The distribution of oxytocin- and neurophysin-stained fibers in the spinal cord of the rat and monkey.

Immunohistochemical methods have been used to describe the distribution of neurophysin I- and oxytocin-stained fibers in the spinal cord. In albino rats of either sex, such fibers descend through the dorsal part of the lateral funiculus to the caudal end of the cord. Fibers leave the lateral funiculus to innervate the marginal zone of the dorsal horn at all levels, and the intermediolateral column at thoracic, lumbar and sacral levels. Some fibers course medially through the intermediate gray to end in the central gray, which is innervated at all levels, and also appears to be innervated by fibers descending in the central gray itself. A semiquantitative analysis shows that the density of terminal fields in the intermediolateral column, marginal zone, and central gray varies significantly at different levels. The intermediolateral column is preferentially innervated between T1-T3, T9-T11, and T13-L2. The nucleus intercalatusspinalis and the dorsal commissural nucleus (between T13 and L2), both of which contain preganglionic neurons, are densely innervated. In the monkey (Macaca fascicularis), neurophysin I- and oxytocin-stained fibers descend through the lateral funiculus to the filum terminale, where some of them enter the pia-arachnoid surrounding the filum. The intermediolateral column is not innervated between T1-T3, is sparsely innervated at sacral levels, and is moderately innervated between T4 and L3; the greatest density of fibers occurs at L3. At most levels, few if any fibers are found in the central gray and marginal zone; the central gray is sparsely innervated between T8 and L2 at S2, and between Co3-Co6, and the marginal zone contains a small number of fibers in the region of S2 and Co6. Neurophysin I- and oxytocin-stained fibers were also found in the intermediolateral column, marginal zone, and central gray of homozygous Brattleboro, spontaneously hypertensive, and Egyptian sand rats. The results suggest that the paraventriculo-spinal pathway is particularly related to specific groups of sympathetic and parasympathetic preganglionic neurons, and to the marginal zone, whick is involved in the relay of ascending nociceptive information through the spinothalamic tract.

Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests.

The divergence of parasites is important for maintenance within an established host and spread to novel host species. In this paper we have carried out phylogenetic analyses of Theileria parasites isolated from different host species. This was performed with small subunit ribosomal RNA sequences available in the data bases and a novel sequence amplified from Theileria lestoquardi DNA. Similar phylogenetic studies were carried out with sequences representing the major merozoite/piroplasm surface antigen (mMPSA) from the data base, and novel sequences representing 2 mMPSA alleles from T. lestoquardi, a full length sequence of a Theileria taurotragi mMPSA gene and partial sequences of two new allelic variants of the Babesia equi mMPSA gene homologue. The analysis indicated that the pathogenic sheep parasite T. lestoquardi has most probably evolved from a common ancestor of T. annulata. Interestingly, the level of mMPSA sequence diversity found for T. lestoquardi was surprisingly low, while diversity between the B. equi sequences was higher than that found within any of the classical Theileria species. The possible implications of these results for the establishment of Theileria parasites within novel species are discussed. Extensive cross-reactivity of a range of antisera was found when tested against recombinant mMPSA polypeptides from different Theileria (including B. equi) species. The cross-reactivity between mMPSA polypeptides and sequence diversity are relevant for the development of species specific diagnostic tests.

Characterisation of an extrachromosomal DNA element from Theileria annulata.

Extrachromosomal nucleic acid elements are found in all organisms, commonly as organelle, viral or plasmid genomes. In this paper we describe the initial characterisation of a novel 6.5-kb linear, double-stranded extrachromosomal element from Theileria annulata, and a 2.6-kb RNA species. The DNA element is present in different stages of the life cycle and in different stocks of the parasite. Northern blots of total RNA isolated from different stages of the parasite, probed with the purified element, detect three major transcripts, of 1.45, 1.05 and 0.24 kb, present in all life-cycle stages examined. The possible origin and function of this element is discussed, together with its possible use as a transfection vector for the introduction of genes into protozoan cells.

Selection of diversity at putative glycosylation sites in the immunodominant merozoite/piroplasm surface antigen of Theileria parasites.

