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Eleven virus isolations were made from the blood of 45 free living healthy African buffaloes by long term cocultivation of their leucocytes with bovine thymus or spleen cells. The isolates were indistinguishable from each other or from herpesviruses isolated from a severely ill buffalo calf and from a dead buffalo. These viruses possessed the characteristics of the bovine herpesvirus-3 (BHV-3) group and were indistinguishable by serology and restriction endonuclease analysis from the BHV-3 type strains Movar 33/63 and DN599. There was a 93.6 per cent prevalence of indirect immunofluorescent antibody to BHV-3 in the sera of 94 buffaloes in the sample population. No clinical signs or viraemia were detected in five cattle inoculated with 10(8.7) log10 TCID50 of the isolate from the sick buffalo calf. Two of three cattle hyperimmunised with this virus resisted challenge with malignant catarrhal fever herpesvirus, which proved fatal for the other immunised animal and for three control cattle.

In the mouse, potent primary in vitro proliferation of T cells can be induced by allelic variants of cell-surface glycoproteins, Ia antigens, the genes for which are located in the I region of the major histocompatibility complex (MHC) on chromosome 17. The only other potent primary proliferative response known is induced by mixing MHC-identical lymphocytes from strains that differ at the locus termed Mls (ref. 1) (for mixed lymphocyte stimulating), which has been mapped to chromosome 1. While it is relatively easy to raise antibodies against Ia antigens, and thus determine both their chemical nature and their role in T-cell stimulation, the nature of the product of the Mls locus has remained obscure. It has been proposed that the Mls locus product is a minor antigen recognized in association with self-Ia antigens2,3, a translocated Ia-like element4, or a mitogenic molecule found on the surface of antigen presenting cells (APC)5. Here, we demonstrate that APCs from mice carrying stimulatory Mls locus alleles present antigen more efficiently to cloned, antigen-specific, Ia-restricted T cells than APCs from mice carrying nonstimulating Mls locus alleles. We propose that the Mls locus does not encode a unique cell-surface antigen at all; we suggest instead that the T-cell proliferative response induced by Mls-locus disparate cells reflects recognition of self-Ia molecules on APCs. If our interpretation is correct, it provides further evidence both for the quantitative nature of self tolerance and for the existence of a distinct recognition site for self-Ia molecules on antigen-specific T lymphocytes.

Lymphoid cells collected from the peripheral blood of 45 free-ranging buffaloes sampled in the Masai Mara Game Reserve in Kenya were cultured in vitro in an attempt to establish cell lines of intralymphocytic theilerial schizonts. Theileria parva lawrencei-infected lymphoblastoid cell lines were established with samples taken from 12 buffaloes, and 11 of these were maintained continuously in vitro. Sixteen of the buffalo samples were contaminated with either trypanosomes or viruses. The successful in vitro isolation of Theileria species from 27 per cent of the buffaloes sampled demonstrates the applicability of this technique for field isolation of T parva lawrencei.

This study compared infection and transformation of peripheral blood mononuclear cells (PBM) of Boran cattle and African buffalo in vitro to determine whether differences occurred which could account for the greater susceptibility of Boran cattle to infection with Theileria parva subsp. parva and T. parva subsp. lawrencei. PBM from buffalo and cattle had a similar percentage of cells which bound T. parva subsp. parva sporozoites (24 to 34%) and in which schizonts developed during the first week after infection (18 to 23%). Using a limiting dilution culture system, it was established, however, that a significantly higher proportion of cattle PBM transformed into continuously replicating cell lines after infection with T. parva subsp. parva than did buffalo PBM. The evidence suggests that the low capacity of T. parva subsp. parva to establish infections in buffalo compared with cattle is related to the lower frequency of buffalo cells which undergo transformation. With T. parva subsp. lawrencei, however, the frequency of transformation of buffalo PBM was higher than that for cattle PBM. The frequency of cells transformed by T. parva subsp. lawrencei, therefore, cannot account for the greater resistance of buffalo to infections with T. parva subsp. lawrencei. Buffalo must have other mechanisms, either innate or acquired, which control infection with T. parva subsp. lawrencei more efficiently than in cattle.

