Beta Blockade Prevents Cardiac Morphological and Molecular Remodelling in Experimental Uremia.

Heart failure and chronic kidney disease (CKD) share several mediators of cardiac pathological remodelling. Akin to heart failure, this remodelling sets in motion a vicious cycle of progressive pathological hypertrophy and myocardial dysfunction in CKD. Several decades of heart failure research have shown that beta blockade is a powerful tool in preventing cardiac remodelling and breaking this vicious cycle. This phenomenon remains hitherto untested in CKD. Therefore, we set out to test the hypothesis that beta blockade prevents cardiac pathological remodelling in experimental uremia. Wistar rats had subtotal nephrectomy or sham surgery and were followed up for 10 weeks. The animals were randomly allocated to the beta blocker metoprolol (10 mg/kg/day) or vehicle. In vivo and in vitro cardiac assessments were performed. Cardiac tissue was extracted, and protein expression was quantified using immunoblotting. Histological analyses were performed to quantify myocardial fibrosis. Beta blockade attenuated cardiac pathological remodelling in nephrectomised animals. The echocardiographic left ventricular mass and the heart weight to tibial length ratio were significantly lower in nephrectomised animals treated with metoprolol. Furthermore, beta blockade attenuated myocardial fibrosis associated with subtotal nephrectomy. In addition, the Ca++- calmodulin-dependent kinase II (CAMKII) pathway was shown to be activated in uremia and attenuated by beta blockade, offering a potential mechanism of action. In conclusion, beta blockade attenuated hypertrophic signalling pathways and ameliorated cardiac pathological remodelling in experimental uremia. The study provides a strong scientific rationale for repurposing beta blockers, a tried and tested treatment in heart failure, for the benefit of patients with CKD.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE-/-) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE-/- and Nox2 (ESMIRO/ApoE-/-/Nox2-/y) were generated and compared with ESMIRO/ApoE-/-/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE-/- mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE-/-/Nox2-/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE-/-/Nox2-/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE-/- mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Fibroblast-specific deletion of interleukin-1 receptor-1 reduces adverse cardiac remodeling following myocardial infarction.

It has been hypothesized that interleukin-1alpha (IL-1α) is released from damaged cardiomyocytes following myocardial infarction (MI) and activates cardiac fibroblasts via its receptor (IL-1R1) to drive the early stages of cardiac remodeling. This study aimed to definitively test this hypothesis using cell type-specific IL-1α and IL-1R1 knockout (KO) mouse models. A floxed Il1α mouse was created and used to generate a cardiomyocyte-specific IL-1α KO mouse line (MIL1AKO). A tamoxifen-inducible fibroblast-specific IL-1R1 hemizygous KO mouse line (FIL1R1KO) was also generated. Mice underwent experimental MI (permanent left anterior descending coronary artery ligation) and cardiac function was determined 4 weeks later by conductance pressure-volume catheter analysis. Molecular markers of remodeling were evaluated at various time points by real-time RT-PCR and histology. MIL1AKO mice showed no difference in cardiac function or molecular markers of remodeling post-MI compared with littermate controls. In contrast, FIL1R1KO mice showed improved cardiac function and reduced remodeling markers post-MI compared with littermate controls. In conclusion, these data highlight a key role for the IL-1R1/cardiac fibroblast signaling axis in regulating post-MI remodeling and provide support for the continued development of anti-IL-1 therapies for improving cardiac function after MI. Cardiomyocyte-derived IL-1α was not an important contributor to post-MI remodeling in this model.

Prevalence and risk factors for IgG antibodies to Neospora spp. in three types of equids from Southern Punjab, Pakistan.

