A hospital-based study on knowledge and attitude related to vitiligo among adults visiting a tertiary health facility of central India.

BACKGROUND: Vitiligo is one of the common stigmatizing dermatosis in the Indian society and the vitiligo patients have to face significant psychological hurt and social neglect. The severity of the stigma is related to the society's attitude and knowledge about it. AIMS AND OBJECTIVES: To document the prevalent knowledge and attitude in general public towards vitiligo patients, and to identify the determinants of good/poor knowledge and attitude. MATERIALS AND METHODS: A systematic random sampling technique was adopted to enroll 700 adult participants visiting an urban tertiary healthcare facility of central India. We developed a questionnaire to collect information on knowledge and attitude of the participants. A composite score was developed for good knowledge and attitude and performance of the participants was compared with the selected determinants. Data analysis was conducted by Stata software version 11. RESULTS: The overall knowledge score was good for 66.3% (95% confidence interval [CI]: 62.8%, 69.8%) of the participants. However, the score for attitude was comparatively poor i.e., only 16.9% (95% CI: 13.9%, 19.5%). None of the studied parameters could be significantly correlated with the knowledge score. Being married and being engaged in a health care related occupation were significant predictors of good attitude levels with P = 0.042 and 0.034 respectively, whereas female gender was the significant predictor for poor attitude with an odds ratio of 0.54 (95% CI: 0.33, 0.9) and P = 0.018. CONCLUSIONS: There were widespread myths prevalent about vitiligo in the studied population. The knowledge scores were better than attitude scores.

Structure of Leishmania donovani coronin coiled coil domain reveals an antiparallel 4 helix bundle with inherent asymmetry.

Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed.

Unconventional actins and actin-binding proteins in human protozoan parasites.

Actin and its regulatory proteins play a key role in several essential cellular processes such as cell movement, intracellular trafficking and cytokinesis in most eukaryotes. While these proteins are highly conserved in higher eukaryotes, a number of unicellular eukaryotic organisms contain divergent forms of these proteins which have highly unusual biochemical and structural properties. Here, we review the biochemical and structural properties of these unconventional actins and their core binding proteins which are present in commonly occurring human protozoan parasites.

Cloning, overexpression, purification and crystallization of the CRN12 coiled-coil domain from Leishmania donovani.

Leishmania donovani coronin CRN12 is an actin-binding protein which consists of two domains: an N-terminal WD repeat domain and a C-terminal coiled-coil domain. The coiled-coil domain is 53 residues in length. Helix-helix interactions in general and coiled coils in particular are ubiquitous in the structure of proteins and play a significant role in the association among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signalling, transport and actin dynamics. The L. donovani coronin CRN12 coiled-coil domain (5.8 kDa) was cloned, overexpressed, purified to homogeneity and the N-terminal 6×His tag was successfully removed by thrombin cleavage. Crystals of recombinant L. donovani coronin CRN12 coiled-coil domain were grown by vapour diffusion using a hanging-drop setup. Diffraction-quality crystals were obtained and data extending to 2.46 Šresolution were collected at 100 K on BM14, ESRF, Grenoble, France. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 118.0, b = 50.6, c = 46.0 Å, ß = 111.0°. Matthews coefficient (VM) calculations suggested the presence of 4-6 molecules in the asymmetric unit, corresponding to a solvent content of ∼33-55%, and are consistent with self-rotation function calculations.

Targeted drug delivery to macrophages in parasitic infections.

Successful homing of drugs to the desired biological compartment of the host usually depends on the intrinsic properties of the drug molecules. However, it can always be manipulated by appropriate designing of the carrier/delivery system, as little can be done to influence the target and its surroundings. Various carrier systems have emerged to deliver drugs to macrophages, albeit the efficacy, reliability and selectivity of these carriers are still in question. To date, the most extensively studied carriers are liposomes and microspheres. In fact, physicochemical properties of these carriers can alter their efficacy and specificity to a great extent. These properties include hydrophilicity, surface charge, composition, concentration, and presence of various target specific ligands on their surface. Incidentally, the particulate nature of these vehicles may facilitate passive homing of the entrapped drug molecules to the macrophages, which may harbour many of the important pathogens in their intracellular compartments, such as Mycobacterium sps, Leishmania and dengue virus etc., belonging to three different major classes of microbes. Moreover, macrophages upon interaction with particulate drug delivery vehicles may act as secondary drug depot, thus helping in localized delivery of the drug at the infected site. In the present article, a comprehensive review of literature is presented on the suitability of some lipid-based and polymeric materials as vehicles in delivery of drugs to macrophages in parasitic infections.

Tuftsin-bearing liposomes as antibiotic carriers in treatment of macrophage infections.

Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) that specifically binds monocytes, macrophages, and polymorphonuclear leukocytes and potentiates their natural killer activity against tumors and pathogens. The antimicrobial activity of this peptide is significantly increased by attaching at the C-terminus a fatty acyl residue through the ethylenediamine spacer arm. This activity is further augmented by incorporating the modified tuftsin in the liposomes. The tuftsin-bearing liposomes not only enhance the host's resistance against a variety of infections but also serve as useful vehicles for the site-specific delivery of drugs in a variety of macrophage-based infections, such as tuberculosis and leishmaniasis.

Traditional remedies for fertility regulation.

Plants have been used worldwide for treatment of various human ailments since antiquity. Their use is still quite prevalent in developing countries in the form of traditional/folkloric system of medicine. Intensive chemical and pharmacological studies on traditional/folkloric medicinal plants during the last 5 decades have led to the validation of traditional claims in many cases and facilitated identification of their active principles. The active principles have provided leads in the development of several life saving drugs, which are in clinical use today. A large number of these plants are used for birth control in different countries. The present review has, therefore, been planned to provide an account of the investigations carried out on traditional/folkloric plants used for fertility regulation. The status of scientific validations of their anti-fertility potential and identification of active principles during the last 28 years are discussed. The literature survey shows that approximately 318 different plants are in traditional/folkloric use worldwide, of which 227 plants are of Indian origin. So far, 74 plants have been screened for their anti-fertility potential, 48 of them have been found to be effective. Active principles of about 15 plants have been identified during the period under review.

Immunomodulator tuftsin augments antifilarial activity of diethylcarbamazine against experimental brugian filariasis.

In the present study, we evaluated the potential of an immunomodulator tuftsin in increasing the efficacy of liposomised diethylcarbamazine (DEC) against experimental filarial infection of Brugia malayi. The liposomised form of DEC, when used at sub-optimal dose of 25 mg/kg body weight, successfully eliminated filarial parasite from systemic circulation in animals inflicted with B. malayi infection. However, the formulation was effective upto 60 days post infection only, followed by recurrence of the infection. In contrast, the co-administration of liposomal formulation of DEC along with an immunomodulator tuftsin was found to be competent enough to suppress microfilarial stage of parasite till 90 days post treatment. Interestingly, tuftsin bearing DEC liposomes were found to be effective against adult parasite as well.

Transbilayer translocation of membrane phosphatidylserine and its role in macrophage invasion in Leishmania promastigotes.

Infectivity of Leishmania promastigotes has been shown to be growth cycle-dependent and restricted to the stationary phase. By using annexin V-FITC binding and procoagulant activity measurement assays, we show here that the promastigotes in the stationary phase contain significantly higher amounts of phosphatidylserine (PS) on their surface as compared to the log phase promastigotes. We also demonstrate that the infectivity of the promastigotes is determined by the presence of PS on their surface. In addition, by using NBD-labelled phospholipids, we show that the promastigote plasma membrane contains ATP-dependent out-to-in and ATP-independent in-to-out PS translocases which regulate the PS localisation in two-halves of the membrane bilayer, and that the greater amounts of external PS observed in the stationary phase promastigotes is perhaps due to the slower ATP-dependent out-to-in PS movements in these cells, as compared to the log phase promastigotes.

Superior chemotherapeutic efficacy of amphotericin B in tuftsin-bearing liposomes against Leishmania donovani infection in hamsters.

Chemotherapeutic efficacy of the amphotericin B (Amp B), which is the drug of choice for treatment of the leishmanial infections (kala-azar) that become resistant to the conventional chemotherapy using antimonials, has been examined in the Leishmania donovani infected hamsters after encapsulating the drug in tuftsin-free as well as tuftsin-bearing liposomes. The activity was significantly increased (p < 0.05) by delivering Amp B in tuftsin-free liposomes. This antileishmanial effect of the liposomized Amp B was further increased (p < 0.05) by grafting the natural macrophage-activator tetrapeptide, tuftsin (Thr-Lys-Pro-Arg), on the liposome's surface. This could possibly be attributed to both the enhanced drug tolerance after liposomization as well as to the increased uptake of tuftsin-bearing Amp B-laden liposomes by the macrophages. In addition to the increased efficacy, encapsulation of Amp B in the tuftsin-bearing liposomes also enhanced the drug accessibility to areas (e.g. bone marrow) that are otherwise inaccessible to the free drug. These results further demonstrate the usefulness of tuftsin-bearing liposomes as drug vehicles in treatment of the macrophage-based infections that have been reviewed recently (Agrawal, A.K. and Gupta, C.M. (2000). Tuftsin-bearing liposomes in treatment of macrophage-based infections, Adv. Drug Deliv. Rev., 41, 135-146).

