Trial geography, pharmacogenetics, and global drug development.

Drug development is increasingly global. The benefits of multiregional trials include worldwide evaluation of safety and efficacy. However, clinical practice, environmental, and genetic factors can vary across geographic regions, significantly influencing trial outcomes within a specific geographic region or the global population relative to the United States (US). Genomic technologies and research discoveries continue to advance at a remarkable pace, offering opportunities to explore intrinsic factors that could account for regional variability in drug pharmacokinetics or response.

The D7 family of salivary proteins in blood sucking diptera.

The D7 subfamily of salivary proteins is widespread in blood sucking Diptera and belongs to the superfamily of pheromone/odourant binding proteins. Although D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies, their role in blood feeding remains elusive. In the present work we report the sequence of seventeen novel D7 proteins, and propose an evolutionary scenario for the appearance of the several forms of this protein, based on a total of twenty-one sequences from Culex quinquefasciatus, Aedes aegypti, Anopheles gambiae, An. arabiensis, An. stephensi, An. darlingi mosquitoes and Lutzomyia longipalpis and Phlebotomus papatasi sand flies.

Mycobacterium bovis BCG but not Mycobacterium leprae induces TNF-alpha secretion in human monocytic THP-1 cells.

In this study, we compared the level of TNF-alpha secretion induced in monocytic THP-1 cells after phagocytosis of Mycobacterium leprae, the causative agent of leprosy, and M. bovis BCG, an attenuated strain used as a vaccine against leprosy and tuberculosis. The presence of M. leprae and BCG was observed in more than 80% of the cells after 24 h of exposure. However, BCG but not M. leprae was able to induce TNF-alpha secretion in these cells. Moreover, THP-1 cells treated simultaneously with BCG and M. leprae secreted lower levels of TNF-alpha compared to cells incubated with BCG alone. M. leprae was able, however, to induce TNF-alpha secretion both in blood-derived monocytes as well as in THP-1 cells pretreated with phorbol myristate acetate. The inclusion of streptomycin in our cultures, together with the fact that the use of both gamma-irradiated M. leprae and heat-killed BCG gave similar results, indicate that the differences observed were not due to differences in viability but in intrinsic properties between M. leprae and BCG. These data suggest that the capacity of M. leprae to induce TNF-alpha is dependent on the stage of cell maturation and emphasize the potential of this model to explore differences in the effects triggered by vaccine strain versus pathogenic species of mycobacteria on the host cell physiology and metabolism.

The salivary adenosine deaminase activity of the mosquitoes Culex quinquefasciatus and Aedes aegypti.

A cDNA coding for a protein with significant similarity to adenosine deaminase (ADA) was found while randomly sequencing a cDNA library constructed from salivary gland extracts of adult female Culex quinquefasciatus. Prompted by this result, we found high ADA activities in two culicine mosquitoes, Culex quinquefasciatus and Aedes aegypti, but not in the anopheline Anopheles gambiae. Homogenates from Culex quinquefasciatus also have an AMP deaminase activity that is three times greater than the ADA activity, whereas in Aedes aegypti the AMP deaminase activity is less than 10% of the ADA activity. Evidence for secretion of ADA during blood feeding by Aedes aegypti includes the presence of ADA activity in warm solutions probed through a membrane by mosquitoes and in serotonin-induced saliva and a statistically significant reduction in the levels of the enzyme in Aedes aegypti following a blood meal. We could not demonstrate, however, that C. quinquefasciatus secrete ADA in their saliva. Male Aedes aegypti and C. quinquefasciatus, which do not feed on blood, have less than 3% of the levels of ADA found in females. We propose that ADA activity in A. aegypti may help blood feeding by removing adenosine, a molecule associated with both the initiation of pain perception and the induction of mast cell degranulation in vertebrates, and by producing inosine, a molecule that potently inhibits the production of inflammatory cytokines. The role of salivary ADA in Culex quinquefasciatus remains unclear.