The immunodominant merozoite/piroplasm surface antigen of Theileria parasites has potential as a diagnostic reagent and as a component of a sub-unit vaccine. This molecule is known to be antigenically diverse, and it is important to determine the nature and extent of this heterogeneity. In the present study nucleotide sequences, representing alleles of the gene (Tams1) encoding this molecule in Theileria annulata were compared to each other and to sequences of homologous genes in Theileria sergenti, Theileria buffeli and Theileria parva. This analysis revealed that a region of the polypeptide which contains putative N-linked glycosylation sites is particularly diverse and, in analogy to retroviral systems, may indicate selection of variable glycosylation sites or amino acid epitopes to evade the bovine immune response. This conclusion was also made from the results of a phylogenetic analysis which compared the variable region of the genes with a second region, which appeared to show no bias for diversity or functional constraint. The results indicated that the variable sequence encoding putative glycosylation sites has diverged, both within and between Theileria species, at a much faster rate than the rest of the molecule. Southern blot analysis of T. annulata populations from within a single geographical region detected six possible variant Tams1 alleles. However, a correlation between restriction-fragment-length polymorphism (RFLP) patterns detected by the Tams1-1 gene probe and geographical location could not be made. In addition, although a high prevalence of one particular RFLP was found, this is unlikely to be the result of a clonal population structure, as we present evidence for significant parasite genotypic variability within a single endemic region.

Temporal co-ordination of macroschizont and merozoite gene expression during stage differentiation of Theileria annulata.

The bovine parasite, Theileria annulata has a complex life-cycle involving the expression and repression of genes during development of its morphologically distinct life-cycle stages. In order to detail the molecular events that occur during differentiation of the intracellular multinucleate macroschizont to the extra-cellular uninucleate merozoite, we have isolated two genes, Tash1 and Tash2 which are differentially expressed during differentiation. Nuclear run on data show that Tash1 gene expression is controlled, at least in part, at the level of transcription. Immunofluorescence data identify the macroschizont as the location for both Tash1 and Tash2 gene products. Northern blot analysis of these genes indicated that their mRNA levels decrease during differentiation in vitro, at a time point coincident with major elevation in the mRNA levels of the merozoite antigen, Tams1, shown previously to be associated with commitment to merozoite production. Furthermore, experiments where cultures were incubated at 41 degrees C for 4 days and replaced at 37 degrees C for 2 days demonstrated that re-expression of Tash1 occurred and is probably linked to reversion to the macroschizont and decreased expression of Tams1. These results imply that the control of macroschizont and merozoite gene expression during differentiation is closely co-ordinated temporally. In addition, a comparison of Tash2 and Tams1 expression has indicated that translational or post-translational control of gene expression may operate in the undifferentiated macroschizont.

Directing differentiation in Theileria annulata: old methods and new possibilities for control of apicomplexan parasites.

Apicomplexan parasites are major pathogens of humans and domesticated animals. The ability of these organisms to evade the host immune response and the emergence of drug-resistant parasites indicates a need for the identification of novel control strategies. Ideally, selected targets should be shared by a range of apicomplexans and fundamental to parasite biology. One process of apicomplexan biology which may provide this type of target is the molecular regulation of stage differentiation. This paper has reviewed studies carried out on differentiation of Theileria annulata and has highlighted general similarities with other apicomplexan differentiation steps. Similarities include asynchrony of differentiation, the loss (attenuation) of differentiation potential and an association between reduced proliferation and differentiation. In addition, novel data are presented assessing a possible role for a signal transduction mechanism or a direct involvement of classical heat-shock polypeptides in regulating differentiation of T. annulata in vitro. These studies, and previously published data, have led to the postulation that progression to the next stage of the life-cycle can be predetermined and involves the attainment of a quantitative threshold by regulators of gene expression. A modification of this model takes into account that for certain in-vitro systems, or differentiation steps in vivo, the process has to be initiated by alteration of the extracellular environment. Work which has shown that the time taken to achieve differentiation can be increased or decreased is also outlined. The ability to change the timing of differentiation suggests that the associated regulatory mechanism could be manipulated directly to significantly influence the outcome of an apicomplexan infection. The observation that a number of existing drugs and control strategies may exert their protective effect by altering differentiation potential supports this possibility.

Selection for antigenic diversity of Tams1, the major merozoite antigen of Theileria annulata.

