Dermotropic Leishmania donovani in Sri Lanka: visceralizing potential in clinical and preclinical studies.

The visceralizing potential of apparently dermotropic Leishmania donovani in Sri Lanka (L. donovani-SL) was investigated through long-term follow-up of cutaneous leishmaniasis (CL) patients and in vivo and in vitro experimental infection models. CL patients (n = 250) treated effectively with intra-lesional antimony therapy were followed-up six monthly for 4 years. There was no clinical evidence of visceralization of infection (VL) during this period. Infection of BALB/c mice with L. donovani-SL (test) through intra-dermal route led to the development of cutaneous lesions at the site of inoculation with no signs of systemic dissemination, in contrast to the observations made in animals similarly infected with a visceralizing strain of L. donovani-1S (control). Cytokine (IL-10, IFN-γ) release patterns of splenocytes and lymph node cell cultures derived from mice primed with experimental infections (with either test or control parasites) revealed significantly high IFN-γ response associated with test mice with CL, while prominent IL-10 levels were observed in association with control mice with VL. Furthermore, diminished infection efficiency, intracellular growth and survival of L. donovani-SL parasites compared with L. donovani-1S were evident through in vitro macrophage infection experiments. These studies confirm, for the first time, the essential dermotropic nature of L. donovani-SL suggesting natural attenuation of virulence of local parasite strains.

Standardized methods to generate mock (spiked) clinical specimens by spiking blood or plasma with cultured pathogens.

AIMS: To demonstrate standardized methods for spiking pathogens into human matrices for evaluation and comparison among diagnostic platforms. METHODS AND RESULTS: This study presents detailed methods for spiking bacteria or protozoan parasites into whole blood and virus into plasma. Proper methods must start with a documented, reproducible pathogen source followed by steps that include standardized culture, preparation of cryopreserved aliquots, quantification of the aliquots by molecular methods, production of sufficient numbers of individual specimens and testing of the platform with multiple mock specimens. Results are presented following the described procedures that showed acceptable reproducibility comparing in-house real-time PCR assays to a commercially available multiplex molecular assay. CONCLUSIONS: A step by step procedure has been described that can be followed by assay developers who are targeting low prevalence pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of diagnostic platforms for detection of low prevalence pathogens such as biothreat or emerging agents is challenged by the lack of clinical specimens for performance evaluation. This deficit can be overcome using mock clinical specimens made by spiking cultured pathogens into human matrices. To facilitate evaluation and comparison among platforms, standardized methods must be followed in the preparation and application of spiked specimens.

Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth.

An endothelial cell inhibitor was purified from supernatant of an Epstein-Barr virus-immortalized cell line and identified as fragments of calreticulin. The purified recombinant NH2-terminal domain of calreticulin (amino acids 1-180) inhibited the proliferation of endothelial cells, but not cells of other lineages, and suppressed angiogenesis in vivo. We have named this NH2-terminal domain of calreticulin vasostatin. When inoculated into athymic mice, vasostatin significantly reduced growth of human Burkitt lymphoma and human colon carcinoma. Compared with other inhibitors of angiogenesis, vasostatin is a small, soluble, and stable molecule that is easy to produce and deliver. As an angiogenesis inhibitor that specifically targets proliferating endothelial cells, vasostatin has a unique potential for cancer treatment.

Protein disulfide isomerase assisted protein folding in malaria parasites.

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.

Methylation and expression of rat kappa-casein gene in normal and neoplastic rat mammary gland.

The kappa-casein mRNA was evaluated in the rat mammary gland during functional differentiation and neoplastic growth. Using a dot-blot assay, the mRNA was barely detectable in the virgin gland; it steadily increased from the onset of gestation and leveled off during lactation. In rat mammary tumors, either primary (7,12-dimethylbenz(a)anthracene-induced) or transplanted (MTW9), the level of kappa-casein mRNA was about 2.5-fold lower than in the lactating gland, but an extensive variation among individual tumors was observed. There was no detectable kappa-casein mRNA in rat liver. In the mammary gland of virgin, 10-day pregnant, and nonlactating females, the DNA sequences within and/or around the kappa-casein gene were found to be hypermethylated at the HpaII-MspI sites as compared to 10-day lactating females. In the two tumors studied, the kappa-casein gene was partially methylated at the same sites. Prolactin treatment induced kappa-casein gene expression in the virgin rat mammary gland but did not result in a change of the methylation status at the HpaII-MspI sites. Under similar conditions of prolactin treatment, however, the methylation of the Sau96I sites was reduced, and an inverse correlation between the onset of kappa-casein gene methylation and kappa-casein gene expression was evident in both the virgin gland and the tumors. Thus, the expression of kappa-casein was found to be inversely correlated with the extent of methylation of the kappa-casein gene, except in the case of the prolactin-stimulated virgin gland.