Intraerythrocytic multiplication of Theileria parva in vitro : an ultrastructural study. International Journal for Parasitology 16 : 223–229. Piroplasms of a Theileria parva isolate from Muguga, Kenya were maintained in short-term stationary erythrocyte cultures and the mode of multiplication studied by light and electron microscopy. Division of single piroplasms into four intraerythrocytic merozoites was observed within the first 24 h in vitro . After 8 days of cultivation, 10–20% of the parasitised erythrocytes contained quadruplet merozoite forms resulting from multiplication in vitro . Electronmicrographs showed that intraerythrocytic multiplication occurred by a schizogonous process that proceeded from the initial formation of rhoptries and electron-dense cytoplasmic plaques beneath the parasite's plasmalemmal membrane, to nuclear division and the final separation of a maximum of four differentiated merozoites. The merozoites formed by intra-erythrocytic schizogony in vitro had ultrastructural features identical to merozoites produced by intra-lymphocytic schizonts of T. parva .

The intraerythrocytic multiplication of two strains of Theileria annulata was studied with parasites maintained in stationary cultures and in the blood of infected cattle. In cultures established with blood from infected cattle 20–60% of single T. annulata piroplasms divided into quadruplet forms by day 6 in vitro. Transmission electron microscopic studies of T. annulata in culture showed that piroplasms possess intracytoplasmic food vacuoles and cytostomes during a pre-division trophozoite stage. The onset of intraerythrocytic multi plication was marked by the appearance of rhoptries and electron-dense plaques beneath the parasite's plasmalemmal membrane. The plaques developed into short segments of subplasmalemmal double membranes which were closely associated with the rhoptries. It was concluded that multiplication of T. annulata in erythrocytes occurred by schizogony, as nuclear division preceded cytoplasmic division and the final separation of merozoites. The four merozoites produced by intraerythrocytic schizogony had the same ultrastructural features as the T. annulata merozoites produced by intralymphocytic schizogony. Clusters of four merozoites, identical to those observed in stationary cultures, were also seen in the erythrocytes of persistently infected cattle and appeared to represent the most significant dividing forms of T. annulata in vivo.

Uptake of tritiated nucleic acid precursors by Babesia bovis in vitro. International Journal for Parasitology 16 : 263–268. The uptake of tritiated nucleic aid precursors in vitro by two Babesia bovis isolates, from Mexico and South Africa, was compared using a semiautomated microdilution method. The purine bases, adenine and hypoxanthine, and the purine nucleosides, adenosine and guanosine, were readily taken up into infected erythrocytes in cultures of both B. bovis isolates over two consecutive 24 h incubation periods. The uptake of adenosine and hypoxanthine was consistently greater than that of the other precursors. Tritiated uridine and cytidine appeared to be taken up into B. bovis infected erythrocytes, but thymidine was not. The ability to establish continuous cultures of B. bovis with blood from infected cattle which have less than a 1% parasitaemia, and to maintain dividing parasites that incorporate tritiated nucleotide precursors during incubation periods of 24–48 h, indicates that the methodology described could be utilized in immunological and biochemical studies on B. bovis , or to evaluate the babesicidal effect of chemotherapeutic agents.

A transmission electron microscopic study of the intra-erythrocytic stages of a pathogenic Theileria parva from cattle and a previously uncharacterized Theileria sp. from waterbuck (Kobus defassa) in Kenya revealed several novel ultrastructural features, associated with feeding and multiplication, in these parasites. In trophozoites a connecting channel was observed between the parasite's cytostome and its intracytoplasmic food vacuole. In some cases the limiting membrane of the food vacuole was seen to be continuous with a close-meshed network of membrane-bounded, anastomosing tubules. This labyrinthine structure, which has not been described previously, may function as a digestive organelle in theilerial trophozoites. Electron micrographs also revealed the mode of intra-erythrocytic multiplication of these parasites in vivo. Prior to division, electron-dense cisternae and rhoptries appeared beneath the parasite's plasmalemmal membrane, marking the sites of merozoite formation. From a single parasite, a maximum of four merozoites were formed by schizogonous division and subsequently separated from a residual body by constriction at the base of each merozoite. In addition, observations on two double-membraned organelles seen in trophozoites and the intra-erythrocytic crystalline structures associated with Theileria sp. in waterbuck are reported.

SUMMARY The response to a novel set of T cell mitogens has been analysed and compared to the response to Mls locus differences. These polyclonal T cell activators, staphylococcal enterotoxins A and B, stimulate T cells in a way that requires an antigen-presenting cell bearing class II MHC products and involves the CD4:T cell receptor complex. However, the specificity of MHC recognition by the T cell receptor is lost in this response. Thus, these mitogens produce a response with characteristics similar to that induced by Mls locus differences. These mitogens can be used to analyse the immunobiology of this response, and may help in understanding and identifying the Mls locus product as well.

