Effects of cholesterol content on activity of P-glycoproteins and membrane physical state, and consequences for anthelmintic resistance in the nematode Haemonchus contortus.

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MßCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.

TITLE: Effets de la teneur en cholestérol sur l'activité des glycoprotéines P et sur l'état physique de la membrane, et conséquences pour la résistance aux anthelminthiques chez le nématode Haemonchus contortus. ABSTRACT: Les membranes plasmiques des eucaryotes protègent les cellules contre les attaques chimiques. Les xénobiotiques, absorbés par diffusion passive, s'accumulent dans les membranes où ils sont capturés par des transporteurs, parmi lesquels les glycoprotéines P (Pgp). Chez les nématodes, les coques des œufs et les cuticules constituent des barrières de protection supplémentaires contre les xénobiotiques. On en sait peu sur le rôle de ces structures dans le transport des molécules chimiques. Les Pgp, membres de la famille des transporteurs ABC, sont présents dans les coques et les cuticules. Des changements dans l'activité de ces protéines ont également été mis en corrélation avec des altérations des lipides, tels que la teneur en cholestérol, des coques des œufs. Cependant, les mécanismes cellulaires sous-jacents à ces effets restent flous. Nous montrons ici que la diminution expérimentale de la teneur en cholestérol des coques des œufs d'Haemonchus contortus, avec la méthyl-beta-cyclodextrine (MßCD), entraîne une augmentation de la fluidité membranaire favorisant l'activité des Pgp et une augmentation de la résistance aux anthelminthiques. Cet effet est modulé par le degré initial de résistance aux anthelminthiques des œufs. Ces résultats suggèrent que la fluidité de la coque joue un rôle majeur dans la modulation de l'activité des Pgp. Ils confirment que l'activité des Pgp est fortement influencée par le microenvironnement local, en particulier les stérols, comme observé dans certains modèles de vertébrés. Ainsi, les barrières de coques des oeufs pourraient jouer un rôle actif dans le transport des xénobiotiques.

Macrocyclic lactones differ in interaction with recombinant P-glycoprotein 9 of the parasitic nematode Cylicocylus elongatus and ketoconazole in a yeast growth assay.

Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the presence of ivermectin, moxidectin, daunorubicin and ketoconazole but not selamectin. These results demonstrate direct effects of MLs on a recombinant nematode Pgp in an ML-specific manner.

Haemonchus contortus P-glycoproteins interact with host eosinophil granules: a novel insight into the role of ABC transporters in host-parasite interaction.

Eosinophils are one of the major mammalian effector cells encountered by helminths during infection. In the present study, we investigated the effects of eosinophil granule exposure on the sheep parasitic nematode Haemonchus contortus as a model. H. contortus eggs exposed to eosinophil granule products showed increased rhodamine 123 efflux and this effect was not due to loss of egg integrity. Rh123 is known to be a specific P-glycoprotein (Pgp) substrate and led to the hypothesis that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as Pgp may also be involved in the detoxification of host cytotoxic products. We showed by quantitative RT-PCR that, among nine different H. contortus Pgp genes, Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitic life stages suggesting a potential involvement of these Pgps in the detoxification of eosinophil granule products. Using exsheathed L3 larvae that mimic the first life stage in contact with the host, we demonstrated that eosinophil granules induced a dose dependent overexpression of Hco-pgp-3 and the closely related Hco-pgp-16. Taken together, our results provide the first evidence that a subset of helminth Pgps interact with, and could be involved in the detoxification of, host products. This opens the way for further studies aiming to explore the role of helminth Pgps in the host-parasite interaction, including evasion of the host immune response.

Plasticity of migrating CD1b+ and CD1b- lymph dendritic cells in the promotion of Th1, Th2 and Th17 in response to Salmonella and helminth secretions.

