Cytotaxonomy, morphology and molecular systematics of the Bioko form of Simulium yahense (Diptera: Simuliidae).

Cytotaxonomic analysis of the polytene chromosomes from larvae of the Simulium damnosum Theobald complex from the island of Bioko in Equatorial Guinea is reported, and a new endemic cytoform is described. Chromosomally this cytoform is close to both S. squamosum (Enderlein) and S. yahense Vajime & Dunbar, but is not identical to either. However, it is morphologically and enzymatically identical to S. yahense. The Bioko form was also found to differ from other cytoforms of the S. damnosum complex in West Africa in the copy number or RFLP pattern of several different repetitive DNA sequences. It is clear that the Bioko form is genetically distinct from other populations of the S. damnosum complex, and whilst it is closest to S. yahense, it shows features that suggest a high degree of geographical and genetic isolation. Such isolation is an important consideration in the assessment of the potential for onchocerciasis vector eradication on Bioko.

Distribution of the Simulium metallicum complex in Mexico in relation to selected environmental variables.

Larvae of the Simulium metallicum complex (Diptera: Simuliidae) were collected from three foci of human onchocerciasis in Mexico. Specimens were separated into five different cytotypes, identified by morphological characteristics (head pattern and body colour) or polytene chromosome features. Differences were found between foci in the cytotype composition of the S. metallicum complex. Nearly all specimens were cytotype A in the Chamula (97%) and Soconusco (86%) foci. In the Oaxaca focus, however, cytotype I predominated (63%) with 14% cytotype A. Cytotype I comprised only 6% of specimens in the Soconusco focus and was very rare in the Chamula focus. Cytotypes B and H occurred only rarely in all three foci. Cytotype X was found only in Oaxaca. Environmental variables were measured at the collection sites and canonical correspondence analysis (CCA) was used to investigate the associations of cytotype distributions with the following factors: stream size, amount of shade, water clarity, pH, temperature and altitude. Members of the S. metallicum species complex were found to be differentially distributed according to stream conditions and there was a significant correlation between their distributions and the environmental variables. The most important factor in canonical axis 1 of the CCA was pH (t-value -4.38) with temperature (-2.48) and altitude (2.19) having some influence, but the other variables were unimportant. In the second canonical axis, pH (-3.52) was the only variable having a significant effect. Thus, cytotype A was associated with high pH, high temperature and low altitude; B was associated with similar sites but with higher temperatures and at lower average elevations. Cytotype H was found at the centres of the ranges of these variables, I at sites with the lowest temperatures and highest altitudes and X in rivers with the lowest pH.

Stimulation and inhibition of bone formation: use of peripheral quantitative computed tomography in the mouse in vivo.

The purpose of this study was to assess peripheral quantitative computed tomography (pQCT) imaging for measurement of volumetric bone mineral density (BMD) in vivo in mouse tibia following ovariectomy, and following treatment with 17 beta-oestradiol (E2). Two studies were undertaken. In study 1, three groups (n = 10) of mature mice were ovariectomized (OVX) or sham operated (SHAM); one of the OVX groups was dosed weekly with E2 (OVX.E2). Images of the proximal tibia were acquired on the day of surgery and at intervals following surgery until week 6. In study 2, four groups (OVX, SHAM, OVX.E2 and a SHAM group dosed with E2, SHAM.E2) of immature mice (n = 10) were imaged weekly up to 10 weeks post-surgery. Precision of pQCT for measurement of total (trabecular plus cortical) BMD was 2.4%, trabecular 5.2% and cortical 2.6%. In mature animals, significantly slower net bone formation was seen in OVX compared with SHAM animals using paired analysis with each animal as its own control. Group analysis detected no significant difference in BMD between SHAM and OVX at any time point. In immature animals, using paired analysis, with each animal as its own control, a significant difference between SHAM and OVX animals was detectable 3 weeks post-surgery (P < 0.05). As in study 1, group analysis of total BMD failed to detect any significant difference between SHAM and OVX at any time point. Treatment with E2 caused an easily-detected increase in BMD and led to osteopetrosis in both groups. The statistical power of this technique is adequate for testing antiresorptive or bone-forming therapies in the mouse.