Equine hosts suffer from neurological disease, congenital infection, and reproductive problems associated with Neospora spp. infection. We conducted a cross sectional study using sera from 631 equids (324 horses, 218 donkeys and 89 mules) from the southern region of Punjab province, Pakistan to determine the prevalence of antibodies against Neospora spp. in this diverse group of equines. Fisk factors associated with seropositivity were evaluated statistically based on equine type, breed, age, husbandry, breeding methods, and reproductive history. Prevalence of antibodies to Neospora spp. was detected using a commercially available competitive ELISA kit. We detected IgG antibodies to Neospora spp. in 23.3% of the equids with prevalence by host being 16.0% in horses, 32.6% in donkeys and 26.9% in 89 mules. Statistically significant (P < 0.05) differences in prevalence were observed among these hosts. Prevalence ranged in breeds of horses from 3.7% in the Morna breed, 12.4% in breeding stock, 26.8% in draughting stock, to 31.4% in unknown breeds. No significant (P > 0.05) difference in prevalence was noted among age groups. Prevalence was significantly (P < 0.05) higher in female equines with a history of abortion than those with no history of abortion. This is the first report documenting Neospora spp. in equines from Pakistan and it provides evidence that Neospora spp. is associated with abortions in female equines in Pakistan.

Prevalence of Toxoplasma gondii, Neospora caninum, and Sarcocystis Species DNA in the Heart and Breast Muscles of Rock Pigeons (Columbia livia).

Little is known about the prevalence of protozoan parasites in the muscles of rock pigeons (Columbia livia). The muscles from 54 (heart from 45 and breast from 54) rock pigeons were examined for DNA of Toxoplasma gondii, Neospora caninum, and Sarcocystis species using PCR. Twenty-four were female and 30 were males. The birds were part of flocks of pigeons housed at the tombs of saints in Lahore, Pakistan. Birds that died or were euthanized due to poor health were submitted for necropsy at the Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan, where DNA isolations and PCR were conducted. Nineteen (35.1%) of the birds were positive for T. gondii DNA. Seven males and 12 females were positive. Breast tissue was always infected in T. gondii positive birds, while the heart was infected in 13 (28.8%) of breast positive birds. Five (9.2%) of the pigeons, 2 males and 3 females, were positive for N. caninum. The distribution of N. caninum DNA was more variable in the muscles of pigeons than T. gondii and was found only in the heart of 1 (female), heart and breast muscle of 2 (male), and only the breast muscle of 2 birds (female). One of the 54 rock pigeons (female) was positive for both T. gondii (heart and breast) and N. caninum (heart only). Two of the positive Neospora caninum amplicons were sequenced and had 97% nucleotide identity with N. caninum isolates. Sarcocystis DNA was not found in any bird. The prevalence of T. gondii in rock pigeons and their predation by cats suggest that they may play an unrecognized role in maintaining environmental contamination with T. gondii oocysts by cats. Our study indicates that rock pigeons are intermediate hosts of N. caninum and this information will aid in understanding the epidemiology of N. caninum.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4.

AIM: To investigate the effect of Tenascin C (TNC) on the expression of pro-inflammatory cytokines and matrix metalloproteinases in human cardiac myofibroblasts (CMF). METHODS: CMF were isolated and cultured from patients undergoing coronary artery bypass grafting. Cultured cells were treated with either TNC (0.1 µmol/L, 24 h) or a recombinant protein corresponding to different domains of the TNC protein; fibrinogen-like globe (FBG) and fibronectin type III-like repeats (TNIII 5-7) (both 1 µmol/L, 24 h). The expression of the pro-inflammatory cytokines; interleukin (IL)-6, IL-1ß, TNFα and the matrix metalloproteinases; MMPs (MMP1, 2, 3, 9, 10, MT1-MMP) was assessed using real time RT-PCR and western blot analysis. RESULTS: TNC increased both IL-6 and MMP3 (P < 0.01) mRNA levels in cultured human CMF but had no significant effect on the other markers studied. The increase in IL-6 mRNA expression was mirrored by an increase in protein secretion as assessed by enzyme-linked immunosorbant assay (P < 0.01). Treating CMF with the recombinant protein FBG increased IL-6 mRNA and protein (P < 0.01) whereas the recombinant protein TNIII 5-7 had no effect. Neither FBG nor TNIII 5-7 had any significant effect on MMP3 expression. The expression of toll-like receptor 4 (TLR4) in human CMF was confirmed by real time RT-PCR, western blot and immunohistochemistry. Pre-incubation of cells with TLR4 neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mRNA and protein expression. CONCLUSION: TNC up-regulates IL-6 expression in human CMF, an effect mediated through the FBG domain of TNC and via the TLR4 receptor.

Entamoeba infections in different populations of dogs in an endemic area of Lahore, Pakistan.

