Local delivery of interleukin 4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system which serves as a model for the human disease multiple sclerosis. We demonstrate here that encephalitogenic T cells, transduced with a retroviral gene, construct to express interleukin 4, and can delay the onset and reduce the severity of EAE when adoptively transferred to myelin basic protein-immunized mice. Thus, T lymphocytes transduced with retroviral vectors can deliver "regulatory cytokines" in a site-specific manner and may represent a viable therapeutic strategy for the treatment of autoimmune disease.

The entry of Theileria parva sporozoites into bovine lymphocytes: evidence for MHC class I involvement.

We have examined the process of Theileria parva sporozoite entry into susceptible bovine lymphocytes and have begun to identify one of the possible molecular interactions involved in the process. The entry process involves a defined series of events and we have used a number of experimental procedures in combination with a method of quantitation to examine various aspects of this process. T. parva sporozoites are nonmotile organisms and the initial sporozoite-lymphocyte interaction is a chance event which can occur at 0-2 degrees C. All subsequent stages in the process are temperature dependent, require the participation of live intact sporozoites and host cells, and involve some cytochalasin-inhibitable rearrangement of the host cell surface membrane or cytoskeleton. Sporozoite entry can be inhibited by antibodies (mAbs) reactive with major histocompatibility complex (MHC) class I molecules (IL-A 19, IL-A 88) and with beta 2 microglobulin (B1G6), whereas mAbs reactive with MHC class II molecules (IL-A 21, J 11), and a common panleucocyte surface antigen, (IL-A 87; a bovine equivalent of CD 11a) have no effect. These results indicate that MHC class I molecules play a role in the process of T. parva sporozoite entry into bovine lymphocytes although as yet the precise role has not been determined. Once internalized within the lymphocyte, a process that takes less than 3 min at 37 degrees C, the sporozoite rapidly escapes from the encapsulating host cell membrane; a process which occurs concurrently with the discharge of the contents of the sporozoite rhoptries and microspheres. The intracytoplasmic parasite is covered by a layer of sporozoite-derived fuzzy material to which host cell microtubules rapidly become associated.

Actin filament cables in Drosophila nurse cells are composed of modules that slide passively past one another during dumping.

At a late stage in Drosophila oogenesis, nurse cells rapidly expel their cytoplasm into the oocyte via intracellular bridges by a process called nurse cell dumping. Before dumping, numerous cables composed of actin filaments appear in the cytoplasm and extend inward from the plasma membrane toward the nucleus. This actin cage prevents the nucleus, which becomes highly lobed, from physically blocking the intracellular bridges during dumping. Each cable is composed of a linear series of modules composed of approximately 25 cross-linked actin filaments. Adjacent modules overlap in the cable like the units of an extension ladder. During cable formation, individual modules are nucleated from the cell surface as microvilli, released, and then cross-linked to an adjacent forming module. The filaments in all the modules in a cable are unidirectionally polarized. During dumping as the volume of the cytoplasm decreases, the nucleus to plasma membrane distance decreases, compressing the actin cables that shorten as adjacent modules slide passively past one another just as the elements of an extension ladder slide past one another for storage. In Drosophila, the modular construction of actin cytoskeletons seems to be a generalized strategy. The behavior of modular actin cytoskeletons has implications for other actin-based cytoskeletal systems, e.g., those involved in Listeria movement, in cell spreading, and in retrograde flow in growth cones and fibroblasts.

The plastid of Toxoplasma gondii is divided by association with the centrosomes.

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.

Why are two different cross-linkers necessary for actin bundle formation in vivo and what does each cross-link contribute?