Effect of unique Mycobacterium leprae phenolic glycolipid-I (PGL-I) on tumour necrosis factor production by human mononuclear cells.

Mycobacterium leprae cell wall-associated components are found in large amounts in the tissues of leprosy patients, particularly those at the lepromatous pole. Among these molecules, the phenolic glycolipid-I (PGL-I), unique to M. leprae, has been involved in the selective anergy observed in the lepromatous patients. Armadillo-derived M. leprae retains only a small proportion of the total PGL-I found in infected tissues. Therefore, the addition of PGL-I to M. leprae in vitro is important for a better understanding of M. leprae effects in vivo. We have studied the influence of PGL-I on TNF production by normal human peripheral blood mononuclear cells (PBMC) and by a human monocytic leukaemia cell line (THP-1) following stimulation with killed M. leprae. PGL-I alone did not induce TNF secretion by PBMC, but when associated with a sub-optimal dose of armadillo-derived M. leprae increased the release of this cytokine. In agreement with these results, M. leprae-exposed THP-1 cells did not secrete detectable levels of TNF unless PGL-I was simultaneously added to the culture. This increase in TNF production suggests that PGL-I plays a role in the induction of TNF during the natural infection. In addition, the modulatory effect of PGL-I on TNF release by THP-1 cells reinforces that monocytes are one of the possible targets of this molecule.

The invertebrate growth factor/CECR1 subfamily of adenosine deaminase proteins.

Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine. Its lack determines severe combined immunodeficiency in mammals, possibly due to accumulation of extracellular adenosine, which induces apoptosis in lymphocytes (Franco et al., 1998). Thus, presence of normal levels of ADA leads to normal growth and proliferation of lymphocytes. Several vertebrate and microbial ADA amino-acid sequences are known, with substantial similarity to each other. On the other hand, there are invertebrate growth factors as well as a candidate gene for the human cat eye syndrome (CECR1) (Riazi et al., 2000. Genomics 64, 277-285), which share substantial similarity to each other, and also to ADA. In this study, we report the expression and ADA enzymatic activity of a cDNA from the salivary glands of Lutzomyia longipalpis, a blood-sucking insect, with substantial similarity to insect growth factors and to human CECR1. We also demonstrate the existence of a subfamily of the adenosine deaminase family characterized by their unique amino-terminal region. Both Drosophila melanogaster and humans have both types of adenosine deaminases. Results indicate that these invertebrate proteins previously annotated as growth factors, as well as the human CECR1 gene product, may exert their actions through adenosine depletion. The different roles played by each type of adenosine deaminase in humans and Drosophila remains to be fully investigated.

Simulium vittatum (Diptera: Simuliidae) and Lutzomyia longipalpis (Diptera: Psychodidae) salivary gland hyaluronidase activity.

Hyaluronidase activity in the salivary gland homogenates of Simulium vittatum (Zetterstedt) is described, and its optimal pH determined. Salivary activity was reduced significantly after a blood meal, indicating that it was secreted after blood feeding. Phlebotomus papatasi (Scopoli) also exhibited salivary hyaluronidase activity. These results indicate that hematophagous pool-feeding insects may secrete this enzyme to help the spread of salivary antihemostatic agents in the vicinity of the feeding lesion, and perhaps to increase the size of the feeding lesion itself. Additionally, this enzyme may affect local host immune reactions and promote arboviral transmission.

The salivary adenosine deaminase from the sand fly Lutzomyia longipalpis.

In the process of sequencing a subtracted cDNA library from the salivary glands of the sand fly Lutzomyia longipalpis, we identified a cDNA with similarities to gene products of the adenosine deaminase family. Prompted by this cDNA finding, we detected adenosine deaminase activity at levels of 1 U/mg protein in salivary gland homogenates. The activity was significantly reduced following a blood meal indicating its apparent secretory fate. The native enzyme has a K(m) of approximately 10 microM, an isoelectric pH between 4.5 and 5.5, and an apparent molecular weight of 52 kDa by size exclusion chromatography. The possible role of this enzyme, which converts adenosine to inosine, in the feeding physiology of L. longipalpis is discussed.

Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis.

The alternative pathway of complement is an important defense against pathogens and in tick rejection reactions. The tick Ixodes scapularis is able to feed repeatedly on its natural host and has a salivary anticomplement activity that presumably facilitates feeding. In this study, we purified and then obtained the amino-terminal sequence of the I. scapularis salivary anticomplement (Isac). We found a full-length clone coding for Isac by random screening of a salivary gland cDNA library. Expressing Isac cDNA in COS cells reproduced the activity found in tick saliva, namely, inhibition of rabbit erythrocyte lysis by human serum in the presence of Mg(2+) and EGTA, inhibition of C3b binding to agarose in the presence of Mg(2+) and EGTA, and acceleration of factor Bb uncoupling from the C3 convertase generated by the alternative pathway. Recombinant Isac had no effect on the recalcification time of human platelet-poor plasma or in the classical complement pathway, indicating that it is a specific inhibitor similar to the regulators of complement activation of the alternative pathway such as factor H. Isac, however, has no similarity to any protein in the GenBank(TM) data base, indicating that it is a novel and relatively small (18.5 kDa) anticomplement molecule.

Salivary amylase activity of the phlebotomine sand fly, Lutzomyia longipalpis.

Both male and female adult stages of the sand fly Lutzomyia longipalpis have detectable amylase activity in their salivary glands, as indicated by formation of p-nitrophenyl-alpha-D-maltoside from p-nitrophenyl-alpha-D-octoside and by hydrolysis of 4-nitrophenyl-alpha-D-maltoheptaoside-4,6,-O-ethylidene. No salivary alpha-glucosidase was detected. Amylase activity was also found in the crop and midgut of female flies, although in a smaller amount. Salivary amylase is significantly reduced from the salivary glands immediately after a blood meal, as is the case with salivary alpha-glucosidases in mosquitoes. Presence of salivary gland amylase in these sand flies, and absence of salivary alpha-glucosidase, indicates that in nature these insects may have a significant intake of carbohydrates in the form of starch, as suggested by their plant-feeding behavior, previously demonstrated by Schlein and Warburg (Schlein, Y., Warburg, A., 1986. Phytophagy and the feeding cycle of Phlebotomus papatasi (Diptera: Psychodidae) under experimental conditions. Journal of Medical Entomology 23, 11-15), and Alexander and Usma (Alexander, B., Usma, M.C., 1994. Potential sources of sugar for the phlebotomine sandfly Lutzomyia youngi (Diptera: Psychodidae) in a Columbia coffee plantation. Ann. Trop. Med. Parasitol. 88, 543-549).

The salivary 5'-nucleotidase/phosphodiesterase of the hematophagus sand fly, Lutzomyia longipalpis [corrected].

Salivary gland homogenates from adult female Lutzomyia longipalpis sand flies contain large amounts of 5'-nucleotidase and phosphodiesterase activities. Phosphodiesterase activity was found to be associated with 5'-nucleotidase in several independent experiments: (i) it coelutes with 5'-nucleotidase on a molecular sieving column, (ii) it coelutes with 5'-nucleotidase on a chromatofocusing column, and (iii) it has the same thermal inactivation kinetics as the 5'-nucleotidase activity. Additionally, both activities are independent of divalent cations, and both are decreased following a blood meal, suggesting that they reside in the same molecule. The role of salivary nucleotidases and purine nucleotides in blood-feeding by sand flies is discussed.

Human immune response to sand fly salivary gland antigens: a useful epidemiological marker?