Entamoeba histolytica, a protozoan parasite that affects humans and other primates all over the world. It is a common waterborne pathogen in endemic areas that have fecal oral transmission cycle. The aim of the present study was to examine the prevalence of E. histolytica and other Entamoeba species cysts in three different dog populations. Fecal samples from 600 dogs were collected and processed to detect Entamoeba cysts using the triple fecal test (light microscopy) and fecal antigens of E. histolytica were detected using a fecal antigen ELISA (TechLab E. histolytica II). Because it is impossible to differentiate E. histolytica from Entamoeba dispar and E. moshkovskii, using light microscopy we referred to all cysts morphologically consistent with E. histolytica as E. histolytica/dispar/moskovskii to reflect this uncertainty. Samples from 197 household dogs without clinical signs, 122 samples from household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting and 281 stray dogs with no specific clinical signs were examined. Entamoeba histolytica-like cysts were observed in 94 (15.6%, 95% CI=±3.88) by triple fecal test microscopy and E. histolytica antigens were demonstrated in 66 (11%, 95% CI=±4.41) by fecal antigen ELISA in 600 fecal samples. Significant differences (P≤0.05) in prevalence were found between the three populations. Twenty (10.1%, 95% CI=±7.86) and 11 (5.6%, 95% CI=±7.70) of 197 fecal samples from household dogs without clinical signs were positive by microscopy and by antigen ELISA, respectively. Twenty-nine (23.8%, 95% CI=±6.58) and 23 (18.8%, 95% CI=±7.81) of 122 the fecal samples from household dogs with clinical signs were positive by microscopy and by antigen ELISA, respectively. Forty-five (16.01%, 95% CI=±5.62) and 32 (11.3%, 95% CI=±6.38) of 281 fecal samples from stray dogs were positive by microscopy and by fecal antigen ELISA, respectively. Dogs from the youngest age group (6 months to 1 year) were more likely to be E. histolytica antigen positive than were dogs from the other two older age groups, with a significant difference (P≤0.05) between all age groups. Statistically, no significant (P≥0.05) difference of prevalence was seen in male and female dogs. The local dogs had the highest prevalence rate of E. histolytica antigens (36 of 246, 14.2%, 95% CI=±6.32) followed by imported breeds (11 of 115, 9.5%, 95% CI=±10.4) and crossbred (19 of 239, 8.3%, 95% CI=±7.47), indicating a significant (P≤0.05) trend of positivity between various breeds of dogs. These findings suggest that dogs may play an important role in the epidemiology of this pathogen.

Trivalent Gd-DOTA reagents for modification of proteins.

The development of novel protein-targeted MRI contrast agents crucially depends on the ability to derivatise suitable targeting moieties with a high payload of relaxation enhancer (e.g., gadolinium(iii) complexes such as Gd-DOTA), without losing affinity for the target proteins. Here, we report robust synthetic procedures for the preparation of trivalent Gd-DOTA reagents with various chemical handles for site-specific modification of biomolecules. The reagents were shown to successfully label proteins through isothiocyanate ligation or through site-specific thiol-maleimide ligation and strain-promoted azide-alkyne cycloaddition.

Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P < 0.05) differences in prevalence were seen between the 3 socioeconomic class groups. Forty-four (15.3%) and 32 (11.1%) of 287 in the fecal samples from the upper socioeconomic class were positive by triple test and by antigen ELISA, respectively. Thirty-nine (22.6%) and 29 (16.8%) of 172 in the fecal samples from the middle socioeconomic class were positive by the triple test and by antigen ELISA, respectively. Fifty-two (36.8%) and 40 (28.3%) of 141 in the fecal samples from the lower socioeconomic class were positive by the triple test and by antigen ELISA, respectively. We accept our hypothesis based on these findings. We also demonstrated that fecal samples collected from the youngest age group (1 mo-5 yr) were more likely to be positive for E. histolytica antigens than were samples from the other 3 age groups, and that prevalence was significantly higher (P < 0.05) in the summer than in the other 3 seasons. These results highlight the importance of surveillance of this relatively ignored pathogen in this developing metropolitan city in Pakistan.

Neospora caninum prevalence in dogs raised under different living conditions.