In developing Drosophila bristles two species of cross-linker, the forked proteins and fascin, connect adjacent actin filaments into bundles. Bundles form in three phases: (a) tiny bundles appear; (b) these bundles aggregate into larger bundles; and (c) the filaments become maximally cross-linked by fascin. In mutants that completely lack forked, aggregation of the bundles does not occur so that the mature bundles consist of <50 filaments versus approximately 700 for wild type. If the forked concentration is genetically reduced to half the wild type, aggregation of the tiny bundles occurs but the filaments are poorly ordered albeit with small patches of fascin cross-linked filaments. In mutants containing an excess of forked, all the bundles tend to aggregate and the filaments are maximally crossbridged by fascin. Alternatively, if fascin is absent, phases 1 and 2 occur normally but the resultant bundles are twisted and the filaments within them are poorly ordered. By extracting fully elongated bristles with potassium iodide which removes fascin but leaves forked, the bundles change from being straight to twisted and the filaments within them become poorly ordered. From these observations we conclude that (a) forked is used early in development to aggregate the tiny bundles into larger bundles; and (b) forked facilitates fascin entry into the bundles to maximally cross-link the actin filaments into straight, compact, rigid bundles. Thus, forked aligns the filaments and then directs fascin binding so that inappropriate cross-linking does not occur.

Regulation of actin filament cross-linking and bundle shape in Drosophila bristles.

Previous studies demonstrate that in developing Drosophila bristles, two cross-linking proteins are required sequentially to bundle the actin filaments that support elongating bristle cells. The forked protein initiates the process and facilitates subsequent cross-linking by fascin. Using cross-linker-specific antibodies, mutants, and drugs we show that fascin and actin are present in excessive amounts throughout bundle elongation. In contrast, the forked cross-linker is limited throughout bundle formation, and accordingly, regulates bundle size and shape. We also show that regulation of cross-linking by phosphorylation can affect bundle size. Specifically, inhibition of phosphorylation by staurosporine results in a failure to form large bundles if added during bundle formation, and leads to a loss of cross-linking by fascin if added after the bundles form. Interestingly, inhibition of dephosphorylation by okadaic acid results in the separation of the actin bundles from the plasma membrane. We further show by thin section electron microscopy analysis of mutant and wild-type bristles that the amount of material that connects the actin bundles to the plasma membrane is also limited throughout bristle elongation. Therefore, overall bundle shape is determined by the number of actin filaments assembled onto the limited area provided by the connector material. We conclude that assembly of actin bundles in Drosophila bristles is controlled in part by the controlled availability of a single cross-linking protein, forked, and in part by controlled phosphorylation of cross-links and membrane actin connector proteins.

Human adenovirus Ad-36 induces adipogenesis via its E4 orf-1 gene.

OBJECTIVE: Understanding the regulation of adipocyte differentiation by cellular and extracellular factors is crucial for better management of chronic conditions such as obesity, insulin resistance and lipodystrophy. Experimental infection of rats with a human adenovirus type 36 (Ad-36) improves insulin sensitivity and promotes adipogenesis, reminiscent of the effect of thiozolinediones. Therefore, we investigated the role of Ad-36 as a novel regulator of the adipogenic process. DESIGN AND RESULTS: Even in the absence of adipogenic inducers, infection of 3T3-L1 preadipocytes and human adipose-derived stem cells (hASC) by Ad-36, but not Ad-2 that is another human adenovirus, modulated regulatory points that spanned the entire adipogenic cascade ranging from the upregulation of cAMP, phosphatidylinositol 3-kinase and p38 signaling pathways, downregulation of Wnt10b expression, and increased expression of CCAAT/enhancer binding protein-beta and peroxisome proliferator-activated receptor gamma2 and consequential lipid accumulation. Next, we identified that E4 open reading frame (orf)-1 gene of the virus is necessary and sufficient for Ad-36-induced adipogenesis. Selective knockdown of E4 orf-1 by RNAi abrogated Ad-36-induced adipogenic signaling cascade in 3T3-L1 cells and hASC. Compared to the null vector, selective expression of Ad-36 E4 orf-1 in 3T3-L1 induced adipogenesis, which was abrogated when the PDZ-binding domain of the protein was deleted. CONCLUSION: Thus, Ad-36 E4 orf-1 is a novel inducer of rodent and human adipocyte differentiation process.

Mobilization of intrasporozoite Ca2+ is essential for Theileria parva sporozoite invasion of bovine lymphocytes.

