Vitamins (A&D) and Isoprenoid (Chenodeoxycholic acid) molecules are accompanied by Th1 immunostimulatory response and therapeutic cure in vivo: possible antileishmanial drugs.

Investigation of immune modulatory anti-leishmanial molecules is now being strongly encouraged to overcome the immunosuppression manifested during visceral leishmaniasis (VL), resistance, toxicity and high cost associated with conventional therapeutics. In the present study, we explored the protective efficacy of vitamin D3, retinoic acid and isoprenoid chenodeoxycholic acid (CDCA) combinations against L. donovani infected BALB/c mice. We also probed the immune modulatory response (Th1 & Th2 cytokines) and infection dynamics following experimental infections with drug treated animals. Our results indicate that Vit.D3/RA and CDCA/RA combination treatment led to significant inhibition of parasite load on days 21 and 28 post treatment. Furthermore, there was a marked inhibition of Th2 type immune responses in IL-4, IL-5 and polarization of Th1 biased immunity along with upregulation of IL-1, IFN-γ, and TNF-α levels on day 28 post treatment. In addition, mice treated with Vit.D3/RA and CDCA/RA demonstrates here that splenic histological recovery against the virulent challenge of L. donovani by day 28 was comparable to control group. The conclusions derived from this study suggests that a combination of vitamin A, D3 and isoprenoids may have a potential immunomodulatory therapeutic role against leishmaniasis.

Downregulation of host tryptophan-aspartate containing coat (TACO) gene restricts the entry and survival of Leishmania donovani in human macrophage model.

Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a crucial role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO) gene has been recognized as playing a central role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the entry/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3 (Vit.D3)/Retinoic acid (RA) and chenodeoxycholic acid (CDCA)/RA combinations in human THP-1 macrophages (in vitro). Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L. donovani replicative niche inside the host. Our study is the first to highlight the important role of the TACO gene in Leishmania entry, survival and to identify TACO gene downregulation as potential drug target against leishmaniasis.

Oral administration of encapsulated bovine lactoferrin protein nanocapsules against intracellular parasite Toxoplasma gondii.

Toxoplasma gondii is a deadly intracellular parasite known to reside in every nucleated cell and known to cause severe complications in immunocompromised host. Standard drugs are cost effective and cause side effects, therefore, there is a necessity for a new drug molecule with immunomodulatory potential. Lactoferrin (Lf) is a natural milk protein, which has shown antimicrobial properties in its nanoformulation using alginate chitosan calcium phosphate bovine lactoferrin nanocapsules (AEC-CCo-CP-bLf-NCs). The present study was aimed to analyze and compare the effect of bovine Lf (bLf) in its native as well as nanoformulation (AEC-CCo-CP-bLf-NC) against coccidian parasite T. gondii. In vitro analysis has shown a significant increase in nitric oxide production and low parasitemia in in vitro cell culture model. In vivo BALB/c mice model have been used to develop human toxoplasmosis model. After treatment with NCs it has substantially increased the bioavailability of the protein and showed comparatively increased levels of reactive oxygen species, nitric oxide production, and Th1 cytokine which helped in parasite clearance. The mechanism of action of NCs has been clarified by immunoreactivity analysis, which showed accumulation of Lf in macrophages of various visceral organs, which is the site of parasite multiplication. Effect of NCs has significantly decreased (P<0.05) the parasite load in various organs and helped survival of mice till day 25 postinfection. Fe metabolism inside the mice has been found to be maintained even after administration of mono form of Lf, this indicates novelty of Lf protein. From the present study we concluded that nanoformulation did not reduce the therapeutic potential of Lf protein; however, nanoformulation has enhanced the stability of the protein and shown anti-toxoplasmal activity. Our study presents for the first time nanoformulation of Lf protein against Toxoplasma, which has advantages over the standard drug therapy without any side effects.

Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction.

BACKGROUND: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs) and macrophages (human monocytic cell line-derived macrophages THP1 cells). METHODS: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections. RESULTS: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene expression and cellular activity depending on the degree of iron saturation of lactoferrin. A significant increase (P<0.05) in production of reactive oxygen species, phagocytic activity, and Toll-like receptor expression was observed in host cells incubated with iron-saturated lactoferrin when compared with an untreated control group. However, there was no significant (P>0.05) change in percentage viability in the different groups of host cells treated, and no downregulation of survivin gene expression was found at 48 hours post-incubation. Upregulation of the Toll-like receptor and downregulation of the P-gp gene confirmed the immunomodulatory potential of lactoferrin protein. CONCLUSION: The present study details the interaction between lactoferrin and parasite host cells, ie, RBCs and macrophages, using various cellular processes and expression studies. The study reveals the possible mechanism of action against various intracellular pathogens such as Toxoplasma, Plasmodium, Leishmania, Trypanosoma, and Mycobacterium. The presence of iron in lactoferrin plays an important role in enhancing the various activities taking place inside these cells. This work provides a lot of information about targeting lactoferrin against many parasitic infections which can rule out the exact pathways for inhibition of diseases caused by intracellular microbes mainly targeting RBCs and macrophages for their survival. Therefore, this initial study can serve as a baseline for further evaluation of the mechanism of action of lactoferrin against parasitic diseases, which is not fully understood to date.

Correlation of molecular markers, Pfmdr1-N86Y and Pfcrt-K76T, with in vitro chloroquine resistant Plasmodium falciparum, isolated in the malaria endemic states of Assam and Arunachal Pradesh, Northeast India.

