response to stimulation with specific antigen was also inhibited in a dose-dependent manner, whereas secretion of type 2 cytokines (IL-4 and IL-10) was not affected by vitamin A. The inhibitory effect was also observed in PMA-stimulated (but not Con A-stimulated) lymphocytes and was noticeable even if the vitamin was added as late as 24 h after initiation of the incubation period. Since PMA does not operate via a receptor-coupled signaling pathway but rather directly affects the protein kinase C (PKC) pathway, we have measured the effect of vitamin A on PKC in situ activation. Incubation of lymphocytes and antigen in the presence of vitamin A caused inhibition of PKC isoenzymes translocation to the particulate cell fraction, as measured by immunoblotting. The results presented indicate that, when added to cell cultures in vitro, vitamin A inhibits only secretion of type 1 but not type 2 cytokines, possibly through an inhibitory effect on protein kinase C activity.

Effective immunization of mice against cutaneous leishmaniasis using an intrinsically adjuvanted synthetic lipopeptide vaccine.

Two peptides representing predicted T-cell epitopes of gp63, a major surface glycoprotein of the parasite Leishmania major, were used in vaccines tested in a murine model of cutaneous leishmaniasis. Either subcutaneous or intraperitoneal immunization in saline with a peptide representing gp63 amino acids 467-482 (p467) significantly protected CBA mice against the development of severe cutaneous lesions only when the peptide was intrinsically adjuvanted by covalently adding a lauryl-cysteine moiety (LC-p467) to its amino terminus during synthesis. In marked contrast, administration of p467 alone, cysteinyl-p467 or gp63 protein in saline resulted in some disease exacerbation. Splenic cells of LC-p467 immunized mice stimulated in vitro with LC-p467 displayed strong proliferative responses and secretion of IL-2, IFN-tau and GM-CSF (but not IL-4 and IL-10) suggesting that immunization with the lipopeptide induced the TH1 type cytokine responses associated with cell-mediated immunity. The safety, efficacy, ease of production and standardization of such lipopeptide vaccines suggest that they have significant potential for the development of vaccines for humans against leishmaniasis or other parasitic or viral diseases that require cell-mediated immunity for protection.

Imbalance of immune responses in patients with chronic and widespread fungal skin infection.

Fungal infections of the skin are caused by dermatophyte fungi. Infections can be acute and inflammatory or chronic and non-inflammatory; it is believed that cell-mediated immunity is the cornerstone of host defence and is instrumental in the eradication of the infection. We describe here parameters of the immune response of a group of patients who although not immunocompromised, suffered from widespread and chronic infections. All patients lacked a specific delayed-type hypersensitivity (DTH) response; however, their in vitro lymphocyte proliferation in response to Trichophyton rubrum extract and to other fungal antigens was normal. The patients were not atopic by clinical history, and yet had high levels of non-specific IgE and of T. rubrum-specific IgG4. Taken together, the results of this study suggest that the group of patients studied suffered from an immune imbalance which has characteristics of a Th2-type response.

Antigen presentation by epidermal Langerhans cells in experimental cutaneous leishmaniasis.

Cutaneous leishmaniasis is a disease induced by intradermal injection of leishmania promastigotes. Since the first cells the parasite encounters are those of the skin, the involvement of this organ in the early immune response might be relevant to the outcome of the disease. In this study we examined the ability of epidermal langerhans cells (LC) to become infected in vivo and to function as antigen presenting cells during the early hours of infection with Leishmania major. Our experiments showed that LC from mice injected with parasites can present antigen to a leishmania-specific T cell line when LC are obtained as early as four h after infection. The stimulation was specific, since LC from leishmania injected mice did not present antigen to an ovalbumin-specific T cell line nor did LC from ovalbumin-injected mice present antigen to the leishmania specific T cell line. Despite the ability of epidermal LC cells to present antigen, no parasites were detected in the epidermis, suggesting that these cells are not directly involved in establishing an infection.

Changing patterns of cutaneous leishmaniasis in Israel and neighbouring territories.

The most frequent form of cutaneous leishmaniasis (CL) in Israel and the neighbouring territories is due to Leishmania major, which is endemic mainly in the Jordan Valley and in the Rift Valley. CL due to L. tropica is much less common, and in the past only sporadic cases have been reported. In this study we present data obtained during the years 1988-1992 regarding CL in the area. Our clinic has diagnosed a total of 371 leishmaniasis cases, most of whom acquired the infection in the Jordan Valley, mainly during June and July. About one-third of the patients had single lesions, and one-third more than 5 lesions. We also describe an outbreak of leishmaniasis in Kfar Adumim, a village 15 km east of Jerusalem, where leishmaniasis was previously unknown. Parasites were characterized by the polymerase chain reaction and by immunostaining, and found to be both L. tropica and L. major. The localization of the homes of the affected people on a slope where hyraxes were abundant suggests that these animals might have been involved in the transmission of L. tropica in this area.