Antibody (IgG) responses to salivary gland homogenate and to a recombinant salivary protein from the sand fly Lutzomyia longipalpis were investigated using sera from children living in an endemic area of visceral leishmaniasis in Brazil. We classified children into four groups according to their responses to Leishmania antigen: (Group I) positive serology and positive delayed type hypersensitivity (DTH), (Group II) positive serology and negative DTH, (Group III) negative serology and positive DTH, and (Group IV) negative serology and negative DTH. A highly significant correlation was found between anti-salivary gland IgG levels and DTH responses. An L. longipalpis salivary recombinant protein used as an antigen in an enzyme-linked immuno sorbent assay (ELISA) gave a significant but different result. A positive correlation was found between anti-Leishmania IgG and anti-recombinant protein IgG titers. The results indicate that sand fly salivary proteins may be of relevance to the study the epidemiology of leishmaniasis.

Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis.

The saliva of blood-sucking arthropods contains powerful pharmacologically active substances and may be a vaccine target against some vector-borne diseases. Subtractive cloning combined with biochemical approaches was used to discover activities in the salivary glands of the hematophagous fly Lutzomyia longipalpis. Sequences of nine full-length cDNA clones were obtained, five of which are possibly associated with blood-meal acquisition, each having cDNA similarity to: (i) the bed bug Cimex lectularius apyrase, (ii) a 5'-nucleotidase/phosphodiesterase, (iii) a hyaluronidase, (iv) a protein containing a carbohydrate-recognition domain (CRD), and (v) a RGD-containing peptide with no significant matches to known proteins in the BLAST databases. Following these findings, we observed that the salivary apyrase activity of L. longipalpis is indeed similar to that of Cimex apyrase in its metal requirements. The predicted isoelectric point of the putative apyrase matches the value found for Lutzomyia salivary apyrase. A 5'-nucleotidase, as well as hyaluronidase activity, was found in the salivary glands, and the CRD-containing cDNA matches the N-terminal sequence of the HPLC-purified salivary anticlotting protein. A cDNA similar to alpha-amylase was discovered and salivary enzymatic activity demonstrated for the first time in a blood-sucking arthropod. Full-length clones were also found coding for three proteins of unknown function matching, respectively, the N-terminal sequence of an abundant salivary protein, having similarity to the CAP superfamily of proteins and the Drosophila yellow protein. Finally, two partial sequences are reported that match possible housekeeping genes. Subtractive cloning will considerably enhance efforts to unravel the salivary pharmacopeia of blood-sucking arthropods.

Purification, cloning, and expression of an apyrase from the bed bug Cimex lectularius. A new type of nucleotide-binding enzyme.

An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying apyrase (EC 3.6.1.5) activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purified C. lectularius apyrase was an acidic protein with a pI of 5.1 and molecular mass of approximately 40 kDa that inhibited ADP-induced platelet aggregation and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectularius cDNA corresponding to the N-terminal sequence of purified apyrase produced a probe that allowed identification of a 1.3 kilobase pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product of the cDNA clone with native C. lectularius apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized both C. lectularius apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the apyrase active band from chromatofocusing gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent apyrase with a pI of 5.1 and immunoreactive material detected by the anti-apyrase serum. C. lectularius apyrase has no significant sequence similarity to any other known apyrases, but homologous sequences have been found in the genome of the nematode C. elegans and in mouse and human expressed sequence tags from fetal and tumor EST libraries.

Hemoglobin: Food for thought in vectors and parasites.

Several reports indicate that hemoglobin can serve as a source of peptides involved in regulatory functions in mammals, including humans. Here, Rosane Charlab and Elói Garcia discuss the potential role of hemoglobin-derived peptides as regulatory molecules in blood-sucking vectors and protozoan parasites.

Leishmania amazonensis: sensitivity of different promastigote morphotypes to salivary gland homogenates of the sand fly Lutzomyia longipalpis.

We have recently demonstrated that Lutzomyia longipalpis salivary gland homogenates (SGH) inhibited the multiplication of Leishmania promastigotes in vitro. The present work shows that Leishmania amazonensis sensitivity to SGH is correlated to the phase of promastigote in vitro growth and can be decreased by the addition of hemin to the culture medium. The possible relevance of these in vitro results is discussed in relation to the development of Leishmania parasites within their sand fly vectors.

Cytostatic effect of Lutzomyia longipalpis salivary gland homogenates on Leishmania parasites.