The entry of Theileria parva sporozoites into bovine lymphocytes occurs rapidly and involves a defined series of events. In the present study the role of calcium in sporozoite entry was examined. Depletion of Ca2+ from the external medium had little effect on sporozoite entry suggesting that the initial sporozoite-host cell interaction is a Ca(2+)-independent process. Sporozoite entry could, however, be inhibited by a range of Ca2+ channel blockers (verapamil, nicardipine, diltiazem) and calmodulin antagonists (TPF, chloropromazine, W7 and calmidazolium). Evidence is also presented that demonstrates that sporozoite entry is dependent on changes in sporozoite cytosolic Ca2+ caused by the release of Ca2+ from intrasporozoite stores. First, reagents that produced an influx of Ca2+ into the parasite (A23187) blocked entry. Second, depletion of intrasporozoite Ca2+ levels (10 microM A23187 + 1.0 mM EGTA) or an increase in the cytoplasmic buffering capacity of the sporozoite cytoplasm (by preloading sporozoites with MAPT/AM) inhibited invasion. Third, sporozoite entry was inhibited by TMB-8 which blocks the release of Ca2+ from intracellular stores. Lastly, treatment of sporozoites with the Ca(2+)-mobilizing agents thapsigargin, cyclopiazonic acid but not InsP3 prevented sporozoite entry. In these cases the premature release of intrasporozoite Ca2+ inhibited sporozoite binding to the host cell surface; sporozoites that bound became internalized at rates comparable to the controls. In contrast, treatment of lymphocytes with these reagents had no significant effect on sporozoite entry. Collectively these results demonstrate that the mobilization of Ca2+ from intrasporozoite stores following sporozoite binding to the host cell surface is essential for successful parasite invasion.

Localization of ribosomal RNA and Pbs21-mRNA in the sexual stages of Plasmodium berghei using electron microscope in situ hybridization.

A reproducible technique for the ultrastructural localization of RNAs in malaria parasites has been developed which combines excellent structural preservation with high hybridization signals. Signals obtained following in situ hybridization with an antisense rRNA probe which recognizes all forms of small subunit (SSU) rRNA correlate with the density of ribosomes in the parasite cytoplasm and show that a) the male gametocyte has only 12 to 25% the ribosomes found in the female cell and asexual parasite and b) the probe did not hybridize with an electron-dense nuclear body previously called a nucleolus. We suggest this structure is either a transcription-, or a replication-factory. Using a probe for the sexual stage-specific protein Pbs21 mRNA, signal was found only in female gametocytes, zygotes and ookinetes and showed a non-random, clumped cytoplasmic distribution. It is not known at present whether the non-random distribution of the Pbs 21 mRNA is critical to the very delayed translation of the Pbs21 message into protein, which occurs only in the zygote and ookinete.

Endocytosed transferrin in African trypanosomes is delivered to lysosomes and may not be recycled.

It has been shown in mammalian systems that the passage of transferrin-colloidal gold (Tf-Au) through the endocytic system is influenced by the size of the gold colloid (Neutra, M. R. et al., J. Histochem. Cytochem. 33, 1134-1144 (1985); Woods, J. W. et al., Eur. J. Cell Biol. 50, 132-143 (1989)). However, in both Trypanosoma brucei brucei and Trypanosoma congolense, widely varying sizes of Tf-Au (Tf-Au5 and Tf-Au15) have been shown to proceed to lysosomes (Webster, P., Eur. J. Cell Biol. 49, 295-302 (1989); Webster, P., D. Grab, J. Cell Biol. 106, 279-288 (1988)). Using an affinity-purified anti-bovine transferrin IgG we have demonstrated that, in both T. brucei and T. congolense, native transferrin, like Tf-Au, is found in the flagellar pocket, coated vesicles, tubular structures, and lysosome-like organelles where it appears to be concentrated. The presence of Tf in the lysosomes was confirmed in colocalization experiments using T. congolense, where native bovine transferrin colocalized with a trypanosome lysosomal marker, a cysteine protease. The data suggest that, unlike the situation in mammalian cells where most transferrin is recycled to the cell surface, in African trypanosomes transferrin is routed into lysosomes and may not, therefore, be recycled.

Directional movement of variable surface glycoprotein-antibody complexes in Trypanosoma brucei.