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of N. caninum because they act as definitive hosts shedding oocysts in the environment. Vertical transmission of the parasite is well recognized as an important aspect of the epidemiology of the parasite but the importance of horizontal transmission has been less studied. A N. caninum competitive ELISA was used to examine serum samples from 600 dogs that were raised under 4 different living conditions. Samples from 138 dogs living on 24 dairies with a prevalence (0-70%) of anti-N. caninum antibodies in the cattle, 294 pet dogs without neurological signs, 76 from pet dogs exhibiting neurological signs, and 92 stray dogs were examined. The overall seroprevalence of N. caninum was 23.5% (95% CI = ± 2.99) in the 600 dogs. Significant (P < 0.05) differences were observed between the 4 different populations of dogs. The number of N. caninum positive samples were: 51 (36.9%, 95% CI = ± 3.09) of 138 dogs from dairies, 31 (10.5%, 95% CI = ± 6.38) of 294 pet dogs without neurological signs, disorders, 22 (28.9%, 95% CI = ± 6.70) of 76 pet dogs with neurological signs, and 37 (40.2%, 95% CI = ± 2.83) of 92 stray dogs. Seropositivity to N. caninum in dogs from dairies was associated with a high prevalence of N. caninum antibodies in the cattle. At the 3 dairies where no dogs were present, the seroprevalence to N. caninum in the cattle was significantly lower (P < 0.05) than in the 21 dairies where dogs were present. Seroprevalence was significantly higher (P < 0.05) in male dogs (97 of 366; 26.5%, 95% CI = ± 3.40) than in female dogs (44 of 234; 18.8%, 95% CI = ± 5.65). Seroprevalence in dogs increased with age suggesting postnatal exposure to N. caninum infection however, this increase was not significant (P > 0.05). The prevalence of N. caninum antibodies was not significantly (P>0.05) different in dogs based on breed. These findings suggest a relationship between N. caninum infection of dogs from dairies and cattle on these dairies. However, further research is required to determine what is the most important way dogs acquire infection and how to prevent dogs from shedding oocysts.

Epidemiology, sero-diagnosis and therapeutic studies on nematodes infection in balochi range-sheep at district quetta, balochistan, pakistan.

BACKGROUND: Among the infectious organisms of parasitic origin, gastrointestinal nematodes are very important as they have been reported worldwide. The main aim of the present research study to highlight the annual epidemiological contributing factors associated with the prevalence of gastrointestinal nematodes and their control in sheep. METHODS: A total 1200 faecal samples (100 per month) were collected from farmers holding Balochi-sheep (either sexes, 1-5 years old) during January-December 2012 and analyzed to determine the prevalence of nematodes based on microscopy and ELISA based diagnostic assay. Therapeutic efficacies of different synthetic and herbal medicines against these nematodes were assessed by field trials. RESULTS: Results showed that 23.92% Balochi-sheep were infected with nematodes. Five nematodes infections were recorded with highest prevalence of Haemonchus (7.75%) followed by Nematodirus (7.58%), Strongyloides (4.42%), Trichostrongylus (2.33%) and Trichuris (1.83%). The younger and older ewes (one and five years) presented higher nematodes prevalence with peak during March/April and August/September. Haemonchus and Trichuris positive samples based on coprological examination were also showed 92-100% positive sensitivity for these nematodes by the ELISA. Sheep treated with Ivermectin showed higher reduction (97.76%) in nematode egg counts followed by Atreefal deedan (96.42%) and Oxfendazole (95.44%), respectively. CONCLUSION: The gastro-intestinal nematodes are prevalent in all age and either sex of Balochi-sheep with peak during summer. The ELISA based diagnosis is more accurte. The synthetic and herbal products are very effective against sheep nematodes.

Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αVß3 and RhoA/ROCK-mediated mechanisms.

Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVß3-dependent, but transforming growth factor ß-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVß3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.

Interleukin-1 has opposing effects on connective tissue growth factor and tenascin-C expression in human cardiac fibroblasts.