Effects of an inhibitor of cathepsin L on bone resorption in thyroparathyroidectomized and ovariectomized rats.

The process of bone resorption by osteoclasts involves the dissolution of mineral salts and enzymatic degradation of the mainly collagenous extracellular matrix. Cysteine proteinases, which can efficiently degrade collagen at acidic pH, have been suggested to play an important role in the bone resorptive process. The cysteine proteinase cathepsin L is secreted by osteoclasts, and inhibitors of this enzyme can prevent bone resorption in vitro. The activity of acetyl-leu-leu-norleucinol (ALLN), a selective inhibitor of cathepsin L, was investigated in two models of bone resorption in vivo. In the first study, the ability of ALLN to inhibit bone resorption was investigated in Ro-13-6298 (arotinoid)-treated thyroparathyroidectomized (TPTX) rats. ALLN [100 mg/kg, intraperitoneally (i.p.)] inhibited hypercalcemia by 62.8% acutely (p < 0.001), compared to 94.9% (p < 0.001) inhibition by salmon calcitonin (sCT) (10 IU/kg, subcutaneously). In rats treated for 3 days with ALLN, arotinoid-induced reduction in cortical bone mineral density measured by peripheral quantitative computed tomography (pQCT) was inhibited by 86.4% (p < 0.05) in rats treated with ALLN 100 mg/kg, i.p., and by 82% in rats treated with 50 mg/kg, i.p. (p < 0.05). In a second study, the efficacy of ALLN was tested in a longitudinal study in ovariectomized (ovx) rats. Bone loss, measured by pQCT, was unaffected by treatment with ALLN. The bisphosphonate alendronate, however, inhibited bone loss in this model. These data demonstrate the ability of a cathepsin L inhibitor to inhibit bone resorption in arotinoid-treated TPTX rats, a process which may be dependent on the activity of cathepsin L-like cysteine proteinases. In contrast to its effects in TPTX rats, ALLN had no inhibitory activity on bone resorption in ovx rats. It is possible that in chronic bone resorption in ovx rats, the activity of other enzymes such as cathepsins OC-2 or K allows the process of resorption to continue even when cathepsin L is inhibited by ALLN. Further studies are required to determine why the activity of ALLN varies between different animal models. These data indicate that there may be variations in the effects of drugs in different animal models of bone resorption which should be considered when investigating novel antiresorptive therapies.

Effect of ovariectomy on magnetic resonance T2* in rat femur.

Measurements have been made in the rat femur in vivo and ex vivo by using an asymmetric spin echo technique of T2(1), the susceptibility contribution to T2*. The trabecular spacing in this study in rat bone is considerably less than in previous studies in the human. A significant increase in T2(1) was seen in vivo 3 mm proximal to the growth plate with ovariectomy (a model of osteopenia), from 8.1 +/- 0.7 to 10.0 +/- 0.6 ms. Parallel changes in trabecular bone mineral density measured by quantitative computed tomography were found. T2(1) was higher in living bones than in the same bones measured post mortem.

Regional analysis of bone mineral density in the distal femur and proximal tibia using peripheral quantitative computed tomography in the rat In vivo.

The use of peripheral quantitative computed tomography (pQCT) was investigated for the measurement of volumetric bone mineral density (BMD) in mg x cm-3. Two studies were undertaken. In the first study, the precision of pQCT in vivo and ex vivo was tested at 14 weeks postovariectomy (OVX). In the second study, the efficacy of a standard antiresorptive treatment, 17beta-estradiol (E2), was tested 6 weeks post-OVX. The precision for total (compact plus trabecular) BMD was 1.3-1.9%, and that for trabecular BMD was 2.4-2. 7%. There was excellent agreement between trabecular BMD measurements in vivo and ex vivo (r = 0.91). Significant reductions in trabecular BMD were observed in vivo at 14 and 6 weeks following ovariectomy in the femur, in each study. The loss of trabecular BMD depended on slice location, and varied from 0 to 22% at 6 weeks, and from 0 to 26% at 14 weeks (P < 0.001, at the affected locations). The antiresorptive effect of treatment was demonstrated in the 6-week study: there was no significant difference in BMD between sham-operated and E2-treated OVX rats.

Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization.

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.