Binding of antibody, antibody fragments (Fab and F(ab)2) and biotin molecules to variable surface glycoprotein (VSG) of Trypanosoma brucei was studied by both light microscopy and fluorescence activated cell sorter (FACS) analysis. Antibodies, antibody fragments and biotin molecules were distributed over the entire parasite surface after incubation at 0 degree C. Upon warming to 37 degrees C, surface bound Fab and F(ab)2 fragments showed different rates of clearance from the parasite surface. Clearance, which in both cases followed double exponential decay kinetics, resulted from a directional movement of VSG-bound antibody complexes from both the surface of the flagellum and the cell body towards the cellular site of active endocytosis, the flagellar pocket (FP), even in the absence of antibody-mediated crosslinking of VSG. Immunofluorescence on trypanosomes permeabilized after binding, clearance and internalization, indicated the location of small amounts of antibody intracellularly, between the nucleus and the flagellar pocket. However, if a cocktail of protease inhibitors was added to the medium, larger amounts of internalized antibody could be detected within vacuoles situated between the nucleus and the flagellar pocket. Movement of antibody-VSG complexes was reversibly inhibited at temperatures below 37 degrees C and by increasing the NaCl concentration in the medium to 200 mM.

The transferrin receptor in African trypanosomes: identification, partial characterization and subcellular localization.

All eukaryotic cells, including African trypanosomes, require iron for growth and division, and this iron is acquired by the receptor-mediated endocytosis of iron-loaded transferrin (diFe(3+)-transferrin). In trypanosomes transferrin (Tf) has been shown to be delivered into lysosomes and may not recycle back to the cell surface as it does in mammalian cells (Grab, D. J., et al., Eur. J. Cell Biol. 59, 398-404 (1992)). Here, we describe for the first time, the characteristics of a Tf-binding protein with receptor-like properties in Trypanosoma brucei brucei. Bloodstream forms of rodent-adapted T. brucei were incubated with [35S]methionine and detergent lysates chromatographed on a Sephacryl S-300 column. Fractions were incubated with anti-Tf serum to immunoprecipitate Tf/Tf-binding protein complexes. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass of the major protein in the immunoprecipitate was 88 to 92 kDa. Tf-binding proteins could also be isolated using diferric Tf-Sepharose. The molecular mass of the major Tf-binding protein, as estimated from Sephacryl S-300 column chromatography, in the presence of detergent, was approximately 90 to 100 kDa and 90 kDa with SDS-PAGE. Each 90 kDa Tf-binding protein was able to bind one molecule of diferric Tf. Since monoclonal antibodies to human and bovine Tf receptors failed to react with any trypanosome proteins, antisera were raised against the T. brucei Tf-binding proteins eluted from Tf-Sepharose at low pH. These antibodies recognized a 90 kDa protein on Western blots of a T. brucei lysate and inhibited the growth of T. brucei in vitro. Immunolocalization studies, using this antiserum showed that the Tf-binding protein was localized in the flagellar pocket and within the early endosomal compartments. In the presence of protease inhibitors there was additional localization in lysosome-like organelles. The Tf-binding characteristics and localization of this 90 kDa protein suggest that this molecule is a strong candidate as a physiological receptor for Tf in these parasites.

DNA replication and daughter cell budding are not tightly linked in the protozoan parasite Toxoplasma gondii.

In the protozoan parasite Toxoplasma gondii, cell division occurs by an unusual internal budding process whereby two daughter cells develop within and eventually subsume the mother cell. We have examined this process using inhibitors targeted at specific events in the cell cycle. By adding inhibitors to newly established parasites we were able to examine the effects of the inhibitors on parasites treated at the start of intracellular development and many hours prior to the onset of daughter cell budding. As with other eukaryotes, inhibitors of nuclear DNA synthesis blocked parasite DNA synthesis and prevented cell division. Examination of parasites treated with the nuclear DNA synthesis inhibitor aphidicolin showed that the formation of daughter apical complexes and the initiation of budding occurred as normal and only the inability of the nucleus to become incorporated into the daughter cells prevented successful cell division. Moreover, these inhibitory effects of aphidicolin were not reversible. The initiation of nuclear DNA synthesis and cell division in newly invaded Toxoplasma required both gene transcription and protein synthesis, although inhibitors of mitochondrial DNA synthesis, transcription and protein synthesis did not block parasite division. Thus, unlike most eukaryotes, Toxoplasma tachyzoites have separated nuclear DNA replication and mitosis from the events associated with cell division (daughter cell budding). This implies that Toxoplasma tachyzoites may have dispensed with specific cell cycle checkpoints present in other eukaryotes with, in particular, a DNA-replication checkpoint control either missing, or downregulated in this stage of the parasite life cycle.