Cardiac fibroblasts (CF) play a central role in the repair and remodeling of the heart following injury and are important regulators of inflammation and extracellular matrix (ECM) turnover. ECM-regulatory matricellular proteins are synthesized by several myocardial cell types including CF. We investigated the effects of pro-inflammatory cytokines on matricellular protein expression in cultured human CF. cDNA array analysis of matricellular proteins revealed that interleukin-1α (IL-1α, 10ng/ml, 6h) down-regulated connective tissue growth factor (CTGF/CCN2) mRNA by 80% and up-regulated tenascin-C (TNC) mRNA levels by 10-fold in human CF, without affecting expression of thrombospondins 1-3, osteonectin or osteopontin. Western blotting confirmed these changes at the protein level. In contrast, tumor necrosis factor α (TNFα) did not modulate CCN2 expression and had only a modest stimulatory effect on TNC levels. Signaling pathway inhibitor studies suggested an important role for the p38 MAPK pathway in suppressing CCN2 expression in response to IL-1α. In contrast, multiple signaling pathways (p38, JNK, PI3K/Akt and NFκB) contributed to IL-1α-induced TNC expression. In conclusion, IL-1α reduced CCN2 expression and increased TNC expression in human CF. These observations are of potential value for understanding how inflammation and ECM regulation are linked at the level of the CF.

Efficacy of decoquinate against Sarcocystis neurona in cell cultures.

Decoquinate is a quinolone anticoccidial approved for use in the prevention of intestinal coccidiosis in farm animals. This compound has good activity against Toxoplasma gondii and Neospora caninum in cell cultures. The drug acts on the parasites' mitochondria. The activity of decoquinate against developing merozoites of 2 isolates of Sarcocystis neurona was examined in cell culture. Merozoite production at 10 days was completely inhibited when decoquinate was used at 20 or 240 nM. The IC50 of decoquinate was 0.5 ± 0.09nM for the Sn6 isolate of S. neurona from a horse and 1.1 ± 0.6 nM for the SnOP15 isolate of S. neurona from an opossum. Levamisole was toxic at 5 µg/ml and no synergism was observed when decoquinate was combined with levamisole and tested against the Sn3YFP isolate of S. neurona. Decoquinate was cidal for developing schizonts of S. neurona at 240 nM.

Novel G protein-coupled oestrogen receptor GPR30 shows changes in mRNA expression in the rat brain over the oestrous cycle.

Oestrogen influences autonomic function via actions at classical nuclear oestrogen receptors α and ß in the brain, and recent evidence suggests the orphan G protein-coupled receptor GPR30 may also function as a cytoplasmic oestrogen receptor. We investigated the expression of GPR30 in female rat brains throughout the oestrous cycle and after ovariectomy to determine whether GPR30 expression in central autonomic nuclei is correlated with circulating oestrogen levels. In the nucleus of the solitary tract (NTS), ventrolateral medulla (VLM) and periaqueductal gray (PAG) GPR30 mRNA, quantified by real-time PCR, was increased in proestrus and oestrus. In ovariectomised (OVX) rats, expression in NTS and VLM appeared increased compared to metoestrus, but in the hypothalamic paraventricular nucleus and PAG lower mRNA levels were seen in OVX. GPR30-like immunoreactivity (GPR30-LI) colocalised with Golgi in neurones in many brain areas associated with autonomic pathways, and analysis of numbers of immunoreactive neurones showed differences consistent with the PCR data. GPR30-LI was found in a variety of transmitter phenotypes, including cholinergic, serotonergic, catecholaminergic and nitrergic neurones in different neuronal groups. These observations support the view that GPR30 could act as a rapid transducer responding to oestrogen levels and thus modulate the activity of central autonomic pathways.

Effects of age and breed on the prevalence of Neospora caninum in commercial dairy cattle from Pakistan.

Neospora caninum is a major cause of bovine abortion worldwide. A serological survey was carried out to determine the seroprevalence of exposure to N. caninum in dairy cattle based on age and breed from Punjab and Sindh provinces, Pakistan. Serum samples from 641 animals from 12 herds from Punjab (n = 7) and Sindh (n = 5) provinces were tested for antibodies against N. caninum using a commercially available competitive enzyme-linked immunosorbent assay. Positive reactions to N. caninum were seen in 277 (43%) of the 641 of the samples. Seropositive animals were present in all 12 herds. Animals over 2 yr of age (47%) and crossbreds (55%) were more likely to be seropositive than the other cattle examined. These results indicate that N. caninum infection is widespread among dairy cattle in Pakistan.