Programmed cell death in the unicellular protozoan parasite Leishmania.

In the present study we have demonstrated some features characterizing programmed cell death (PCD) in the unicellular protozoan parasite Leishmania donovani, the causative agent of visceral Leishmaniasis. We report that PCD is initiated in stationary phase cultures of promastigotes and both in actively growing cultures of axenic amastigotes and promastigotes upon treatment with anti Leishmanial drugs (Pentostam and amphotericin B). However, the two cell types respond to antileishmanial drugs differently. The features of PCD in L. donovani promastigotes are nuclear condensation, nicked DNA in the nucleus, DNA ladder formation, increase in plasma membrane permeability, decrease in the mitochondrial membrane potential (DeltaPsi m) and induction of a PhiPhiLux (PPL)-cleavage activity. PCD in both stationary phase culture and upon induction by amphotericin B resulted first in the decrease of mitochondrial membrane potential followed by simultaneous change in plasma membrane permeability and induction of PPL-cleavage activity. Of the total PPL-cleavage activity, several caspase inhibitors inhibited a significant amount (21-34%). Inhibitors of cathepsin or calpain did not inhibit PPL-cleavage activity. Taken together this study demonstrates that the characteristic features of PCD exist in unicellular protozoan Leishmania donovani. The implication of PCD on the Leishmania pathogenesis is discussed.

Bifunctional activity of epidermal growth factor on alpha- and kappa-casein gene expression in rodent mammary glands in vitro.

Explants from mammary glands of virgin rats and pregnant rats and mice were cultured under serum-free conditions in the presence of various combinations of the hormones insulin (I), aldosterone (A), corticosterone (C), PRL, and epidermal growth factor (EGF). Bifunctional activity of EGF was found on expression of the alpha- and kappa-casein genes. In the presence of I, A, and C, EGF increased the level of alpha-casein mRNA in pregnant mouse mammary gland explants, but not in rats. kappa-Casein mRNA sequences in mouse mammary gland explants were also significantly increased by EGF in the presence of I, A, and C, but in rat mammary gland explants, the increase was less. In contrast, in the presence of I, A, C, and PRL, EGF inhibited the induction of both alpha- and kappa-casein mRNA sequences in tissue from rats and mice. This bifunctionality of EGF in terminal differentiation of the rodent mammary gland was also reflected in the levels of synthesis of total casein and alpha-lactalbumin.

La autoantigen binding to a 5' cis-element of rubella virus RNA correlates with element function in vivo.

Rubella virus genomic RNA contains a 5' stem-loop (5'(+) SL) which is required for efficient translation and replication. The La autoantigen previously was shown to bind this RNA sequence in vitro. Results reported here demonstrate that this cellular RNA-binding protein binds to the RV 5' SL RNA with sufficient specificity for the binding to occur in the presence of excess total cellular RNA. Further, the affinity of purified La for the RV sequence is similar to its affinity for known cellular substrates. To address the functional significance of La binding, mutant forms of the RV 5'(+) SL were analysed which bind La weaker or stronger than the native form. These three forms of the RV 5' SL were incorporated into RV-luciferase constructs which expressed luciferase activity in transient transfection. The level of expression from each construct correlated with the ability of its RV sequence to bind La. The detection of La/RV RNA complexes in infected cells further supported the physiological relevance of this interaction. Possible implications of autoantigen La interaction with RV RNA for rubella virus pathology and vaccine associated adverse reactions are discussed.

Nucleotide sequence of the 24S subgenomic messenger RNA of a vaccine strain (HPV77) of rubella virus: comparison with a wild-type strain (M33).