Differential expression of the oligomycin-sensitive ATPase in bloodstream forms of Trypanosoma brucei brucei.

We report the differential expression of the oligomycin-sensitive mitochondrial ATPase in pleomorphic bloodstream forms of Trypanosoma brucei brucei as observed with enzymatic assays and electron microscope histochemistry. As the cells differentiate from long slender to short stumpy forms, total specific activity of the mitochondrial ATPase in a crude mitochondrial fraction doubles and the oligomycin-sensitive specific activity increases 5-fold. Upon in vitro differentiation to procyclic forms, there is a further doubling of total specific activity and a further tripling of oligomycin-sensitive specific activity. The oligomycin-insensitive ATPase activity remained essentially constant throughout differentiation. We have attempted to characterize this oligomycin-insensitive activity utilizing inhibitors of several other ATPases.

Inhibitors of cholesterol biosynthesis. 4. trans-6-[2-(substituted-quinolinyl)ethenyl/ethyl]tetrahydro-4-hydroxy-2 H-pyran-2-ones, a novel series of HMG-CoA reductase inhibitors.

A series of substituted quinoline mevalonolactones were prepared and evaluated for their ability to inhibit the enzyme HMG-CoA reductase both in vitro and (cholesterol biosynthesis) in vivo. Since previous studies suggested that the 4-(4-fluorophenyl) and 2-(1-methylethyl) substituents afforded optimum potency, attention was focused on variations at position 6 of the quinoline ring. Biological evaluation of a small number of analogues bearing a variety of 6-substituents showed that modification at this position had little effect on potency. Several compounds (8b, 8e, and 11) were identified that showed comparable potency to compactin and mevinolin in both the in vitro and in vivo assays.

Inhibitors of cholesterol biosynthesis. 6. trans-6-[2-(2-N-heteroaryl-3,5-disubstituted- pyrazol-4-yl)ethyl/ethenyl]tetrahydro-4-hydroxy-2H-pyran-2-ones.

A series of N-heteroaryl-substituted mevalonolactones were prepared and evaluated for their ability to inhibit the enzyme HMG-CoA reductase both in vitro and in vivo, and to lower plasma cholesterol in a hypercholesterolemic dog model. The goal of the strategy employed was to design an inhibitor which possessed the pharmacological properties of lovastatin (1), and the physicochemical properties (increased hydrophilicity) of pravastatin (2). Two compounds 20a and 20b, were more potent than lovastatin at inhibiting cholesterol biosynthesis both in vitro and in vivo. In terms of plasma cholesterol lowering, 20a was much more efficacious than lovastatin. In addition to possessing increased biological activity, these compounds are significantly less lipophilic than lovastatin, in fact, 20b has a CLOGP value comparable to pravastatin.

Inhibitors of acyl-Coa:cholesterol acyltransferase. 4. A novel series of urea ACAT inhibitors as potential hypocholesterolemic agents.