MMP-3 (5A/6A) polymorphism does not influence human smooth muscle cell invasion.

BACKGROUND: Stromelysin (MMP-3) is an important regulator of vascular smooth muscle cell (SMC) invasion, a key contributor to saphenous vein (SV) bypass graft failure. The 5A allele of the common -1612 MMP-3 5A/6A promoter polymorphism reportedly confers increased promoter activity, MMP-3 tissue expression, and susceptibility to a number of vascular pathologies. The aim of this study was to determine whether the MMP-3 5A/6A polymorphism directly influences endogenous MMP-3 expression levels and, consequently, cell invasion, in SV-derived SMC cultured from patients with different genotypes. MATERIAL AND METHODS: Genotyping of 226 patients revealed -1612 MMP-3 5A/6A genotype frequencies of 20.8% 5A/5A, 52.7% 5A/6A, and 26.5% 6A/6A. Using a standardized, controlled protocol, we investigated cytokine- and growth factor-induced MMP-3 expression (real-time polymerase chain reaction [RT-PCR], ELISA) and SV-SMC invasion (Boyden chamber with Matrigel barrier) using cultured SV-SMC from patients with different MMP-3 genotypes. RESULTS: Despite observing a strong correlation between MMP-3 mRNA levels and MMP-3 protein secretion, no significant differences were apparent in MMP-3 expression levels or cell invasion between cells with different MMP-3 5A/6A genotypes. CONCLUSIONS: Our data suggest that the MMP-3 5A/6A promoter polymorphism in isolation does not influence levels of MMP-3 secretion or cellular invasion in human SV-SMC.

The α(2C)-Del322-325 adrenoceptor polymorphism and the occurrence of left ventricular hypertrophy in hypertensives.

OBJECTIVES: Sympathetic activation has a role in the development of left ventricular hypertrophy (LVH). The presynaptic α(2C)-adrenoceptor inhibits the release of norepinephrine from sympathetic nerve terminals in the heart. A deletion polymorphism in the α(2C)-adrenoceptor (α(2C)Del322-325) generates a hypofunctional α(2C)-adrenoceptor, which may result in chronic adrenergic signalling. This study aimed to investigate whether the α(2C)Del322-325 polymorphism was associated with an increased prevalence of LVH in patients with systemic hypertension. METHODS: Left ventricular mass was measured in 205 patients with systemic hypertension and 60 normal volunteers using a 1.5-T Philips MRI system. Genotyping was performed using a restriction fragment length polymorphism assay. RESULTS: No significant difference was observed between the distribution of the α(2C)Del322-325 genotypes in hypertensive patients with LVH compared with those without LVH. Adjusting for confounding variables the odds ratio (OR) of being ins/del for the α(2C)Del322-325 and having LVH was 0.49 (95% CI 0.14-1.69, p = 0.256). CONCLUSIONS: These observations suggest that there is little evidence for an association between α(2C)Del322-325 polymorphism and an increased prevalence of LVH in patients with systemic hypertension.

Seroprevalence of Neospora caninum and Brucella abortus in dairy cattle herds with high abortion rates.

The protozoan Neospora caninum and the bacterium Brucella abortus are well-recognized causes of abortion in dairy cattle. Serum samples (n  =  240) from aborting (n  =  141) and at-risk (n  =  99) animals from 5 herds with high abortion rates in Punjab Province, Pakistan, were tested for antibodies to N. caninum using monoclonal antibody-based ELISA and for antibodies to B. abortus using the serum agglutination test. Antibodies to N. caninum and B. abortus were detected in 105 (43.8%) and 135 (56.3%) cattle, respectively. Prevalences of antibodies to N. caninum and B. abortus were higher in aborting cows (46.8% and 76.6%, P < 0.05) than in animals at risk (39.4% and 27.3%, P > 0.05). Sixty-six animals (27.5%) were seropositive to both N. caninum and B. abortus , and results showed no significant difference (P > 0.05) with respect to geographical district, breed, and age. This is the first report of N. caninum infection among dairy cattle herds in Pakistan.