Dendritic cells (DCs) are pivotal in the development of specific T-cell responses to control pathogens, as they govern both the initiation and the polarization of adaptive immunity. To investigate the capacities of migrating DCs to respond to pathogens, we used physiologically generated lymph DCs (L-DCs). The flexible polarization of L-DCs was analysed in response to Salmonella or helminth secretions known to induce different T cell responses. Mature conventional CD1b(+) L-DCs showed a predisposition to promote pro-inflammatory (IL-6), pro-Th1 (IL-12p40) and anti-inflammatory (IL-10) responses which were amplified by Salmonella, and limited to only IL-6 induction by helminth secretions. The other major population of L-DCs did not express the CD1b molecule and displayed phenotypic features of immaturity compared to CD1b(+) L-DCs. Salmonella infection reduced the constitutive expression of TNF-α and IL-4 mRNA in CD1b(-) L-DCs, whereas this expression was not affected by helminth secretions. The cytokine response of T cells promoted by L-DCs was analysed in T cell subsets after co-culture with Salmonella or helminth secretion-driven CD1b(+) or CD1b(-) L-DCs. T cells preferentially expressed the IL-17 gene, and to a lesser extent the IFN-γ and IL-10 genes, in response to Salmonella-driven CD1b(+) L-DCs, whereas a preferential IL-10, IFN-γ and IL-17 gene expression was observed in response to Salmonella-driven CD1b(-) L-DCs. In contrast, a predominant IL-4 and IL-13 gene expression by CD4(+) and CD8(+) T cells was observed after stimulation of CD1b(+) and CD1b(-) L-DCs with helminth secretions. These results show that mature conventional CD1b(+) L-DCs maintain a flexible capacity to respond differently to pathogens, that the predisposition of CD1b(-) L-DCs to promote a Th2 response can be oriented towards other Th responses, and finally that the modulation of migrating L-DCs responses is controlled more by the pathogen encountered than the L-DC subsets.

Potential contribution of P-glycoproteins to macrocyclic lactone resistance in the cattle parasitic nematode Cooperia oncophora.

Resistance against macrocyclic lactones such as ivermectin is widespread among parasitic gastrointestinal nematodes of small ruminants and is rapidly increasing in cattle parasites. ABC transporters of the subfamily B, the so-called P-glycoproteins (Pgps) have been frequently implicated in ivermectin resistance and are a major cause of multi-drug resistance in protozoa and helminths. The Pgp inhibitor verapamil (VPL) dramatically enhanced susceptibility of the cattle parasitic nematode Cooperia oncophora to ivermectin in vitro as measured in a larval developmental assay and a larval migration inhibition assay using third stage larvae. Moreover, VPL completely restored susceptibility to ivermectin in a resistant isolate resulting in virtually identical dose-response curves of susceptible and resistant isolates in the presence of VPL. Further characterisation of the molecular mechanisms resulting in Pgp-mediated ivermectin resistance is still hampered by the lack of molecular and biochemical information for Pgps of parasitic nematodes. Using PCR with degenerate primers, fragments of four different C. oncophora Pgps could be amplified and the Conpgp-2, previously implicated in macrocyclic lactone resistance in Haemonchus contortus, and Conpgp-3 full-length cDNAs were obtained by RACE PCR. The pgp sequences presented here contribute important data required to systematically screen resistant C. oncophora isolates for up- or down-regulation of Pgps and for the detection of single nucleotide polymorphisms in Pgps to detect selection of specific Pgp alleles by anthelmintics as early as possible.

Membrane lipidomics for the discovery of new antiparasitic drug targets.

Advances in lipid separation methods and mass spectrometry technologies allow the fine characterization of the lipidome of parasites, ranging from unicellular protists to worms, which cause threatening infections in vertebrates, including humans. Specific lipid structures or lipid metabolic pathways can inspire the development of novel antiparasitic drugs. Changes in the lipid balance in membranes of parasites can also provide clues on the dynamics of drugs and some mechanisms of drug resistance. This review highlights recent trends in parasite lipidomics, combined with functional analyses, for the discovery of novel targets and the development of novel drugs.

Expression of Toll-like receptor 4 and downstream effectors in selected cecal cell subpopulations of chicks resistant or susceptible to Salmonella carrier state.

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1ß (IL-1ß), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1ß, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ.

Anthelmintics are substrates and activators of nematode P glycoprotein.