We have synthesized a series of N-phenyl-N'-aralkyl and N-phenyl-N'-(1-phenylcycloalkyl)ureas as inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). This intracellular enzyme is thought to be responsible for the esterification of dietary cholesterol; hence inhibition of this enzyme could reduce diet-induced hypercholesterolemia. For this series of compounds, the in vitro ACAT inhibitory activity was improved by increasing the bulk of the 2,6-substituents on the phenyl ring. Additionally, we found that spacing of the aromatic rings was critical for ACAT inhibitory activity. A phenyl ring five atoms away from the requisite 2,6-diisopropylphenyl moiety was optimal for in vitro activity. Substitution alpha to the N'-phenyl moiety enhanced in vitro potency. In the case of phenylcycloalkyl ureas, ACAT inhibitory activity was independent of the size of the cycloalkyl ring. From this series of analogs, compound 25, which had excellent in vitro potency for inhibiting ACAT, was found to lower plasma cholesterol by 73% in vivo when administered in the diet at 50 mg/kg in an animal model of hypercholesterolemia. In this model, compound 25 lowered plasma cholesterol dose dependently and was as efficacious as the Lederle ACAT inhibitor CL 277082.

Identification and characterization of the antigen-specific subpopulation of alloreactive CD4+ T cells in vitro and in vivo.

We report the identification and characterization of the small subpopulation of alloantigen-specific T cells in vitro and in vivo. This subpopulation of T cells was distinguished by up-regulation of cell surface CD4 expression. These CD4high T cells were alloantigen specific in proliferation assays in vitro, and they expressed memory/activation markers, including CD44high and CD69high. Further studies demonstrated that these allospecific CD4high cells were also present (< or = 1% of CD4+ T cells) in vivo in BALB/c (H-2d) recipients of C57BL/6 (H-2b) skin allografts. CD4high T cells isolated from regional draining lymph nodes in these skin graft recipients reacted in a donor-specific fashion to C57BL/6 splenocyte stimulator cells in mixed lymphocyte culture. Adoptive transfer of CD4high, but not CD4normal T cells, just before skin engraftment in CD4 knockout mice, reconstituted rejection. The discovery that a small subpopulation of CD4high lymph node cells contained all of the alloantigen-specific T cells may allow study of tissue-specificity and subsequent alloantigen identification in transplantation.

A combination of adoptive transfer and antigenic challenge induces consistent murine experimental autoimmune encephalomyelitis in C57BL/6 mice and other reputed resistant strains.

Adoptive EAE was induced in SJL mice by the transfer of MBP-primed and in vitro-stimulated donor lymph node cells into naive syngeneic recipients. Priming donor mice with OVA instead of restimulating MBP-primed donor cell with OVA resulted in no transfer of EAE. This apparent lack of disease, however, could be overcome if the recipients were subsequently challenged with MBP. When this transfer-challenge technique was applied to BALB/c and C57BL/6 mice, these reputed (MBP)EAE-resistant strains developed consistent and severe disease similar to that seen in susceptible strains. In fact, a survey of eleven (MBP)EAE-resistant strains, defined on the basis of their inability to mount an encephalitogenic response in recipient mice following the transfer of MBP-primed and in vitro activated lymph node cells, revealed that EAE could be induced in all these strains. Since the surveyed strains represented a wide spectrum of genetic backgrounds as well as the common MHC congenic haplotypes (H-2b,d,k,m,r,s,v), it is concluded that the machinery for recognition of MBP, i.e. MHC genes and the appropriate T cell receptors, is functionally intact in these resistant mice. While MHC and T cell receptor genes are required for T cell responses, they are not the limiting factors that confer resistance in murine EAE.

The same but different: the biology of Theileria sporozoite entry into bovine cells.

Theileria are important tick-transmitted protozoan parasites that infect wild Bovidae and domestic animals throughout much of the world. Much of our understanding of Theileria sporozoite invasion of bovine cells is based on work on T. parva, the causative agent of East Coast fever in cattle throughout east, central and southern Africa. Sporozoite entry involves a defined series of sequential but separable steps that differ in important details from the invasion process in other apicomplexans such as Plasmodium and Toxoplasma. While the morphological features of invasion are fairly well documented, the detailed biology of the individual steps is only now becoming clear. This review summarizes much of this recent work on the biology of sporozoite entry. In particular, recent studies on the role of Ca2+ and cell activation processes in sporozoite entry suggest that the initial sporozoite binding event triggers the mobilization of intrasporozoite Ca2+ and the activation of both kinase and G-protein associated signalling processes in the parasite. These processes in turn regulate the invasive capacity of the sporozoite although the identity of these parasite molecules and how they contribute to the invasion process remain to be determined.