Study on the safety and efficacy of miltefosine for the treatment of children and adolescents with post-kala-azar dermal leishmaniasis in Bangladesh, and an association of serum vitamin E and exposure to arsenic with post-kala-azar dermal leishmaniasis: an open clinical trial and case-control study protocol.

INTRODUCTION: Post-kala-azar dermal leishmaniasis (PKDL) is a dermatological complication that occurs primarily among treated visceral leishmaniasis (VL) patients, and sporadically in a few without a history of VL. It mostly affects children and adolescents but is also common in adults. The conventional treatment with 120 intramuscular injections of sodium stibogluconate (SSG) is phasing out. Miltefosine (MF) is the only eventual alternative to SSG; however, its efficacy and safety profiles for treatment of children and adolescents with PKDL are lacking. In addition, risk factors for PKDL are poorly investigated. Host genetic, nutritional and environmental factors could be potential risk factors. As such, here we propose to evaluate the efficacy and safety of MF for 12 weeks at an allometric dose for children and adolescents with PKDL, and also to explore potential risk factors for PKDL. METHODS AND ANALYSIS: A cross-sectional survey will look for suspected participants with PKDL among treated VL children and adolescents, a subsequent open clinical trial with MF at allometric dose, with a follow-up at 12 months. A case-control study will be carried out for PKDL risk factors. Assuming 95% cure rate, 95% CI and α=0.05, a sample size of 73 children with PKDL is needed. Considering an attrition rate of 10%, the final sample size is 80 children in each group. Descriptive and analytical analyses will be performed. Primary outcome is safety and cure rate of 12 weeks of treatment with MF. ETHICS AND DISSEMINATION: International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) Ethical Review Committee (ERC) approved the protocol (PR#013045). Written informed consent will be taken from all participants and their guardians (in case of minor). A Data and Safety Monitoring Board (DSMB) of ICDDR,B ERC will monitor all study activities to ensure the safety of the participants. TRIAL REGISTRATION NUMBER: NCT02193022; Pre-results.

Randomized, double-blind clinical trial of topical imiquimod 5% with parenteral meglumine antimoniate in the treatment of cutaneous leishmaniasis in Peru.

BACKGROUND: Current treatments for cutaneous leishmaniasis are limited by their toxicity, high cost, and discomfort and the emergence of drug resistance. New approaches, including combination therapies, are urgently needed. We performed a double-blind, randomized trial of therapy with parenteral antimony plus topical imiquimod, an innate immune-response modulator, versus therapy with antimony alone, in subjects with cutaneous leishmaniasis for whom an initial course of antimony therapy had failed. METHODS: Forty subjects with clinical resistance to antimony were recruited in Lima, Peru, between February 2001 and December 2002. All subjects received meglumine antimoniate (20 mg/kg/day im or iv) and were randomized to receive either topical imiquimod 5% cream (Aldara; 3M Pharmaceuticals) or vehicle control every other day for 20 days. Lesions and adverse events were evaluated during treatment and at 1, 2, 3, 6, and 12 months after the treatment period. RESULTS: The mean number of lesions was 1.2 per person; 71% of the lesions were facial and 76% were ulcerative. There were no major differences between the groups, and all but 2 subjects completed therapy. Mild adverse events were reported by 73% of the subjects, but only erythema occurred more commonly in the imiquimod group (P < or = .02). Lesions resolved more rapidly in the imiquimod group: 50% of the imiquimod group achieved cure at 1 month after the treatment period versus 15% of the vehicle cream group (P < or = .02); 61% of the imiquimod group at 2 months versus 25% of the vehicle cream group (P < or = .03); and 72% of the imiquimod group at 3 months versus 35% of the vehicle cream group (P < or = .02). Residual scarring in the imiquimod group was less prominent than in the vehicle cream group. CONCLUSIONS: Combined antimony plus imiquimod treatment was well tolerated, accelerated healing of lesions, and improved scar quality. This therapy may have particular advantages for subjects with facial lesions.

Successful treatment of drug-resistant cutaneous leishmaniasis in humans by use of imiquimod, an immunomodulator.

Treatment failures for leishmaniasis with pentavalent antimonials, including meglumine antimonate, are increasingly common in many endemic areas. Imiquimod (Aldara; 3M Pharmaceuticals) is a novel immune response-activating compound, approved by the United States Food and Drug Administration, that is currently used to treat cervical warts and has been shown to activate macrophage killing of Leishmania species. Therefore, an open-label, prospective study was conducted of combined imiquimod plus meglumine antimonate therapy in 12 patients with cutaneous leishmaniasis who had previously not responded to meglumine antimonate therapy. All of the patients responded well to this combination therapy, and 90% were found to be cured at the 6-month follow-up period.