A full-length cDNA clone for the 24S subgenomic mRNA of the vaccine strain (HPV77) of rubella virus has been isolated from a cDNA library made from the RNAs of infected cells. Starting from the first Met start codon, the 24S mRNA codes for a precursor protein of 1063 amino acids (aa). This precursor encodes a capsid protein of 300 aa, and two envelope proteins, E1 (481 aa) and E2 (282 aa). Both the E1 and E2 proteins are preceded by a stretch of 21 hydrophobic aa, characteristic of a signal peptide, and each has three putative glycosylation sites in the polypeptide chains. Comparison between the structural proteins of the vaccine and the wild-type (wt; M33) strains of rubella virus, revealed that the E2 protein of the vaccine strain differs, in its apparent Mr, by approx. 3 kDa, from the wt strain. The difference could be due to decreased glycosylation of the vaccine strain E2 protein, as revealed by [3H]mannose incorporation studies. Five single-aa changes in the structural proteins occurred during the attenuation process, one each in the capsid and the E1 protein and three in the E2 protein. The change of Thr-412----Ile in the E2 protein results in the loss of a putative glycosylation site at Asn-410, which offers a plausible explanation for decreased glycosylation of the E2 protein from the vaccine strain of rubella virus.

The cis-acting 3'-element of rubella virus RNA has DNA promoter activity.

The 3'-terminal region of the rubella virus (RV) positive-strand RNA, referred to here as the cis-acting element (CAE), is implicated in the initiation of negative-strand RNA synthesis. Sequence analysis of the 3'-CAE shows that there is a putative TATA box which is surrounded by G + C-rich sequences. To determine whether this element, in a DNA form, has the capability to initiate transcription, a 3'-end 165-bp NarI-EcoRI fragment from the RV cDNA was cloned upstream from a cat reporter gene. The level of CAT activity was dependent on the presence of the 3'-CAE and the SV40 enhancer. Primer extension analysis of the CAT mRNA showed that the transcription start point is in the RV 3'-CAE, 34 bp downstream from the putative TATA box. DNA-gel shift analysis revealed that three nucleoprotein-specific complexes were formed with the 3'-CAE and the binding sites for these proteins were between bp -64 to -108. The possible promoter function of the RV 3'-CAE is discussed in context to RV persistence.

Identification of differentially expressed Leishmania donovani genes using arbitrarily primed polymerase chain reactions.

Arbitrarily primed polymerase chain reactions (AP-PCR) were used to amplify polymorphic DNA fragments from the genomes of a variety of geographic isolates of Leishmania donovani (Ld). From the latter, five polymorphic DNA fragments were cloned and sequence analysis identified 15 unique clones. Northern blot analysis showed that 13 of the 15 clones hybridized to transcribed RNAs isolated from Ld. Eight of these 13 AP-PCR clones specifically hybridized to Ld RNAs that were differentially expressed in promastigote and 'amastigote' cells. Comparative Northern analysis of four differentially expressed AP-PCR clones indicated that two clones, LdS-14-14 and LdI-9-7, were expressed in Ld and several other Leishmania species. However, RNAs corresponding to two other AP-PCR clones, LdE-6-1 and LdI-9-5, were detected only in members of the Ld complex, and not in L. major (Lm) or L. tropica (Lt). Comparative Southern blot analysis of the LdS-14-14 locus revealed numerous restriction-fragment length polymorphisms (RFLP) distinguishing Lm and Lt from the Ld isolates and L. infantum. However, the LdS-14-14 loci were mapped to similar-sized chromosomes observed among all Old World Leishmania species tested, indicating that localized nucleotide divergence, not chromosomal rearrangement, was responsible for altered Southern blot patterns. These results demonstrate that AP-PCR is a very useful method for identifying expressed gene sequences in organisms of relatively low-complexity genomes. Interestingly, the majority of these sequences identified in this study correspond to differentially expressed genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a cDNA clone for a rare mRNA modulated by ovariectomy in mammary carcinomas.

A complementary DNA (cDNA) clone (p13) for a rare mRNA was isolated from a cDNA library generated from total polyA+ RNA of 14-day lactating rat mammary gland. In vitro translation of the positively selected mRNA from p13 cDNA revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a polypeptide of 24 kDa. The p13 cDNA clone hybridized on northern blots predominantly to approximately 1100 base size RNA and weakly to approximately 3800 base size RNA from lactating mammary gland. It hybridized only to approximately 3800 base size RNA from rat liver. Southern blot analysis of genomic DNA showed differences in gene organization in mammary gland and liver. The mRNA level for the 24 kDa polypeptide was higher in 7-12 DMBA-induced tumor and lower in the MTW9 carcinoma as compared to lactating mammary gland. After ovariectomy, the mRNA level in mid pregnant gland increased but was reduced in the 7-12 DMBA tumors.