Protein and glycoprotein changes induced in bovine lymphoblasts by infection with Theileria parva were analyzed by high-resolution two-dimensional gel electrophoresis. Uninfected and infected cloned bovine T and B lymphoblasts were biosynthetically labeled with [35S]methionine and their two-dimensional autoradiographic patterns were compared with each other and with the pattern obtained using purified labeled schizonts. Ten proteins were found in infected cells which were not present in uninfected cells, and seven of these were detected in preparations of purified schizonts. Four glycoproteins were detected on the surface of infected cells labeled with [3H]borohydride while a major glycoprotein present on uninfected cells disappeared or was reduced in infected cells. Other minor changes in protein and glycoprotein patterns were also observed.

The expression of polymorphic determinants on I-E molecules is largely dependent on allelic variation in the E beta chain. We have previously analyzed the expression of Ek beta and Eb beta chains in F1 hybrid mice by a combination of techniques, and have shown that functional variation detected by the responsiveness of cloned T-cell lines specific for these molecules correlates well with serological determination of E beta expression. In the present study, we have extended our analysis to Ed beta expression in F1 hybrid mice. We show that Ed beta is relatively poorly expressed in three F1 combinations: H-2d X H-2b, H-2d X H-2s, and H-2d X H-2u. The former two crosses express E alpha chains from the H-2d parent only; when recombinant strains carrying Eb beta or Es beta and an active E alpha gene are used, Ed beta expression is significantly increased. On the other hand, H-2u mice synthesize E alpha chains; the poor expression of Ed beta chains in this F1 hybrid apparently reflects the strong preferential association of Eu beta chains with all E alpha molecules thus far analyzed. These results confirm that E beta chains compete for binding to E alpha chains and that preferential association of different allelic forms of E beta chains with E alpha chains is a generalized phenomenon. They also illustrate the importance of the rate of biosynthesis of Ia chains for cell-surface expression.

The growth of T lymphocytes is dependent on the T-cell growth factor interleukin 2 (IL-2), which causes T cells bearing high-affinity receptors for IL-2 to proliferate. Most cloned helper-T-cell lines can be shown to both produce and respond to IL-2; thus, growth of such cells is by an autocrine mechanism. We report that the failure of the cloned murine T-cell line D10.G4.1 to respond to its own IL-2 results from the secretion, by the same cells, of a potent inhibitor of the IL-2-driven T-cell proliferative response. This inhibition can be overcome by increasing the number of IL-2 receptors expressed by the target cell. In the cloned T-cell line producing the inhibitory substance, this increase in IL-2 receptors is driven by the monokine interleukin-1. We propose that this inhibitor of IL-2 responses may play a role in preventing "bystander" activation of T cells by IL-2 released in vivo and could be a potent pharmacologic agent.

SUMMARYThe characteristics of intra-lymphocytic Theileria isolated from African buffalo and from cattle that were infected with buffalo-derived parasites were evaluated using anti-schizont monoclonal antibodies (mAbs) and DNA probes. Antigenic differences were revealed by the reactivities of 27 mAbs with the buffalo-derived parasites isolated from different animals. Antigenic diversity was also seen with Theileria-infected lymphoblastoid cell isolates taken from the lymph nodes and blood of the same animals. Two DNA probes, selected from a genomic library of T. parva piroplasm DNA cloned in λgt11, showed specific hybridization to parasite DNA in Southern blots of restriction enzyme-digested, lymphoblastoid cells infected with buffalo-derived theilerial parasites. Genotypic differences between the buffalo-derived parasites were revealed by the restriction fragment length polymorphisms seen with hybridization of those probes to DNA from cloned and uncloned Theileria-infected cell lines. The evaluation of theilerial parasites derived from buffalo and from cattle which underwent typical T. p. lawrencei reactions, after being infected with buffalo-derived theilerial parasites, did not show any specific phenotypic or genotypic characteristics of these parasites that would distinguish them from T. p. parva and T. p. bovis parasites. The validity of these subspecies distinctions is discussed.