The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum is not clearly understood. However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1. These genes encode for digestive vacuole transmembrane proteins Pfcrt and Pgh1, respectively. The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood). All the CQ resistant isolates showed the presence of Pfmdr1 and Pfcrt mutations. CQ sensitive isolates were negative for these mutations. Strong linkage disequilibrium was observed between the alleles at these two loci (Pfmdr1-N86Y and Pfcrt-K76T). The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum. The result necessitates the evaluation of CQ in vivo therapeutic efficacy in endemic areas for more effective malaria control strategies.

Anti-Toxoplasma gondii antibody detection in serum and urine samples by enzyme-linked immunosorbent assay in HIV-infected patients.

BACKGROUND: Toxoplasmosis is a common parasitic infection of man, and reactivation of latent disease in HIV-infected patients can cause fatal encephalitis. Diagnosis depends on demonstration of parasite-specific antibodies in serum. In HIV-infected patients, IgM is often undetectable, whereas IgG remains detectable in the majority. Urine sample is very easily available and has not been evaluated for immunodiagnosis of toxoplasmosis. AIM: The study was an effort to find whether urine sample can be used in place of serum for immunodiagnosis of toxoplasmosis. MATERIALS AND METHODS: Enzyme-linked immunosorbent assay (ELISA) was carried out in serum and urine samples collected from 100 HIV-infected patients to detect anti-toxoplasma IgG and IgM antibodies and whether positivity correlated with the CD4 T-cell counts of patients. RESULTS: In this study, we observed that there was no significant difference in positivity of anti-toxoplasma IgM and IgG between serum and urine samples of HIV-infected patients by ELISA. There was a negative correlation between CD4 count and seropositivity. CONCLUSION: Urine sample can be satisfactorily used in place of serum for immunodiagnosis of toxoplasmosis.

Extensive genetic diversity, unique population structure and evidence of genetic exchange in the sexually transmitted parasite Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasite's genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages. CONCLUSIONS/SIGNIFICANCE: Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.

Atypical trichinellosis without eosinophilia associated with osteomyelitis.

Human trichinellosis is an important food-borne zoonosis caused by a nematode worm, Trichinella. The symptoms of the disease vary widely depending on the infection load, stage of infection and host immunity and include nausea, vomiting, abdominal pain, fever, facial edema and muscle pain. The disease is usually characterized by moderate to high eosinophilia. We hereby discuss an atypical case of trichinellosis, which presented with myositis of the thigh muscles but had no eosinophilia and no facial or periorbital edema and was associated with osteomyelitis of the femur. The diagnosis was made by the demonstration of anti-trichinella antibodies and later confirmed by the presence of larvae of Trichinella in the digested muscle biopsy. Physicians must be aware of trichinosis and should include it in their differential diagnosis when examining patients with fever and myositis with or without eosinophilia.

Immunogenicity and efficacy of single antigen Gp63, polytope and polytopeHSP70 DNA vaccines against visceral Leishmaniasis in experimental mouse model.

Polytope approach of genetic immunization is a promising strategy for the prevention of infectious disease as it is capable of generating effective cell mediated immunity by delivering the T cell epitopes assembled in series. Leishmaniasis is a significant world wide health problem for which no vaccine exists. In this study we have compared immunogenicity and efficacy of three types of DNA vaccines: single antigen Gp63 (Gp63/pcDNA), polytope (Poly/pcDNA) and Polytope fused with hsp70 (Poly/hsp/pcDNA) against visceral leishmaniasis in susceptible BALB/c mice. Mice vaccinated with these plasmids generated strong Th1 immune response as seen by dominating IFN-gamma over IL-10 cytokine. Interestingly, cytotoxic responses generated by polytope DNA plasmid fused with hsp70 of Leishmania donovani were significantly higher when compared to polytope and single antigen Gp63 vaccine. Challenge studies revealed that the parasite load in liver and spleen was significantly lower with Poly/hsp/pcDNA vaccination compared to other vaccines. Therefore, our study indicates that polytope DNA vaccine is a feasible, practical and effective approach for visceral leishmaniasis.

Utilization of monoclonal antibodies for detection of Plasmodium falciparum antigen in cerebrospinal fluid of cerebral malaria patients.

A uniform protein profile of bands at 34, 43, and 52 kDa was obtained with all the cerebrospinal fluid (CSF) samples of malaria (10 in number) and non-malaria patients (31 in number) by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). An immunoreactive band was observed at 43 kDa in CSF samples of cerebral malaria patients but not in non-malaria cases when tested with rabbit anti-Plasmodium falciparum antibodies by Western blot analysis. Eleven reactive monoclonal antibodies against P. falciparum were stabilized and expanded. Nine monoclonal antibodies were reactive to CSF samples of cerebral malaria and non-malaria and P. falciparum antigen by dot-ELISA and a common immunoreactive band at 43 kDa by Western blot. One clone Cl-2 was reactive at 43 kDa with CSF of the cerebral malaria patients and also in P. falciparum antigen but at 66 kDa with non-malarial CSF samples in Western blot. The other two clones (Cl-6 and 14) reacted with 3/31 (90% specific) and 8/31 (74%) CSF samples of non-malaria patients, respectively. The monoclonal antibody based ELISA reported in the present study using clone-6 can therefore offer another possibility for developing rapid, easy-to-perform, low-cost tests for diagnosis of cerebral malaria in CSF samples. Western blot using clone-2 might be useful for the detection of cerebral malaria antigen in CSF.