Clearance of infection with Mycobacterium bovis BCG in mice is enhanced by treatment with S28463 (R-848), and its efficiency depends on expression of wild-type Nramp1 (resistance allele).

The mouse bcg host resistance gene is known to control the activation of host macrophages for killing of intracellular parasites like Leishmania donovani as well as intracellular bacteria, including Mycobacterium bovis BCG and Salmonella enterica serovar Typhimurium. The Nramp1 gene has been mapped to this locus and affects the efficiency of macrophage activation. It has been shown that imidazoquinoline compounds, including S28463, are able to improve the clearance of a number of intracellular pathogens such as herpes simplex virus 2, human papillomavirus, and Leishmania. The goal of this study was to determine whether S28463 is efficient against infection with another intracellular pathogen, M. bovis BCG, and to determine the molecular basis underlying this effect. To achieve this, B10A.Nramp1(r) and B10A.Nramp1(-/-) mice were infected with M. bovis BCG and treated with S28463. The bacterial content in the spleen from these mice was assayed by a colony-forming assay. In addition, in vitro experiments were performed using bone marrow-derived macrophage cell lines from these mice. These cells were treated with S28463 and/or gamma interferon (IFN-gamma), and nitric oxide (NO) production was measured. Our study was able to show that S28463 acts in synergy with IFN-gamma to increase the production of NO in vitro. We were also able to demonstrate that mice that carried the resistant allele of the Nramp1 gene and were infected with M. bovis BCG responded to treatment with S28463, resulting in a decreased bacterial load after 2 weeks of treatment. Mice that do not express the Nramp1 gene responded only to a very large dose of S28463, and the response was not as efficient as that observed in mice carrying a wild-type Nramp1 allele. Our data provide evidence for the potential of S28463 as an immunomodulator that may be helpful in designing efficient strategies to improve host defense against mycobacterial infection.

Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response which protects mice against Leishmania donovani infections.

The A2 genes of Leishmania donovani encode amastigote-specific A2 proteins, which are considered to be virulence factors required for the survival of this protozoan parasite in the mammalian host. The A2 genes are present within a multigene family and corresponding A2 proteins are composed predominantly of multiple copies of a 10 amino acid repeat sequences. A2-specific antibodies have been detected in the sera of patients suffering from visceral leishmaniasis (VL) and it has been shown that generation of A2 deficient L. donovani resulted in an avirulent phenotype. In this report, we show that immunization of mice with recombinant A2 protein conferred significant protection against challenge infection with L. donovani. The protection correlated with in vitro splenocyte proliferation, production of IFN-gamma in response to A2 protein and the presence of A2-specific antibodies in the sera of immunized mice. These data demonstrate that A2 represents a potential antigen for protection against infection with L. donovani and VL.

Protection against Leishmania donovani infection by DNA vaccination: increased DNA vaccination efficiency through inhibiting the cellular p53 response.

DNA-vaccination holds great promise for the future of vaccine development against infectious diseases, especially in developing countries. We therefore investigated the possibility of using DNA-vaccination against Leishmania donovani infection with the A2 virulence gene and whether inhibiting the cellular p53 response could increase the effectiveness of the A2 DNA vaccine. p53, also known as the guardian of the genome, is activated following DNA transfection and has pleotropic effects on cells, which could have adverse effects on the effectiveness of DNA-vaccination. Two major observations are reported within. First, vaccination with the A2 gene induced both humoral and cellular immune responses against A2 which provided significant protection against infection with L. donovani. Second, inhibition of p53 with human papillomavirus E6 resulted in higher expression of heterologous transfected genes in vitro and more efficient DNA-vaccination in vivo. These results have important implications for DNA vaccination against leishmaniasis and potentially against other infectious diseases.

Control of interferon signaling in human papillomavirus infection.