Tams1, the major merozoite/piroplasm surface antigen of Theileria annulata has the potential to be a component of a diagnostic ELISA test and be included in a recombinant subunit vaccine. However, the observation that this antigen displays diversity could constrain these applications. In this paper we have extensively characterized Tams1 diversity at the DNA level, using a PCR/sequencing strategy. Up to 44 alleles have been cloned and sequenced. The comparison of these alleles has identified regions of sequence conservation, variability and hyper-variability. Computer analysis of these alleles has indicated that positive selection may operate on certain regions of Tams1. Expression and Western blot analysis of selected alleles has indicated that sequence diversity is reflected in altered antigenicity and a continuum of relatedness and antibody cross recognition may exist. The possible function of the sequence conservation and polymorphism within Tams1 is discussed in relation to protein structure, host cell invasion and immune evasion.

Treatment of infected left ventricular assist device using antibiotic-impregnated beads.

There is no well-established therapy for treating infections of heart-assist or artificial heart devices, a serious problem with life-threatening consequences. We used a promising new approach in which antibiotic-impregnated polymethylmethacrylate beads were placed around an implanted left ventricular assist device to control an external blood pump infection in a bridge-to-transplant patient. In this case report, we describe the potential of antimicrobial-impregnated polymethylmethacrylate beads for in situ control of infections involving external surfaces of cardiovascular devices.

Serotonergic projections from the ventral medulla to the intermediolateral cell column in the rat.

Serotonergic neurons of the ventral medulla (B1 and B3 cell groups are retrogradely labeled after HRP injection in the T1-T2 levels of the spinal cord. Following stereotaxic injections of 3H-labeled amino acids in this region, a direct projection to th intermediolateral cell column and ventral horn can be demonstrated.

Organization of some brain stem afferents to the paraventricular nucleus of the hypothalamus in the rat.

Brain stem afferents to subnuclei of the paraventricular nucleus of the hypothalamus were studied by the anterograde autoradiographic technique in the rat. The parabrachial nuclei and locus coeruleus project to the posterior, periventricular, parvocellular and dorsal divisions. The ventral medulla projects to the posterior, medial, lateral, parvocellular and dorsal divisions. The A1 catecholamine cell group projects to the posterior, medial, lateral, parvocellular and dorsal divisions, and the nucleus of the solitary tract to the parvocellular and dorsal divisions. The A1 region and the ventral medulla also project to the supraoptic nucleus.

Efferent projections of the A1 catecholamine cell group in the rat: an autoradiographic study.

Efferent connections of the region of the A1 catecholamine cell group were investigated by the anterograde autoradiographic method in rats, some of which had been pretreated with intraventricular injections of 6-hydroxydopamine (6-OHDA). Spinal projections were further studied by combining histofluorescence and horseradish peroxidase staining in the same sections. Projections from the A1 region ascend through the lateral hypothalamus to the bed nucleus of the stria terminalis, the medial preoptic area, and several hypothalamic nuclei: the dorsomedial nucleus, the dorsal hypothalamic area, the paraventricular and supraoptic nuclei, and the median eminence. These projections are predominantly ipsilateral. Since they are sensitive to 6-OHDA, they presumably arise from catecholamine cells of the A1 group. Other pathways are not noticeably affected by 6-OHDA. These include projections through the reticular formation to the contralateral nucleus ambiguus, the ipsilateral facial nucleus, and the Kölliker-Fuse nuclei, the parabrachial nuclei and the periaqueductal gray on both sides, as well as the intralaminar nuclei of the thalamus. These pathways probably do not arise from the A1 group, although a minor noradrenergic component cannot be ruled out. Spinal projections extend to the intermediolateral cell column and the ventral horn, and especially to the phrenic motor nucleus. However, these projections arise almost exclusively from non-catecholamine cells. Only a small minority of the fluorescent A1 cells are retrogradely labeled after injections of HRP into the upper thoracic spinal cord. They lie at the level of the pyramidal decussation. Likewise only a few fluorescent cells of the A2 group are labeled. Although the A1 and A2 groups were long thought to be principal sources of spinal norepinephrine, a review of the literature shows that this belief was based on equivocal evidence.

Measuring fruit and vegetable intake: is five-a-day enough?