P glycoproteins (Pgp), members of the ABC transporter superfamily, play a major role in chemoresistance. In nematodes, Pgp are responsible for resistance to anthelmintics, suggesting that they are Pgp substrates, as they are in mammalian cells. However, their binding to nematode Pgp and the functional consequences of this interaction have not been investigated. Our study showed that levamisole and most of the macrocyclic lactones (MLs) are Pgp substrates in nematodes. Ivermectin, although a very good substrate in mammalian cells, is poorly transported. In contrast to their inhibitory effect on mammalian Pgp, these drugs had a stimulatory effect on the transport activity of the reference Pgp substrate rhodamine 123 (R123) in the nematode. This may be due to a specific sequence of nematode Pgp, which shares only 44% identity with mammalian Pgp. Other factors, such as the affinity of anthelmintics for Pgp and their concentration in the Pgp microenvironment, could also differ in nematodes, as suggested by the specific relationship observed between the octanol-water partition coefficient (log P) of MLs and R123 efflux. Nevertheless, some similarities were also observed in the functional activities of the mammalian and nematode Pgp. As in mammalian cells, substrates known to bind the H site (Hoechst 33342 and colchicine) activated the R site, resulting in an increased R123 efflux. Our findings thus show that ML anthelmintics, which inhibit Pgp-mediated efflux in mammals, activate transport activity in nematodes and suggest that several substituents in the ML structure are involved in modulating the stimulatory effect.

Drug resistance is affected by colocalization of P-glycoproteins in raft-like structures unexpected in eggshells of the nematode Haemonchus contortus.

In nematodes as in other eukaryotes, there is increasing evidence that drug resistance depends on both changes in the drug cellular targets and in nonspecific mechanisms, involving cellular detoxification by efflux pumps. In vertebrates, P-glycoproteins (Pgp) are membrane efflux pumps responsible for the elimination of xenobiotic agents, especially drugs. We previously reported the presence of Pgp pumps in eggshells and cuticles of the nematode Haemonchus contortus. Eggshells and cuticles are different from cell membranes, in particular they include a chitin layer. Nevertheless these structures present some common biological features with cell membranes and play a role in xenobiotic transport. Pgp activity has been shown to depend on the lipid environment and, in particular, on the cholesterol content in both vertebrate and nematode models. In vertebrates, Pgp is in part located in membrane cholesterol-enriched microdomains, the rafts. We describe here, for the first time, lipid microdomains in eggshells that could correspond with raft-like structures (RLSs). Moreover, a large proportion of the Pgp was shown to colocalize with these RLSs. The functional consequences of the colocalization for xenobiotic transport and thus drug resistance in nematodes were analyzed and compared with results obtained in vertebrates. An understanding of such mechanisms is crucial in overcoming the failure of drug treatments due to the development of resistance.

Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes.

The ability to reliably detect anthelmintic resistance is a crucial part of resistance management. If data between countries are to be compared, the same test should give the same results in each laboratory. As the egg hatch test for benzimidazole resistance is used for both research and surveys, the ability of different laboratories to obtain similar results was studied through testing of known isolates of cyathostomins, Haemonchus contortus, Ostertagia ostertagi, and Cooperia oncophora in programs supported by the EU (Cost B16 and FP6-PARASOL). Initial results showed difficulties in obtaining reproducible and similar data within and between laboratories. A series of ring tests, i.e., simultaneous and coordinated rounds of testing of nematode isolates in different laboratories was subsequently performed. By adopting identical protocols, especially the use of deionized water and making dilutions of thiabendazole in dimethyl sulfoxide in the final ring test, laboratories correctly identified both susceptible and resistant isolates. The protocols for the test and preparation of solutions of thiabendazole are described.

CD40 engagement strongly induces CD25 expression on porcine dendritic cells and polarizes the T cell immune response toward Th1.