Human papillomaviruses (HPV) infect mucosal and cutaneous epithelium resulting in several types of pathologies, most notably, cervical cancer. Persistent infection with sexually transmitted oncogenic HPV types represents the major risk factor for the development of cervical cancer. The development of HPV-associated cervical cancer has been closely linked to the expression of the viral oncogenes E6 and E7 in the tumor cells. The major viral oncoproteins, E6 and E7, target the cellular tumor suppressor gene products p53 and Rb, respectively. As detailed within, these interactions result in the stimulation of proliferation and the inhibition of apoptosis, thus representing major oncogenic insults to the infected cell. In addition to mediating transformation, the E6 and E7 genes also play significant roles in altering the immune response against infected cells by suppressing interferon (IFN) expression and signaling. At the clinical level, IFNs have been used in the treatment of HPV-associated cervical intraepithelial neoplasia (CIN) or cervical cancers with mixed results. The success of the treatment is largely dependent on the subtype of HPV and the immune response of the patients. Despite this inefficiency, the increasing knowledge about the regulation of IFN signaling pathways at molecular level may hold a promise for the use of new therapeutic strategies against HPV infection. Studies on the regulation of the function of IFN-inducible gene products by the E6 and E7 may lead to the development of new therapeutic approaches based on strategies that modify the function of the HPV oncoproteins and restore IFN-signaling pathways through endogenous control mechanisms.

Characterization of the A2-A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection.

The A2 gene family is present in Leishmania donovani, which causes fatal visceral leishmaniasis in human patients, but is not present in Leishmania major, which causes cutaneous leishmaniasis infections. The A2 genes in L. donovani are stage specific and are expressed at high levels in the amastigote stage in the mammalian host, but are not expressed in the promastigote stage in the insect sandfly vector. The A2 genes are tandem repeated with a distinct gene family termed the A2rel genes. In order to characterize the structure and function of the A2-A2rel gene clusters, the 5' and 3' DNA sequences flanking the A2-A2rel cluster were isolated, sequenced and used to generate mutants through gene targeting. Although it was possible to generate partial A2-A2rel gene clusters knock-out mutants, it was not possible to delete all the A2-A2rel gene clusters completely from the L. donovani genome, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A2 and A2rel gene expression was compensated by amplifying the remaining intact A2 and A2rel genes, and the proliferation of these mutants in culture and their virulence in BALB/c mice were compromised. In order to explore further the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2-expressing L. major parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralization of infection associated with L. donovani.

Identification of genes induced by a macrophage activator, S-28463, using gene expression array analysis.

S-28463 and imiquimod are imidazoquinoline compounds which stimulate microbicidal activity by inducing a local immune response at the site of application. Imiquimod-containing cream is an effective clinical treatment against cervical warts caused by human papillomavirus infection. Imiquimod also induces leishmanicidal activity both in vitro in macrophages and in vivo in a mouse model for cutaneous leishmaniasis. The major target cells of S-28463 and imiquimod are macrophages. To explore the molecular basis in which imidazoquinolines generate macrophage microbicidal activity, a cDNA gene array analysis was undertaken to identify genes induced by S-28463. Out of 588 genes screened in this assay, only 13 genes were significantly induced by S-28463. Remarkably, virtually all of the induced genes are involved in macrophage activation and inflammatory response. This experimental approach defines the mechanism of action of this clinically relevant compound in the induction of microbicidal activity in macrophages and also potentially identifies novel genes associated with microbicidal activity in this cell type.

General suppression of macrophage gene expression during Leishmania donovani infection.

Within the mammalian host, Leishmania donovani is an obligatory intracellular protozoan that resides and multiplies exclusively in the phagolysosomes of macrophages. The outcome of this infection is governed by the interaction between Leishmania and macrophage molecules that ultimately effect the expression of genes within both cells. To explore the effect of this intracellular infection on macrophage gene expression, a cDNA expression array analysis was performed to compare gene expression profiles in noninfected and L. donovani-infected macrophages. In this manner, it was possible to examine the effect of infection on the expression of several hundred well-characterized host cell genes in an unbiased manner. Interestingly, approximately 40% of the genes whose expression was detected in macrophages were down-regulated during infection with L. donovani. However, several genes were also induced during the infection process, some of which could play a role in recruitment of additional macrophages to the site of infection. Taken together, the general suppression of gene expression in addition to the selective induction of key genes is likely to play an important role in allowing the parasite to survive and proliferate within its host macrophage cell.

Development of a p53 responsive GFP reporter; identification of live cells with p53 activity.

p53 is among the most intensely studied human proteins because of its vital role as the prototype tumor suppressor. As a result, there are widespread applications for p53 functional analysis in biotechnology as it relates to cancer research. p53 is a potent sequence specific transcription factor, which induces the expression of a number of genes whose products mediate cell cycle arrest and apoptosis. Because the tumor suppressor activity of p53 is dependent on its transcription regulatory function, we have undertaken to develop a p53-responsive green fluorescent protein reporter strategy to enable the identification of live cells containing p53 transcriptional transactivation activity. We demonstrate within the use of GFP fluorescence to monitor both endogenous and plasmid derived p53 biochemical and biological activity. Identifying live cells with p53 activity through GFP fluorescence will have wide application for both in vitro and in vivo studies of the p53 tumor suppressor protein.