Serial study of the immune response of an individual with exacerbated simple cutaneous leishmaniasis.

The immunological responses of a patient with exacerbated cutaneous leishmaniasis, measured during the course of the disease, are described. Except for skin lesions the patient was healthy and showed no signs of immunosuppression. Three immunological parameters were measured: specific lymphocyte proliferation (LPA), monocyte effector activity (MEA), and antibody levels. LPA was positive early in the course of the disease, became negative as the lesions enlarged, and was positive again as the lesions healed 28 weeks after initiation of the study. In the MEA test, in which the mononuclear cells of the patient were incubated in the presence of Leishmania major promastigotes in a 3-day assay, the number of amastigotes per 100 monocytes remained constant until week 28 and then decreased significantly. Antibody levels remained elevated until week 28 and then decreased to background levels. The results indicate that the cell-mediated immune response parallels the course of the disease while circulating antibodies show an inverse relationship.

In vitro secretion of cytokines by human mononuclear cells of individuals during and after cutaneous leishmaniasis infection.

Human peripheral blood mononuclear cells were incubated in the presence of Leishmania major promastigotes. The culture supernatants were collected after 24 and 72 h and the cytokine content was measured. It was found that mononuclear cells from cured individuals, in the presence of L. major promastigotes, had increased leishmanicidal activity and secreted increased levels of interferon gamma (IFN gamma), interleukin-2 (IL-2) and tumour necrosis factor alpha (TNF). The levels of IL-6, on the other hand, did not differ from the levels secreted by cells from unexposed individuals. The secretion of cytokines by cells from patients in the course of the disease was sometimes elevated, but the pattern they presented was more heterogeneous than that of cured individuals, and showed less correlation with leishmanicidal effector activity. Addition of IFN and IL-2, but not TNF, to cultures from unexposed individuals caused increased leishmanicidal activity, suggesting that these factors play a role in controlling the parasite infection; the role of TNF is not yet clear, and is probably concentration-dependent.

Gorging response of culicine mosquitoes (Diptera: Culicidae) to blood fractions.

Although anopheline mosquitoes will ingest plasma without blood cells, Culex spp. and Aedes spp. require the phagostimulatory effect of blood cells; this effect can be duplicated by the addition of adenine-nucleotides to plasma. Because activation of platelets released ADP and ATP into the plasma, they were suspected as the major source of the phagostimulant. This paper describes quantitatively the role of platelets in ingestion by Aedes aegypti (L.) and Culex univittatus Theobald. We found that about 10(6)/mm3 inactivated platelets are required to induce engorgement by 80-90% of the mosquitoes of both species. Thrombin activation of the platelets reduced the effective dose to < 2 x 10(4)/mm3. Other blood fractions also were tested as possible sources of stimulation. A series of washed red blood cells (RBC) dilutions was tested; 5 x 10(5) RBC/mm3 were required to induce 90% engorgement. Several types of leukocytes derived from blood by standard methods also induced engorgement at their physiological concentrations. Macrophages and cultured lymphocytes that do not contain any platelets induced gorging in Cx. univittatus, but not in Ae. aegypti. Because RBC and leukocytes do not release nucleotides unless broken, we suggest that their phagostimulatory effect is due to platelet contamination, which invariably occurs during standard methods of blood fractionation.

Development of in vitro parameters of cell-mediated immunity in the course of human cutaneous leishmaniasis infection.

The goal of the present study was to determine whether a correlation between the clinical stage of cutaneous leishmaniasis lesions and in vitro parameters of cell-mediated immunity could be established. For this purpose, we measured lymphocyte proliferation, using a total lymphocyte proliferation (TLP) blood assay, and leishmanicidal effector activity using peripheral blood mononuclear cells (PBMC) in a three-day assay. The parameters of leishmanicidal activity measured included percent infected monocytes and number of amastigotes per 100 infected monocytes 24 and 72 hr after infection. Three groups of people were studied: a group of patients in the course of the disease, a group of immune individuals, and unexposed controls. The results of the study suggested that the ability of PBMC to kill parasites increased in patients as the lesions cured, and was highest in immune individuals. In contrast, the TLP response once positive, did not increase after cure. In approximately 30% of the patients who were retested on several occasions during the course of the disease, a positive response reversed to negative both in the TLP and the effector assays while the lesions were still active. In approximately 50% of these cases, the response eventually became positive again. The data presented show that effector activity and proliferation correlate with immunity, and suggest that marked heterogeneity characterizes the immune response in the course of active disease.