Increase in cell-surface N-acetylglucosaminide beta (1----4)galactosyltransferase activity with retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

Exposure of F9 cells to all-trans-retinoic acid over a period of 6 days resulted in 4-fold induction of cell surface N-acetylglucosaminide beta (1----4)galactosyltransferase (GT) activity. The retinoic acid-induced GT activity was further enhanced by treatment of the cells with 8-bromo cyclic AMP. The ability of retinoic acid alone, or retinoic acid in combination with 8-bromo cyclic AMP, to induce GT activity was inhibited by both actinomycin D and cycloheximide. These findings indicate that the induction of galactosyltransferase activity noted with differentiation of F9 cells involves de novo synthesis of new enzyme protein.

Cloning and characterization of differentially expressed genes from in vitro-grown 'amastigotes' of Leishmania donovani.

Leishmanial parasites routinely undergo cyclic differentiation from promastigotes to amastigotes during their life cycle. This process involves both morphological and macromolecular changes. To study such changes, we used a axenic culture system which permits the continuous generation and cycling of Leishmania donovani from promastigotes to 'amastigotes' in vitro. cDNA libraries were constructed from poly(A)+ RNA isolated from both the pro- and amastigote forms. Using differential cDNA hybridization techniques, 3 unique cDNAs clones (P17, A41 and A45) were isolated from the amastigote library. To assess whether these clones were differentially expressed by the pro-or 'amastigotes' forms, they were hybridized to RNA isolated from each of these parasite forms in Northern and slot-blots. Results of these analyses showed that 'amastigotes' had approx. 2-fold higher levels of the A41 and A45 RNAs compared to the promastigotes. Conversely, promastigotes showed approx. 2-fold higher levels of the P17 RNA than 'amastigotes'. Nucleotide sequence analysis and comparison with those in Gene bank, revealed that the 3 cDNAs represent unique leishmanial genes. Comparison of the deduced amino acid sequences revealed that P17 open reading frame (ORF) had significant similarity with a soybean ribosomal protein S11; A41 ORF with a Bacillus subtilis spore germination gene (gerC) and A45 ORF with yeast stress-inducible protein (STI1). It is of interest to note that, of the 3 cDNAs identified, the A45-encoded protein was recognized by sera from patients with clinically active visceral leishmaniasis and was encoded by a single copy gene.

Conservation of low-copy gene loci in Old World leishmanias identifies mechanisms of parasite evolution and diagnostic markers.

Genome plasticity has been hypothesized to be a driving force behind parasite speciation. We have evaluated divergence in single and low-copy genes in terms of locus organization, chromosomal localization and gene expression in Leishmania infantum, L. major, L. tropica and three widely divergent geographic isolates of L. donovani. Seventeen genes of low to moderate copy number (1-4 copies/haploid genome) were analyzed to identify restriction fragment length polymorphisms (RFLPs) providing heritable markers distinguishing Old World (OW) leishmanias. These RFLP markers were conserved in parasite isolates from primary infections demonstrating their utility as diagnostic tools. The species designations established by RFLP analysis of field isolates was confirmed by use of monoclonal antibodies. All 17 genes were present in each OW leishmania analyzed except LSIP (A45), which was absent from L. infantum. The 17 genes were found to be distributed among 9 distinct chromosomes. However, in spite of variations in chromosome karyotypes among the various OW leishmanias, individual gene probes localized to a similar sized chromosome from each isolate. These observations coupled with a molecular tree derived from RFLP data suggest that the OW leishmanias comprise a monophyletic lineage, with species associated with cutaneous disease exhibiting the greatest level of divergence. Data from this study supports previous observations that species causing cutaneous and visceral disease have diverged primarily by nucleotide substitutions. Such nucleotide divergence may not only lead to changes in protein function and antigenicity, but may also alter gene regulation programs as exemplified by the finding that the LdI-9-5 and LdE-6-1 genes were expressed only in visceralizing leishmanias.

Isolation and characterization of Leishmania donovani calreticulin gene and its conservation of the RNA binding activity.