Leishmania infection and virulence.

Leishmaniasis is among the most important infectious diseases of the developing world. With the advent of effective culture conditions for the various stages of the life cycle, together with state of the art biochemical, genomics, and reverse genetic approaches, it has been possible to identify virulence factors required for the infection process. Several of these virulence factors are discussed within including lipophosphoglygan, A2, cysteine proteinases, and gp63.

P53 polymorphism in codon 72 and risk of human papillomavirus-induced cervical cancer: effect of inter-laboratory variation.

An association between codon-72 p53 polymorphism and risk of human papillomavirus (HPV)-induced cervical cancer has been found recently, but it has been difficult to replicate. In this study, we assess the impact of inter-laboratory variation in p53 genotyping on the validity of the proposed association. DNA specimens were randomly selected from 54 invasive, squamous cell carcinoma cases, 52 HPV-negative, and 39 HPV-positive controls from a previous case-control study in Brazil. Codon-72 polymorphism was blindly analyzed in three different laboratories. We calculated age- and race-adjusted odds ratios (OR) and 95% confidence intervals (CI) using logistic regression for gauging the association between p53 polymorphism and cervical cancer risk. The proportions of the Arg/Arg, Arg/Pro, and Pro/Pro genotypes varied substantially among laboratories with Kappa coefficients in the 0.49-0.63 range. When disagreement between labs was allowed, the OR for the Arg/Arg genotype, compared to other forms, was as low as 1.5 (95% CI: 0.5-3. 9). In contrast, the OR increased to 8.0 (95% CI: 2.3-28.5) after exclusion of discordant genotypes. Restricting the comparison to HPV-positive controls increased the magnitude of the relation appreciably. After exclusion of all discordant diagnoses, the OR was 21.5 (95% CI: 3.4-137.8), whereas with disagreed genotypes the association was not significant (OR = 2.9, 95% CI: 0.7-11.9). Homozygous codon-72 p53-Arg apparently confers a higher susceptibility to HPV-associated cervical tumorigenesis. However, exposure misclassification consequent to inter-laboratory variation in protocols may affect the ability to detect the association.

Design and methods of the Ludwig-McGill longitudinal study of the natural history of human papillomavirus infection and cervical neoplasia in Brazil. Ludwig-McGill Study Group.

This article reports on a large longitudinal study, begun in 1993, of the natural history of human papillomavirus (HPV) infection and cervical neoplasia in a population of low-income women in São Paulo, Brazil, a city with one of the highest risks worldwide for cervical cancer. Known as the Ludwig-McGill cohort study, the epidemiological investigation focuses on persistent infection with oncogenic HPV types as the precursor event leading to cervical neoplasia. The objectives of this study are to: 1) study the epidemiology of persistent cervical HPV infection in asymptomatic women, 2) investigate whether persistent HPV infection increases risk of low-grade and high-grade cervical lesions, 3) search for determinants of persistent HPV infection, 4) search for molecular variants of HPV that may be associated with an increased risk of lesions, 5) investigate whether viral burden is correlated with persistent infections and with lesion risk, 6) study the antibody response to HPV as a predictor of persistence and lesion progression, and 7) examine the role of HLA typing and codon 72 p53 gene polymorphism in mediating HPV persistence and lesion severity. The study accrued 2,528 female subjects through March 1997. Subjects were followed up every 4 months in the first year, with twice-yearly return visits to take place in subsequent years. Participants undergo a questionnaire-based interview, have a cervical specimen taken for Pap cytology and HPV testing, and have a blood sample drawn for HPV antibody testing. A cervicography is performed once in the first year and every two years thereafter. In this article we describe the design and methods of the study, provide baseline cohort characteristics, and present a preliminary assessment of the prognostic value of baseline HPV status.

The human papilloma virus (HPV)-18 E6 oncoprotein physically associates with Tyk2 and impairs Jak-STAT activation by interferon-alpha.