Orientation of the immune response toward Th1, Th2, Th17 or Treg plays an important role in self-tolerance and defence against pathogens and tumors. However, this orientation has not been fully characterised in the pig and little is known about the influence of maturation stimulus on the capacity of dendritic cells selectively to direct different types of Th cell responses. Dendritic cell (DC) maturation can be induced by different agents such as inflammatory cytokines, TLR ligands and CD40L. However, the role of the latter in the maturation of pig DC has never been reported. In this study we analysed how different maturation agents influence the capacity of DC to skew the immune response. Monocyte-derived porcine DCs were matured with human CD40L-transfected L-cells, Lipopolysaccharide (LPS) alone or LPS in combination with Tumor necrosis factor-alpha (TNFalpha) and interferon-alpha (IFNalpha). We found that human CD40L induced DC maturation characterised by increased expression of co-stimulatory CD80/86 molecules, high production of IL-12p40 in DC and induction of IFNgamma and t-bet mRNA in T cells, suggesting a Th1 orientation. Moreover we report for the first time the appearance of CD25 after activation of porcine DC. Furthermore, DC activated with TNF+LPS+IFN showed the highest allo-stimulatory capacity of allogeneic lymphocytes and induced IL-17 mRNA in T lymphocytes, suggesting a Th17 orientation that has never been previously reported in the pig. We also showed that immature DCs did not produce any IL-10 or IL-12 and induced both GATA-3 and IL-13 transcription in allogeneic MLR suggesting a Th2 orientation. This study therefore underlines that the nature of the stimulus strongly influences the capacity of DC to steer the immune response in the pig.

Evidence that the choroids plexus in female sheep express P-glycoprotein.

OBJECTIVES: Previous studies in the female sheep have shown that photoperiod modulates the passage of sex steroids between the blood and the cerebrospinal fluid (CSF) and have suggested the involvement of efflux transport. The objective of the present work was to assess the presence of P-glycoprotein (Pgp), which could be responsible for this transport at the level of the choroid plexuses (CP). METHODS: We used flow cytometry and the UIC2 monoclonal antibody against Pgp, to demonstrate the presence of Pgp in the epithelial cell fraction isolated from the sheep choroid plexuses (CPEC). Thanks to the size of the brain structures in sheep, we analyse separately the CP from 4th ventricle (4V) and the CP from the lateral ventricle (LV). RESULTS: In the whole population isolated from the CP, the 4V contained a higher percentage of living, epithelial cells than the LV. The immunoreactive cells to the UIC2 antibody i.e. bearing activated form of Pgp, represented 26.8% of the CPEC in the 4V, and 39.3% in the LV (P<0.05). CONCLUSION: Pgp is expressed in the CPEC from the sheep. Differences in the expression of Pgp between CPEC from LV and 4V might suggest in vivo functional differences between LV and 4V CP in sheep.

Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells.

We have studied the plasma membrane protein phenotype of human culture-amplified and native bone marrow mesenchymal stem cells (BM MSCs). We have found, using microarrays and flow cytometry, that cultured cells express specifically 113 transcripts and 17 proteins that were not detected in hematopoietic cells. These antigens define a lineage-homogenous cell population of mesenchymal cells, clearly distinct from the hematopoietic lineages, and distinguishable from other cultured skeletal mesenchymal cells (periosteal cells and synovial fibroblasts). Among the specific membrane proteins present on cultured MSCs, 9 allowed the isolation from BM mononuclear cells of a minute population of native MSCs. The enrichment in colony-forming units-fibroblasts was low for CD49b, CD90, and CD105, but high for CD73, CD130, CD146, CD200, and integrin alphaV/beta5. In addition, the expression of CD73, CD146, and CD200 was down-regulated in differentiated cells. The new marker CD200, because of its specificity and immunomodulatory properties, deserves further in-depth studies.

Relationships between sterol/phospholipid composition and xenobiotic transport in nematodes.

Therapeutic failure limits prophylaxis of nematode diseases and has been mainly attributed to mutations in cellular targets of anthelmintics. Besides these specific mechanisms, alterations of drug transport also occur in parasites resistant to anthelmintics and depend on both the presence of membrane pumps such as P-glycoprotein (Pgp) and on the lipid composition of membranes. We recently showed in the nematode Haemonchus contortus, using eggs as a model, that the total cholesterol (TC) concentration alters the transport of lipophilic molecules due to membrane pumps such as P-glycoprotein and the resistance to anthelmintics. The effect of TC may depend on the presence of other lipids interacting with TC. Therefore, we analysed the lipid composition and its relationship with Pgp and resistance to anthelmintics. Better correlations were found between Pgp and free cholesterol (FC) than with TC. We also showed that the relationships between lipid composition and resistance to anthelmintics or Pgp depended on the equilibrium between FC and phospholipids (PLs), mainly PLs known to be present primarily in either the external leaflets of cell membranes or the internal leaflets. The PLs phosphatidylcholine and phosphatidylethanolamine played the most significant role, but phosphatidic acid also influenced drug resistance.