Different stocks of Theileria parva were analysed for restriction fragment length polymorphisms by agarose gel electrophoresis, orthogonal-field-alternation gel electrophoresis (OFAGE) and Southern hybridization with DNA probes. Polymorphisms seen with DNA from purified piroplasms of different T. parva stocks, after digestion with restriction enzymes, were more clearly apparent with OFAGE than with standard agarose gel electrophoresis. Genomic differences between these theilerial parasites were investigated further using three DNA probes, which were selected from a genomic library of T. parva (Muguga) piroplasm DNA cloned in λgt11. All three clones hybridized to T. parva DNA in preparations from schizont-infected bovine lymphoblastoid cells and to DNA from intraerythrocytic piroplasms. These probes did not, however, hybridize under high stringency conditions to DNA prepared from uninfected bovine lymphoblasts, T. mutans piroplasms, or bovine lymphoblasts infected with T. annulata or T. taurotragi . The five Kenyan stocks of T. parva that were tested showed characteristic hybridization patterns with these DNA probes. Our results show that DNA probes can be used to distinguish selected stocks of T. parva by hybridization to DNA either from intraerythrocytic piroplasms taken from infected cattle, or from isolates of schizont-infected bovine lymphoblastoid cells that are maintained continuously in vitro.

Objectives. To establish, implement, and evaluate a drug use management and policy residency program within the context of a service-learning framework. Design. Residents completed a 4-month term in which they were paired with a preceptor (health care manager or policy analyst) to complete a project designed to assist their work and to provide the resident with an understanding of policy formulation related to pharmaceuticals. Assessment. A formative evaluation of the first 2 years of the residency was conducted using semi-structured interviews with key stakeholders and an examination of program documents. Recurring themes were identified and a set of “lessons learned” was generated. Conclusion. The Drug Use Management and Policy Residency Program adhered to service learning tenets and was a practical educational experience for residents.

The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cyclic manner as the leading edge of a cell slowly advances. We used video-enhanced contrast microscopy (VEC) to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and established protrusions (Fisher et al.: Cell Motility and the Cytoskeleton 11:235–247, 1988), and to monitor the fixation of cells. Multiple parameter fluorescence imaging methods (DeBiasio et al.: Journal of Cell Biology 105:1613–1622, 1987; Waggoner et al.: Methods in Cell Biology, Vol. 30, Part B, pp. 449–478, 1989) were then used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusions. Initial protrustions exhibited no substructure as evidenced by VEC and actin was diffusely arranged, while myosin and microtubules were absent. Newly established protrusions contained diffuse actin as well as actin in microspikes. There was a delay in the appearance of myosin into established protrusions relative to the presence of actin. Microtubules were found in established protrusions after myosin was detected, and they were oriented parallel to the direction of migration. Actin and myosin were also localized in fibers transverse to the direction of migration at the base of initial and established protrusions. Image analysis was used to quantify the orientation of actin fibers relative to the leading edge of motile cells. The combined use of VEC, multiple parameter immunofluorescence, and image analysis should have a major impact on defining complex relationships within cells.

Background PI3K and mTOR are key components of signal transduction pathways critical for cell survival. Numerous PI3K inhibitors have entered clinical trials, while mTOR is the target of approved drugs for metastatic renal cell carcinoma (RCC). We characterized expression of p85 and p110α PI3K subunits and mTOR in RCC specimens and assessed pharmacologic co-targeting of these molecules in vitro. Methods We employed tissue microarrays containing 330 nephrectomy cases using a novel immunofluorescence-based method of Automated Quantitative Analysis (AQUA) of in situ protein expression. In RCC cell lines we assessed synergism between PI3K and mTOR inhibitors and activity of NVP-BEZ235, which co-targets PI3K and mTOR. Results p85 expression was associated with high stage and grade (P < 0.0001 for both). High p85 and high mTOR expression were strongly associated with decreased survival, and high p85 was independently prognostic on multi-variable analysis. Strong co-expression of both PI3K subunits and mTOR was found in the human specimens. The PI3K inhibitor LY294002 and rapamycin were highly synergistic in all six RCC cell lines studied. Similar synergism was seen with all rapamycin concentrations used. NVP-BEZ235 inhibited RCC cell growth in vitro with IC50s in the low ηM range and resultant PARP cleavage. Conclusions High PI3K and mTOR expression in RCC defines populations with decreased survival, suggesting that they are good drug targets in RCC. These targets tend to be co-expressed, and co-targeting these molecules is synergistic. NVP-BEZ235 is active in RCC cells in vitro; suggesting that concurrent PI3K and mTOR targeting in RCC warrants further investigation.