We have examined the effects of human papilloma virus (HPV) E6 proteins on interferon (IFN) signaling. Here we show that expression of the 'malignant' HPV-18 E6 in human HT1080 cells results in inhibition of Jak-STAT activation in response to IFN-alpha but not IFN-gamma. This inhibitory effect is not shared by the 'benign' HPV-11 E6. The DNA-binding and transactivation capacities of the transcription factor ISGF3 are diminished in cells expressing HPV-18 E6 after IFN-alpha treatment as a result of decreased tyrosine phosphorylation of Tyk2, STAT2 and STAT1. However, HPV-18 E6 does not affect the induction of tyrosine phosphorylation and DNA-binding of STAT1 by IFN-gamma. In addition, HPV E6 proteins physically interact with Tyk2. This interaction takes place preferably with HPV-18 E6 and to a lesser extent with HPV-11 E6. The E6/Tyk2 interaction requires the JH6-JH7 domains of Tyk2, which are important for Tyk2 binding to the cytoplasmic portion of IFN-alpha receptor 1 (IFNAR1). These findings demonstrate an inhibitory role of HPV-18 E6 in the IFN-alpha-induced Jak-STAT pathway, which may be explained, at least in part, by the ability of E6 to interact with and impair Tyk2 activation.

Treatment of experimental leishmaniasis with the immunomodulators imiquimod and S-28463: efficacy and mode of action.

There is a need for new, effective, and less toxic treatments for leishmaniasis, an infectious disease caused by Leishmania protozoa and is a major cause of suffering and morbidity in much of the developing world. Imiquimod, an immune-response modifier, has recently been approved by the Food and Drug Administration for the treatment of genital warts caused by human papillomaviruses. Imiquimod initiates a local immune reaction, including the stimulation of macrophages, resulting in resolution of human papillomavirus infection and regression of the viral lesion. Since imiquimod activates a number of immune cells, including macrophages, which are the only host cells of Leishmania species, an investigation was done to determine whether it induces leishmanicidal properties in infected macrophages in vitro and in vivo in a mouse model. Imiquimod and a related compound, S-28463, effectively stimulated leishmanicidal activity in macrophages; moreover, imiquimod stimulated signal transduction associated with inducing nitric oxide synthesis in macrophages.

Two polymorphic variants of wild-type p53 differ biochemically and biologically.

The wild-type p53 protein exhibits a common polymorphism at amino acid 72, resulting in either a proline residue (p53Pro) or an arginine residue (p53Arg) at this position. Despite the difference that this change makes in the primary structure of the protein resulting in a difference in migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no differences in the biochemical or biological characteristics of these wild-type p53 variants have been reported. We have recently shown that p53Arg is significantly more susceptible than p53Pro to the degradation induced by human papillomavirus (HPV) E6 protein. Moreover, this may result in an increased susceptibility to HPV-induced tumors in homozygous p53Arg individuals. In further investigating the characteristics of these p53 variants, we now show that both forms are morphologically wild type and do not differ in their ability to bind to DNA in a sequence-specific manner. However, there are a number of differences between the p53 variants in their abilities to bind components of the transcriptional machinery, to activate transcription, to induce apoptosis, and to repress the transformation of primary cells. These observations may have implications for the development of cancers which harbor wild-type p53 sequences and possibly for the ability of such tumors to respond to therapy, depending on their p53 genotype.

Inducible expression of suicide genes in Leishmania donovani amastigotes.

This study tests the feasibility of using the A2 gene regulatory system to create a Leishmania cell line in which attenuation is developmentally regulated when the parasite differentiates from promastigotes to amastigotes. The Leishmania donovani- inducible A2 gene regulatory system was used to differentially express in amastigotes two potential suicide genes: a truncated version of the L. donovani 3'-nucleotidase/nuclease expressed in the cytoplasm and the herpes simplex virus thymidine kinase gene. These genes were inserted between A2 noncoding regulatory sequences for up-regulation of expression in amastigotes. The accumulation of toxic products affected L. donovani cell replication and viability both in vitro and in vivo. The inducible expression of toxic gene products represents a valuable tool for the development of safe and effective vaccines.

A2rel: a constitutively expressed Leishmania gene linked to an amastigote-stage-specific gene.

The A2-A2rel gene copies are arranged in tandem arrays on a 850 kb chromosome in Leishmania donovani. Contrary to A2 mRNA which displays amastigote-stage-specific expression, A2rel gene expression is constitutive throughout the L. donovani life cycle. The A2rel sequence was found to be conserved in all Leishmania species tested, while the A2 sequence is specific to L. donovani and L. mexicana. The A2rel full length cDNA is of 2.3 kb and it contains one open reading frame coding for a putative protein of 436 amino acids.