Calreticulin has been implicated in multiple cell functions. Recently, we have shown that both human and simian calreticulin are RNA binding proteins and that their binding activity is due to phosphorylation. To demonstrate that the RNA binding property of calreticulin is an intrinsic part of this multi-functional molecule and is evolutionarily conserved, we isolated and characterized the calreticulin gene from the unicellular parasite, Leishmania donovani. Amino acid sequence homology between human and Leishmania calreticulin (L. d. cal) is limited, but like the human homologue, L. d. cal binds Ca+2, can be phosphorylated in vitro and binds certain RNA sequences in a phosphorylation-dependent manner. Unlike human calreticulin, L. d. cal is glycosylated and its binding to endogenous Leishmania RNA is phosphorylation-independent. The binding of L. d. cal to Leishmania RNA suggests that the RNA binding activity of calreticulin has remained evolutionarily conserved.

Effect of site-directed asparagine to isoleucine substitutions at the N-linked E1 glycosylation sites on rubella virus viability.

The role of three N-linked glycosylation sites in rubella virus (RV) E1 protein on virion release was analyzed by transfecting Vero 76 cells with infectious RV RNA (Robo302WT) containing isoleucine substitutions at N76, N177, and N209 (individually and in combinations). RV RNAs were detected and found to retain substitutions in the transfected cells, but RV capsid indicative of infection was undetectable, except for in Robo302WT and Robo302-N177I transfected cells. Only culture supernatants of Robo302WT and Robo302-N177I RNA transfected cells were positive for RV, suggestive of the virion release into the culture medium. Further, detection of intracellular RV E1 and newly released virion-associated E1 was possible only from cells previously incubated with Robo302-N177I and Robo302WT culture supernatants, suggesting that N177I substituted virus retained infectivity. These results suggest that while glycosylation at N177 is not critical, N76I and N209I mutations are lethal to RV viability.

Rubella virus replication: effect of interferons and actinomycin D.

The effect of alpha and gamma interferon (IFN alpha, IFN gamma) and actinomycin D on the expression of wild type rubella virus in African green monkey kidney cells (Vero 76) was studied. Viral protein synthesis in the infected cells was significantly reduced upon treatment of the cells with IFN alpha or IFN gamma, which is accompanied by the reduction in the level of both the (+) stranded and the (-) stranded viral RNAs. The residual rubella viral RNA from interferon-treated cells, however, was structurally intact as judged by Northern blot analysis and in vitro translation. These results suggest that the effect of IFN alpha and IFN gamma on rubella viral protein synthesis is both at the transcriptional and the translational level. The effect of actinomycin D on rubella virus replication was found to be time-dependent. It is much more pronounced during the eclipse phase of the viral growth (first 4 h) than after 8 h at which time actinomycin D had lesser effect. A similar effect on rubella virus replication was observed when alpha-amanitin was used instead of actinomycin D. These results were taken to indicate that during the viral infection, host cell DNA directs the synthesis of a cellular factor(s) which is essential for the viral replication. When the synthesis of this cellular factor(s) is terminated at an early stage of viral infection by actinomycin D or by alpha-amanitin, viral replication is impaired.

Rubella virus: mechanism of attenuation in the vaccine strain (HPV77).

The vaccine type (HPV77 strain) of rubella virus replicates slower and manifests a delayed appearance of cytopathic effect in Vero-76 cells as compared to wild-type virus (M33). The change in cytopathic effect coincides with the delayed appearance of both genomic and subgenomic RNA as well as viral structural proteins in the cell. The delay in the appearance of the viral proteins in the cells was also evident when the cells infected with the vaccine-type virus were treated with the lysosomotropic agent such as chloroquine. Binding studies using [35S]methionine-labeled virus showed that the vaccine-type virus bound to the cells poorly and the binding was not completely competed out with the cold virus.

Identification and characterization of host factor interactions with cis-acting elements of rubella virus RNA.

We have analyzed the function of cis-acting elements of rubella virus RNA and the components which interact with these elements in viral RNA replication. We demonstrated that the 5'- and 3'-terminal sequences from RV RNA promote translation and negative-strand RNA synthesis of chimeric chloroamphenicol acetyltransferase (CAT) RNAs. These sequences have a potential to form stem-loop (SL) structures and bind cellular proteins specifically in RNA gel-shift and UV cross-linking assays. The 5' end binding proteins were identified to be Ro/SSA-associated antigens by virtue of being recognized in an RNA complex by an autoimmune patient serum with Ro antigen type specificity. Purification and sequence analysis of the 3' end binding protein revealed that it is a homologue of human calreticulin. The role of host protein in RV replication is discussed.