Association of variants in renal salt reabsorption-related gene SLC12A3 with essential hypertension in a Mongolian population.

Mounting evidence has implicated the SLC12A3 gene in essential hypertension. Here, we examined the potential associations of common variants of the SLC12A3 gene with blood pressure traits in Mongolians in China. Genomic DNA was extracted from 508 unrelated Mongolian patients with essential hypertension and 246 normotensive Mongolian subjects for genotyping. The genotype distributions of all selected polymorphisms were consistent with Hardy-Weinberg equilibrium. The presence of the G allele in the rs7187932 polymorphism was found to be associated with an increased risk of hypertension (OR: 1.30; 95%CI = 1.00-1.38; P = 0.048), whereas the rs2399594 G allele was associated with a reduced risk for hypertension (OR: 0.76; 95%CI = 0.60-0.97; P = 0.030). No significant difference was observed for other alleles. Haplotype analysis revealed an association of the rs2399594 and rs711746 GG haplotype with a reduced risk for hypertension (OR: 0.76; 95%CI = 0.60-0.97; P = 0.029). No significant association was observed between other haplotypes and hypertension. These results suggest that the SLC12A3 gene is a susceptibility gene for hypertension in the Mongolian population.

Evolution of a strain of CJD that induces BSE-like plaques.

Bovine spongiform encephalopathy (BSE) has become a public health issue because a recently evolved BSE agent has infected people, yielding an unusual form of Creutzfeld-Jakob disease (CJD). A new CJD agent that provokes similar amyloid plaques and cerebellar pathology was serially propagated. First-passage rats showed obvious clinical signs and activated microglia but had negligible PrP-res (the more protease-resistant form of host PrP) or cerebellar lesions. Microglia and astrocytes may participate in strain selection because the agent evolved, stabilized, and reproducibly provoked BSE-like disease in subsequent passages. Early vacuolar change involving activated microglia and astrocytes preceded significant PrP-res accumulation by more than 50 days. These studies reveal several inflammatory host reactions to an exogenous agent.

Small virus-like structure in brains from cases of sporadic and familial Creutzfeldt-Jakob disease.

We have previously observed small virus-like particles in the brain of hamsters with experimental scrapie. Here we report that small virus-like structures can be isolated from brains of patients with Creutzfeldt-Jakob disease and identified by electronmicroscopy.

Detection of proteinase-resistant protein (PrP) in small brain tissue samples from Creutzfeldt-Jakob disease patients.

We describe a short and a sensitive method to isolate PrP in small samples of brain tissue using a one day procedure. The tissue was homogenized in sarkosyl, cleared by low-speed centrifugation, and then ultracentrifuged. The pellet was suspended in 10 mM Tris-HCl, 10% NaCl, 1% sarkosyl, precipitated by centrifugation and re-suspended in the above solution with proteinase K. After digestion, PrP was spun down, electrophoresed on a 15% SDS-polyacrylamide minigel and then electro-transferred to a nitrocellulose membrane. The blots were processed with rabbit polyclonal antibody against hamster PrP27-30. Four bands of PrP with molecular weights of 28-30 kDa, 24-26 kDa, 19-20 kDa, and 16 kDa were clearly detected by Western blot in two samples obtained by brain biopsy. To test the sensitivity and the specificity of our method we also purified PrP from 20, 50 and 100 mg of cerebral cortical tissues taken from six frozen CJD brains and one Alzheimer's disease brain of our collection. All the CJD samples, but not the Alzheimer's disease one, resulted positive by Western blot. In the smallest sample tested (20 mg), there was at least one band (about 25 kDa) of PrP detectable by Western blot. Thus, this is a valid and efficient method for the diagnosis of CJD in small brain tissue samples.

Characterisation of antisera raised against species-specific peptide sequences from scrapie-associated fibril protein and their application for post-mortem immunodiagnosis of spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSE), such as scrapie or Creutzfeldt-Jakob disease (CJD), are fatal neurodegenerative diseases of the central nervous system caused by a yet unidentified virus. They are accompanied by a brain specific amyloidosis, during which a host coded protein irreversibly aggregates to form the scrapie-associated fibrils. The diagnosis of TSE relies on histopathological detection of spongiform lesions, on electron microscopical detection of fibrils, or on the immunological detection of SAF protein, which is the most specific diagnostic marker. In order to improve the diagnosis of TSE, we developed a protocol for rapid tissue fractionation and enrichment of SAF protein which subsequently allows the specific detection of SAF protein by western blotting and immunodetection. Using some new antisera raised against synthetic peptides with sequences specific for the hamster, sheep, cattle and human SAF protein, several samples can be diagnosed for TSE within 24 hours, starting with only 10-100 mg of brain tissue from different species.

A new point mutation of the prion protein gene in Creutzfeldt-Jakob disease.