Two-hybrid-based analysis of protein-protein interactions of the yeast multidrug resistance protein, Pdr5p.

The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes. The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood. In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae. Four of the major cytosolic loops that have been predicted for this protein [including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region] were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system. Results of these studies have revealed that the first cytosolic loop (CL1)--containing the first NBD domain--and also the C-terminal region of Pdr5p, interact with several candidate proteins. The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein.

ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators.

We have used fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-tagged phospholipid analogues, NBD-PE (phosphatidylethanolamine), NBD-PC (phosphatidylcholine) and NBD-PS (phosphatidylserine), to demonstrate that Cdr1p and its other homologues, Cdr2p and Cdr3p, belonging to the ATP-binding cassette (ABC) superfamily behave as general phospholipid translocators. Interestingly, CDR1 and CDR2, whose overexpression leads to azole resistance in C. albicans, elicit in-to-out transbilayer phospholipid movement, while CDR3, which is not involved in drug resistance, carries out-to-in translocation of phospholipids between the two monolayers of plasma membrane. Cdr1p, Cdr2p and Cdr3p could be further distinguished on the basis of their sensitivities to different inhibitors. For example, the in-to-out activity associated with Cdr1p and Cdr2p is energy-dependent and sensitive to sulphydryl blocking agents such as N-ethylmaleimide (NEM) and cytoskeleton disrupting agent cytochalasin E, while Cdr3p-associated out-to-in activity is energy-dependent but insensitive to NEM and cytochalasin E. We found that certain drugs, such as fluconazole, cycloheximide and miconazole, to which Cdr1p confers resistance could also affect in-to-out transbilayer movement of NBD-PE, while the same drugs had no effect on Cdr3p-mediated out-to-in translocation of NBD-PE. The ineffectiveness of these drugs to affect Cdr3p mediated out-to-in phospholipid translocation further confirms the inherent difference in the directionality of phospholipid translocation between these pumps. Notwithstanding the role of some of the Cdrps in drug resistance, this study clearly demonstrates that these ABC transporters of C. albicans are phospholipid translocators and this function could represent one of the physiological functions of such large family of proteins.

Enhanced immunostimulant activity and protective effect of a synthetic lipopeptide after liposomization against Plasmodium berghei infection in mice.

The immunostimulant activity of non-pyrogenic, sugar-free immunomodulator lipopeptide, Ala-D-Glu(Gly-Lys-CO.C11H23)-NH2 (comp. no 84/201), and its liposomized formulation has been studied. Liposomization of this lipopeptide significantly enhanced its antigen specific as well as nonspecific immune responses, as compared to the free lipopeptide. The liposomized formulation of lipopeptide significantly stimulated both the antibody and delayed-type hypersensitivity responses in Balb/c mice, and also enhanced nonspecifically the macrophage migration index, phagocytic activity and incorporation of 14C glucosamine in peritoneal macrophages of the mice that received pretreatment with this preparation. Further, the mice that received pretreatment with the liposomized preparation strongly resisted lethal P. berghei infection and consequently survived for longer period of times. These results indicate that liposomization of the compound no 84/201 significantly improves its ability to enhance not only antigen-specific immune response but also the nonspecific host's resistance against infections.

Use of liposomes as an immunopotentiating delivery system: in perspective of vaccine development.

Liposomes have been widely used to deliver antigens to the antigen-presenting cells (APCs) and also to modify their immunological behaviour in model animals. We recently demonstrated the potential of yeast lipid liposomes to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells. Interestingly, studies in the present report revealed that antigen encapsulated in yeast lipid liposomes could be successfully delivered simultaneously into the cytosolic as well as endosomal processing pathways of APCs, leading to the generation of both CD4+ T helper and CD8+ cytotoxic T cells. In contrast, encapsulation of same antigen in egg phosphatidyl-choline (PC) liposomes, just like its free form, has inefficient access to the cytosolic pathway of major histocompatibility complex (MHC) I dependent antigen presentation and failed to generate antigen specific CD8+ cytotoxic T-cell response. However, both egg PC as well as yeast lipid liposomes have elicited strong antigen specific antibody responses in immunized animals. These results imply usage of liposome encapsulated antigen as potential candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses.

Clinical and bacteriological study of urethral discharge.