OBJECTIVE: Validation of a self-monitoring "portions' measurement of fruit and vegetable (F&V) consumption against a standard of weighed intakes. DESIGN: Component of a randomized controlled trial. SETTING: Subjects attended research centres in Reading and Glasgow for instruction and monitoring but undertook free-living dietary changes at home. SUBJECTS: A study sample of 42 adult men and women fulfilling the main recruitment criterion of eating less than five F&V portions/day but contemplating increasing intakes and providing weighted baseline reported energy intakes exceeding (estimated basal metabolic rate x 1.1). INTERVENTIONS: Subjects attended an intensive group advice session which included the specific relationship of high F&V intake with reduced risk of disease; practicalities; portion definition and measurement recording. The target was to exceed five F&V portions/day for 8 weeks. MAIN OUTCOME MEASURES: Self-recorded simultaneous weighed inventories and F&V portion measures. RESULTS: Data from subjects who were not evident under-recorders showed correlations between portion and weighed intakes of r = 0.73, (P < 0.000), although the portions measure tended to under-estimate intakes. Using 80 g/portion the "5-a-day' concept tends to create false negatives (namely consumption could be greater than 400 g whilst recording fewer than five discrete portions) but rarely false positives (namely recorded consumption of less than 400 g did not give measures of more than five discrete portions). CONCLUSIONS: The data suggest that the five portions F&V/day health message, if used in conjunction with defined discrete portions, would encourage desirable consumption exceeding 400 g.

The maturation of Theileria annulata in Hyalomma anatolicum anatolicum stimulated by incubation or feeding to produce sporozoites.

Adult Hyalomma anatolicum anatolicum ticks infected with Theileria annulata (Hissar strain) were incubated at 36 degrees C or fed on rabbits. Tick salivary glands were stained whole with methyl green pyronin or ground up and deposited on microscope slides and stained with Giemsa's solution. Separate batches of ticks from both treatments were ground up, centrifuged and filtered to produce sporozoite suspensions. The suspensions were examined as deposits on microscope slides stained with Giemsa's solution. The Theileria in the salivary glands of the fed ticks matured more completely and rapidly than in the incubated ticks. The peak numbers of sporozoites from the fed ticks was greater by at least tenfold than the peak from the incubated ticks. This peak was on the third day of feeding or on the fourth day of incubation. It was confirmed that fed ticks will be more suitable for sporozoite production for infection of cattle and production of stabilates.

Class-specific circulating antibodies in infections with Eimeria tenella.

Using a three-layer immunofluorescent test, class-specific, parasite-specific circulating antibody responses to Eimeria tenella were investigated following oocyst infection, drug-truncated oocyst infection and the injection of non-living antigens. Following all three means of antigenic stimulation, IgG, IgM and IgA antibodies were detected. The response to drug-truncated infections was dose-dependent. The sequence of appearance of antibody was IgM, IgA, IgG, whilst the relative quantities were IgG greater than or equal to IgM greater than IgA.

Artificial infection of the tick Rhipicephalus appendiculatus with Theileria parva.

Methods for infecting Rhipicephalus appendiculatus ticks with Theileria parva by injection and by artificial feeding were confirmed and compared. The injection method proved simpler and at best as effective and suggested improvements are described.

Failed venture: Gordon Murray and the W. P. Caven Memorial Research Foundation, 1949-74.

This paper examines the founding and operation of the W. P. Caven Memorial Research Foundation, a private Toronto laboratory, which existed from 1949 to 1974. The Caven Foundation's Research Director was celebrated Toronto surgeon Gordon Murray (1894-1976), who, in 1949, accepted this position amidst personal and public expectations of great medical discoveries and innovations to come. For 25 years Murray carried on his research at the Caven Foundation, generating more controversy and disappointment than medical cures, before the laboratory was closed for financial reasons. What might have been a successful alternative to the University-based medical research structure in Canada resulted in a failed venture. The Foundation did not become a viable research centre largely because of its Research Director and his inability to adapt to the many changes occurring in the conduct and funding of clinical research. The history of the Caven Foundation is explored here within the context of increasingly specialized research techniques and methodology, the rising predominance of the interdisciplinary research team, and the new system of grantsmanship.