Salivary gland homogenates (SGH) of female Lutzomyia longipalpis, in concentrations as small as 0.05 pairs of glands/ml, inhibit the in vitro multiplication of promastigotes of Leishmania mexicana amazonensis. The effect seems to be cytostatic since promastigote viability 24 hr after exposure ranged from 55% to 100% in different experiments. The cells cultivated in the presence of SGH were characterized by a very slender shape, with cell bodies that were almost two times as long as controls. The promastigote growth inhibitory activity was not present in Anopheles albimanus SGH or in the gut extracts of Lu. longipalpis sand flies. Additionally, the salivary gland homogenates of Lu. longipalpis did not inhibit the growth of other cell types such as Escherichia coli or a monkey kidney cell line (LLCMK2), suggesting that the activity had a specific range of action. The SGH activity was sensitive to both trypsinization and boiling, partially resistant to heating at 56 degrees C for 30 min, and had a molecular weight of approximately 20 kD as determined by size exclusion high-performance liquid chromatography. The results suggest that vector saliva could influence the development of Leishmania parasites within the vector by inhibiting their growth and triggering them to a differentiation pathway.

Granulocyte-macrophage colony-stimulating factor increases the infectivity of Leishmania amazonensis by protecting promastigotes from heat-induced death.

We have studied the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the infectivity of promastigotes of Leishmania amazonensis, an obligate intramacrophage parasite. We measured the capacity of the promastigotes to infect macrophages after preincubation at different temperatures (28, 34, and 37 degrees C) with recombinant murine GM-CSF, as well as the effect of an anti-murine GM-CSF antibody on the in vitro and in vivo infectivity of the parasite. GM-CSF increases the capacity of the promastigotes to infect cells when preincubated at 34 and 37 degrees C, whereas the anti-GM-CSF antibody exerts the opposite effect: it decreases the internalization rate and the progression of infection in macrophage cultures and slows the growth of the lesion in infected BALB/c mice. Neither of the described effects were observed when the in vitro and in vivo infections were made with amastigotes. Promastigotes die in a time-dependent manner when incubated at temperatures higher than 28 degrees C in the absence of GM-CSF. They are protected from this heat-induced death by incubation with the recombinant hormone. Our interpretation of these data is that the increase in the infectivity of promastigotes when incubated with GM-CSF at the temperatures at which infection occurs (34 and 37 degrees C) is due to the larger number of surviving forms within the infecting population. The decrease in infectivity when they are incubated with the antibody is due to inhibition of the protection conferred by the GM-CSF produced by the macrophages during the in vitro and in vivo infections.

Granulocyte-macrophage colony-stimulating factor is a growth-factor for promastigotes of Leishmania mexicana amazonensis.

In this paper we show that murine lung conditioned medium (LCM) displays, in addition to its already described colony-stimulating activity on bone marrow cells, a potent growth-stimulating activity on promastigotes of Leishmania mexicana amazonesis. Immunoprecipitation of LCM with an antibody specific for murine granulocyte-macrophage colony stimulating factor (GM-CSF) abrogates both activities, indicating that the leishmanial growth-promoting activity is due to the presence of GM-CSF on LCM. Furthermore, recombinant GM-CSF (rGM-CSF) added to the culture medium or to the immunoprecipitated LCM is able to respectively induce or to partially recover the growth-promoting activity of the LCM. Sequential in vitro passages of the parasite induces a progressive loss of sensitivity to the growth-factor. Parasite forms recently collected from lesions are significantly more responsive to the growth-factor than forms already adapted to grow in culture. Since it has been shown that several different microorganisms display receptors for vertebrate-like hormones and that GM-CSF is able to enhance a cutaneous leishmanial lesion, our results permit us to raise the hypothesis that a direct interaction between a host-derived hormone and a pathogenic microorganism can be of importance in defining the fate of an infection. The fact that GM-CSF is produced by cells that actively participate in a leishmanial infection (T-lymphocytes and macrophages) reinforces our hypothesis.