Further development of the locus control region/murine erythroleukemia expression system: high level expression and characterization of recombinant human calcitonin receptor.

In order to demonstrate the potential of the LCR/MEL expression system for the expression of seven-transmembrane helix receptors the human calcitonin receptor (hCTR) has been cloned and expressed at high levels in this system. Using the newly developed single-step expression vectors pEV and pNV, stable recombinant MEL cells which express the hCTR at approximately 6 pmol of receptor/mg of total cell protein (60 pmol/mg of membrane protein), or 1.9 x 10(6) receptors per cell, have been generated. Ligand binding studies have shown that the cloned receptor expressed in MEL cells has properties which are indistinguishable from those of the native receptor in T47D cells. It is suggested that MEL cells expressing seven-transmembrane helix receptors provide an efficient and readily attainable source of material for ligand binding experiments using these receptors.

Sustained release of salmon calcitonin in vivo from lactide: glycolide copolymer depots.

Studies were carried out to determine whether monolithic depot formulations, prepared using lactide:glycolide copolymers, could be used to administer salmon calcitonin (sCT) to rats in vivo. Formulations containing 2, 5, or 10% (w/w) sCT were administered subcutaneously to female Wistar strain rats. Release of sCT was determined by measurement of peptide in plasma using a specific radioimmunoassay and by measurement of residual sCT in the depots after recovery at postmortem. Plasma calcium concentrations and cumulative weight gain of the animals were used to measure pharmacological effects of the released sCT. Release of sCT from the depots was controlled by the copolymer and was sustained for periods up to 10 days. However, the release of sCT from the depots did not significantly alter plasma calcium concentrations, and effects on cumulative weight gain were small and transient. Peptide loading of the formulations was shown to modify sCT release. Maximal release of sCT from depots containing 10% peptide occurred over a 7 to 14-day period postadministration, with 5% sCT release occurred between days 11 and 14, and with 2% sCT, the period of maximal release was between days 11 and 18. Release of peptide from the depots was essentially complete by 21 days postadministration irrespective of the peptide loading. These data suggest that lactide:glycolide copolymer depots may have application for the convenient clinical administration of sCT in metabolic bone diseases.

Identification of members of the Simulium ochraceum species complex in the three onchocerciasis foci in Mexico.

Larval collections of Simulium ochraceum Walker (Diptera: Simuliidae) were made in a variety of streams in the three onchocerciasis foci in Mexico and identified by cytotaxonomic criteria, based on the banding pattern of polytene chromosomes in larval salivary gland nucleii. S. ochraceum cytotype A was found in the Soconusco focus, cytotype B in the Oaxaca focus and cytotype C (not previously recorded in Mexico) in the Chamula focus. Attempts to analyse chromosomes of adult specimens were unsuccessful. A preliminary study of the cuticular hydrocarbons of adult specimens by gas liquid chromatography provided evidence that this technique may be suitable for separating the cytotypes.

Immunization with calcitonin gene-related peptide reduces the inflammatory response to adjuvant arthritis in the rat.

The influence of circulating antibodies to calcitonin gene-related peptide on the inflammatory response was examined in rats with adjuvant-induced arthritis. Rats were immunized with alpha calcitonin gene-related peptide conjugated to thyroglobulin, and circulating antibodies were identified by their capacity to bind radiolabelled rat alpha or human calcitonin gene-related peptide. In unimmunized rats and rats immunized with thyroglobulin alone, the secondary lesions (characterized as paw swelling, nodules on ears and tail, and inflamed nose) produced after adjuvant-induced arthritis were similar. However, at 21 days, when these lesions were maximal, the animals immunized with calcitonin gene-related peptide showed decreased numbers of lesions. An additional marker of disease activity, namely alpha 1 glycoprotein levels in plasma, was also measured. Again, plasma alpha 1 glycoprotein levels were similar in rats that were unimmunized or received thyroglobulin alone, but at 21 days were significantly reduced in animals immunized with calcitonin gene-related peptide. In contrast, the initial foot swelling seen in the first few days after injection of adjuvant was not significantly different in the various groups. The results suggest that antibodies to calcitonin gene-related peptide are able to reduce the severity of the adjuvant arthritis syndrome, and that this peptide contributes to the inflammatory response seen in the later stages of the disease model.