Complete sequencing of the prion protein open reading frame of a 68-year-old woman affected by a familial form of Creutzfeldt-Jakob disease (CJD) revealed a new mutation at codon 210 resulting in the substitution of isoleucine for valine. Moreover, a new 24-bp deletion encompassing codons 54 to 61 or 62 to 69 was found in the other allele. Four of the 17 asymptomatic relatives tested carry the 210 mutation. Two of them were 81 and 82 years old. Four of 22 patients with CJD whose recorded familial history was negative for demented illnesses, but none of 103 healthy control subjects, tested positive for the 210 mutation. These data suggest that the 210 mutation is associated with CJD, but that environmental factors or incomplete penetrance may contribute to the development of the disease. This finding also suggests that in Italy, familial CJD is more common than previously reported.

Amphotericin B treatment dissociates in vivo replication of the scrapie agent from PrP accumulation.

Scrapie and related animal and human disorders are neurodegenerative diseases characterized by the formation of a modified, partly proteinase-resistant protein (PrP) of the host, which tends to aggregate as amyloid fibrils and accumulate in the brain of infected individuals. There is a general consensus that the pathological form of PrP (PrPSc) is essential for the clinical appearance of the disease, but whether it is part of the scrapie agent or a by-product of viral infection is still controversial. Here we report that treatment of scrapie-infected hamsters with amphotericin B delays the accumulation in the brain of the proteinase-resistant portion of PrPSc by about 30 days without affecting scrapie replication. The consequence is that hamsters treated with amphotericin B developed clinical signs of disease later than infected controls. We argue that the proteinase-resistant portion of PrPSc is necessary for the development of the disease but that it is unlikely to be essential for scrapie replication.

Sulphate polyanions prolong the incubation period of scrapie-infected hamsters.

The effect of the organic sulphated polyanions, pentosan sulphate (SP54), dextran sulphate 500 (DS500) and suramin, have been tested on golden Syrian hamsters infected with the 263K strain of scrapie by the intraperitoneal (i.p.) or the intracerebral route. SP54 had the greatest effect in prolonging the incubation period of the disease when administered within 2 h of the i.p. inoculum. The same amount of SP54 given 24 h after scrapie inoculation had a potent effect in some animals and no effect in others. This result suggests that SP54 inhibits the uptake of the scrapie agent into the nerve endings and/or carrier cells at the site of the inoculum, i.e. the peritoneum, and that this event occurs in about 24 h. DS500 had a similar although less potent effect (22.4 days delay during the incubation period) than SP54 (54.4 days) when administered within 2 h of scrapie injection by the i.p. route, and suramin had only a minimal effect (10 days). This study suggests that treatment of scrapie and related spongiform encephalopathies of animals and man is possible only before the agent has reached the clinical target areas of the brain.

Experimental drug treatment of scrapie: a pathogenetic basis for rationale therapeutics.

Pharmacological treatment with polyanions or amphotericin B in hamsters with experimental scrapie reveals that it is possible to delay the appearance of the disease only when the drug is given before the invasion of the agent into the clinical target areas of the brain. We suggest such early treatment may be possible for individuals at high risk of acquiring the disease, such as healthy mutation-positive relatives of patients with familial Creutzfeldt-Jakob disease or Gerstmann-Sträussler syndrome, or recipients of potentially contaminated pituitary-extracted human growth hormone.

Measurement of the concentration of amphotericin B in brain tissue of scrapie-infected hamsters with a simple and sensitive method.

A simple, sensitive, and reproducible assay for the measurement of the amphotericin B concentration in tissue extracts was developed by using the fourth derivative of the absorption spectrum of amphotericin B between wavelengths of 330 and 430 nm. The amphotericin B concentration in spleen and brain was proportional to the total amount administered. The amphotericin B concentration in the brain was highly correlated with the increase in the mean incubation period of intracerebrally scrapie-infected hamsters.

Combination ultrafiltration and 6 M urea treatment of human growth hormone effectively minimizes risk from potential Creutzfeldt-Jakob disease virus contamination.

Although genetically engineered human growth hormone (hGH) is now commercially available, native pituitary-derived hGH is still used by physicians in many countries for the treatment of hormone deficiency states. We describe a method using ultrafiltration and 6 M urea that reduced infectivity in human pituitary tissue that had been deliberately contaminated with scrapie virus (an animal analogue of human Creutzfeldt-Jakob disease virus) from an initial level of 10(9.7) infectious units to just 5 infectious units. Based on estimates of the frequency of contamination and infectivity levels in batches of human pituitaries, the use of this protocol to prepare GH from cadaveric human glands yields a calculated probability of exposure to a contaminated vial of not greater than 1 in 3.2 million recipients; therefore, native hormone prepared by this method may be considered to be essentially risk-free. The same methodology may be useful in the preparation of other hormones, such as prolactin, for which no synthetic substitutes are currently available, as well as biological products derived from sheep or cattle, that may be infected with scrapie or bovine spongiform encephalopathy.

Levels of infectivity in the blood throughout the incubation period of hamsters peripherally injected with scrapie.

Viremia is found in intraperitoneally scrapie-injected hamsters. The absence of a viremic peak before the beginning of scrapie replication in the brain suggests either that the spread of the agent to the brain is not via the blood or that early after infection, circulating monocytes carry the agent to the brain where it remains silent until the neural cells start replicating it.