Anthelmintic resistance: the state of play revisited.

Helminthosis is one of the major constraints in the successful wool and mutton industry throughout the world. Anthelmintic Resistance (AR) is said to have been established when previously effective drug ceases to kill exposed parasitic population at the therapeutically recommended dosages. Anthelmintic resistance is almost cosmopolitan in distribution and it has been reported in almost all species of domestic animals and even in some parasites of human beings. Some of the most important species of parasites of small ruminants in which AR has been reported include: Haemonchus spp., Trichostrongylus spp. Teladorsagia spp., Cooperia spp. Nematodirus spp., and Oesophagostomum spp. All the major groups of anthelmintics have been reported for development of variable degrees of resistance in different species of gastrointestinal nematodes. This paper describes the global scenario of prevalence and methods used for detection of AR in small ruminants. Different mechanisms and contributory factors for the development of AR are discussed. Various options and alternate strategies for the control and/or delay in the onset of AR are suggested in the light of available information.

Immunolocalisation of an ABC transporter, P-glycoprotein, in the eggshells and cuticles of free-living and parasitic stages of Haemonchus contortus.

Recent data have suggested that P-glycoprotein (Pgp), working as membrane efflux "pumps", plays a major role in the transport of anthelmintic drugs in parasitic nematodes of ruminants. Flow cytometry analyses has shown that active Pgp is probably present in the external layers of Haemonchus contortus eggshells, following staining with the mouse monoclonal anti-human MDR1 antibody UIC2, which binds to Pgp in its active conformation. We evaluated the presence and distribution of this protein in the envelopes (eggshells and cuticles) of H. contortus and compared the various stages (eggs, L1-L2 larvae, L3 larvae, adult male and female worms). Electrophoresis revealed a 170-kDa band, corresponding to the molecular weight of Pgp in all stages. Indirect immunofluorescence staining with UIC2 showed Pgp to be located in the external layer of eggshells or cuticles. Transmission electron microscopy was used to localise Pgp more accurately in the three layers of the eggshells and cuticles. The conformation and biological functions of this protein, which we did not expect to find in such structures, remain to be determined.

Increased resistance to anthelmintics of Haemonchus contortus eggs associated with changes in membrane fluidity of eggshells during embryonation.

The embryonation of nematode eggs has been shown to increase their resistance to anthelmintics when parasites are submitted to egg hatch assays. Nevertheless, no mechanism has been suggested to explain this phenomenon. Earlier observations by other authors showed that the biochemical composition of eggshells is altered during the embryonation of eggs. The functional consequences of these changes have not been identified. We studied the changes in membrane environment (eggshells) of Haemonchus contortus eggs during the embryonation by fluidity measurements and their effects on nonspecific mechanisms of resistance to anthelmintics. We previously demonstrated that these mechanisms imply P-glycoproteins (Pgp) belonging to the multi-drug resistance (MDR) system and that the Pgp activity is very susceptible to their lipidic environment. The results obtained here show that the embryonation induced a significant and gradual increase in eggshell fluidity which was associated with an increased resistance to anthelmintics. Differences were observed between H. contortus isolates with various levels of resistance which might result from their specific biology and/or membrane composition. The membrane environment changes could act both on the solubilization of anthelmintics into the eggs and on the efflux of these lipophilic molecules by Pgp.

P-glycoprotein in helminths: function and perspectives for anthelmintic treatment and reversal of resistance.