A clinics-bacteriological study of 52 patients with urethritis was carried out. All the patients were evaluated clinically and bacteriologically at the beginning and end of the treatment. Majority of the patients belonged to the age group 21-30 years (58%). Incidence of gonococcal urethritis was 65% and Non gonococcal Urethritis (NGU) 35%. The common organisms causing NGU were chlamydia (28%) ureaplasma (11%) and mycoplasma (11%). Neisseria gonorrhoea was resistant to penicillin in (38%), ciprolloxacin in 67, and to noriloxaein in 6% cases. High incidence of HIV positivity was found in gonorrhoea (21%).

Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development.

In the majority of bacterial and viral infections the generation of cytotoxic T cells is of particular interest because such pathogens are able to escape the host defence mechanisms by surviving intracellularly within the phagocytic cells. To generate a CD8+ T lymphocyte response against exogenous antigens, the prerequisite is their delivery into the cytosol followed by processing and presentation along with class I major histocompatibility complex (MHC-I) molecules. In the present study we describe the method of liposome-based delivery of antigens and other macromolecules into the cytosol of target cells. To develop safe and effective methods for generating CD8+ T lymphocytes, we exploited the fusogenic character of lipids derived from lower organisms, that is baker's yeast (Saccharomyces cerevisiae). The degree of fusion with model membrane systems using yeast lipid liposomes varied from 40-70%, as opposed to 1-8% observed with egg PtdCho liposomes, depending on the assay system used. The fusion of yeast lipid liposomes with macrophages resulted in effective delivery of the entrapped solutes into the cytoplasmic compartment. This was further supported by the inhibition of cellular protein synthesis in J774 A1 cells by ricin A, encapsulated in the yeast lipid liposomes. Interestingly, the model antigen ovalbumin, when entrapped in the yeast lipid liposomes, successfully elicited antigen reactive CD8+ T cell responses. It may be concluded that the liposomes made of lipids derived from S. cerevisiae can spontaneously fuse with macrophages, delivering a significant portion of their contents into the cytoplasmic compartment of the cells.

Tuftsin-bearing liposomes in treatment of macrophage-based infections.

The use of liposomes as drug carriers in treatment of various diseases has been explored extensively for more than 20 years. 'Conventional' liposomes, when administered in vivo by a variety of routes, rapidly accumulate in the mononuclear phagocyte system (MPS). The inherent tendency of the liposomes to concentrate in MPS can be exploited in enhancing the non-specific host defence against infections by entrapping in them the macrophage modulators, and as carriers of antibiotics in treatment of intracellular infections that reside in MPS. This must further be enhanced by grafting on the liposome surface the ligands, e.g. tuftsin, that not only binds specifically to the MPS cells but also enhances their natural killer activity. Keeping this in view, we designed and developed tuftsin-bearing liposomes as drug carriers for the treatment of macrophage-based infections and outline these studies in this overview.

Antibody-bearing liposomes as chloroquine vehicles in treatment of murine malaria.

Malaria is a serious public health problem that affects about 300-500 million people and claims 1.5-2.7 million deaths every year. One-third of all humans live in zones where they risk catching it (1). The situation is aggravated because the malarial parasites are rapidly developing resistance to the existing antimalarial drugs, like chloroquine (2), when given in classical pharmaceutical forms. Studies on the molecular basis of chloroquine resistance suggest that enhanced active efflux of the drug from the cells infected with resistant parasite strain prevents drug accumulation to toxic levels within the cytosol of the infected erythrocytes (3-5). It has been shown that erythrocytes infected with chloroquine-resistant parasite accumulate less chloroquine than those with sensitive parasites (6,7). Furthermore, inhibiting the chloroquine efflux by Ca(2+)-channel blockers render the resistant cells fully sensitive to chloroquine (8), indicating that the antimalarial activity of the chloroquine is directly related to its concentration within the parasite food vacuole (9). The mechanism by which this concentration effect is achieved is unclear, but it is believed to involve binding to a putative chloroquine receptor (10).

Probing the role of C-1 ester group in Naja naja phospholipase A2-phospholipid interactions using butanetriol-containing phosphatidylcholine analogues.

To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A2-glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A2. Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A2-phospholipid interactions.

Asymmetric distribution of phosphatidylethanolamine in C. albicans: possible mediation by CDR1, a multidrug transporter belonging to ATP binding cassette (ABC) superfamily.

By using two molecular probes, we demonstrate that only 4% of total phosphatidylethanolamine (PtdEtn) in the plasma membrane (PM) of a human pathogenic yeast, Candida albicans, is present in its external half. Evidence is presented to show that the availability of PtdEtn could be related to the expression of a multidrug transporter CDR1 of C. albicans, and the process is energy-dependent. A homozygous CDR1 disruptant strain of C. albicans shows almost 23% reduction in the external labelling of PtdEtn. This report shows that, similar to human MDRs, yeast multidrug transporter could also be involved in aminophospholipid translocation.