Infestation with parasitic helminths is a common problem in human populations of third world countries and is ubiquitous in livestock and other domestic animals. The cell-membrane efflux pump, P-glycoprotein (Pgp), appears to contribute to anthelmintic resistance. Pgp have been identified from both phyla of parasitic helminths, Platyhelmintha and Nematoda, and alterations in expression levels and allele frequencies of Pgp in anthelmintic-resistant populations have been observed in nematodes. Localisation of Pgp has been studied in the free-living nematode Caenorhabditis elegans and in the sheep parasite Haemonchus contortus using specific monoclonal antibodies or lectins. Reversing agents used in human studies, such as the calcium-channel blocker verapamil (VPL), appear to have similar effects in helminths as they do in human cancer cells: the efficacy of drug treatment is increased in drug-resistant parasites when reversing agents are co-administered with the anthelmintic. The functional role of the Pgp glycosylation was also studied using a lectin specific for the alpha-mannosyl residues and showed that resistance can be associated with a decreased affinity of the lectin for Pgp sites and that up to 50% reversion in the resistance to benzimidazoles (BZ) can be obtained using this lectin. Furthermore, the current knowledge on the role of Pgp in molecular mechanisms of drug resistance in the parasitic protozoan genus Trypanosoma is discussed. In some Trypanosoma species it was shown that drug resistance was associated with reduced uptake and in other ones with increased efflux. Several trypanosome Pgp-coding sequences have been described. In contrast to earlier data, most recent observations, based on experimentally overexpressed Pgp in Trypanosoma brucei, indicate a possible involvement in the mechanism of drug resistance in this parasite.

Modulation of the multidrug resistance (MDR) system in the nematode Haemonchus contortus by changing cholesterol content: effects on resistance to anthelmintics.

OBJECTIVES: The efficiency of the anthelmintics used to treat small domestic ruminants infected with nematodes is compromised by the emergence of resistant parasites. Both specific and non-specific mechanisms of resistance exist. The non-specific mechanisms involve multiple resistance phenomena and are dependent on the multidrug resistance (MDR) system, which is also responsible for the development of chemotherapy-resistant tumour cells. We showed previously that the system also exists in nematodes. Membrane 'pumps', known as P-glycoproteins (Pgp), are activated in the MDR system. The nature of the membrane, in particular the lipids, appears to condition the activity of the pumps. Thus, we studied the effects of cholesterol on drug transport activity in the nematode Haemonchus contortus. MATERIALS AND METHODS: We used methyl-beta-cyclodextrin to carry out cholesterol depletion and cholesterol loading experiments. The resulting changes in resistance were estimated by measuring changes in drug transport (a) by means of in vitro egg hatch assays in the presence of a benzimidazole anthelmintic, thiabendazole and (b) by measuring the transport of rhodamine 123 (R123), a specific substrate of Pgp. We used biochemical assays to estimate the cholesterol concentration in the parasites. RESULTS: Changes in the cholesterol content induced changes in anthelmintic resistance; cholesterol depletion gave increased resistance and cholesterol loading gave decreased resistance. These changes also altered the transport of R123. CONCLUSION: Cholesterol depletion or cholesterol loading allow modulation of xenobiotic resistance in nematode eggs as they do in tumour cells. The effect appears to be correlated with changes in the function of membrane P-glycoproteins. The lipid environment thus influences the nematode Pgp activity.

Development of the meiotic step in testes of pubertal rats: comparison between the in vivo situation and under in vitro conditions.

The present work aimed to compare some features of the meiotic process which develops in the testis of pubertal rats, in vivo and in vitro, paying special attention to the time-course of the phenomenon. The differentiation of spermatocytes was assessed in testes of 20- to 46-day-old rats and in tubule segments of 20- or 28-day-old rats cultured over a 4-week period. Very similar results were obtained in vivo and in vitro, during the first week of culture, when considering the changes in the cell populations of different ploidy, the gene expression of germ cells, the kinetics of differentiation of BrdU-labeled early or middle pachytene spermatocytes and the levels of apoptosis in the different cell populations. However, during the second week of culture, the decrease in the proportion of the 4C cell population which was only slightly more marked than that observed in vivo between 27 and 34 days, was not associated with an increase in the 1C cell population as large as in vivo. This result could be explained partly by a high proportion of apoptotic 1C cells beyond one week of culture. Concomitantly, the rate of in vitro differentiation of BrdU-labeled spermatocytes slowed down when reaching the stage of middle pachytene spermatocytes and BrdU-labeled round spermatids were observed 6-11 days later than when BrdU-labeled spermatocytes differentiated in vivo. Taken together, our results indicate that the bottleneck for the development of the meiotic cells in vitro is at the transition from middle to late pachytene spermatocytes. Hence, comparing the expression of locally produced regulatory molecules in vivo and in vitro at different days of culture should allow to identify key regulators of the meiotic step of spermatogenesis.