Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum

Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97?bp) showed Tm of 78°C for both biovars. The pSG amplicon (273?bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260?bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 10⁸–10¹ CFU of these biovars.     

A Comparison of Mental Health Hospital Admissions in a Cohort of Heroin Users Prior to and After Rapid Opiate Detoxification and Oral Naltrexone Maintenance

Mental health (MH) hospital admissions were investigated in a cohort (N = 1184) of heroin dependent persons using linked health records. All MH in-patient admissions were extracted 36 months before to 36 months after commencing rapid opioid detoxification (ROD) and oral naltrexone. Results show that the incidence rate ratio (IRR) of drug-related and other MH admissions peaked in the 3 months immediately prior to treatment. All categories subsequently declined to baseline levels by 36 months following treatment. The authors conclude that treatment for heroin dependence reduces risk of MH admissions.     

Our Tiny Organization Packs a National Wallop

The relationships between drinking history, housing history, several clinical variables and treatment retention after detoxification were evaluated in 109 inner-city alcoholics. Compared to men with a fisxed abode, homeless subjects (N = 50) reported a history of alcoholism of ealry onset and increased severity. No differences in aftercare retention were found between homeless and non-homeless subjects.     

Efficacy and safety of long-term oral falecalcitriol treatment in patients with renal osteodystrophy

We investigated, in a multicenter study, the efficacy and safety of long-term administration of falecalcitriol, a new active vitamin D₃, in patients with renal osteodystrophy of the osteitis fibrosa type associated with secondary hyperparathyroidism caused by chronic renal failure. Falecalcitriol was orally administered every day for 48 weeks. Administration was started at a dosage of 0.3 μg/day, and the dosage was changed whenever necessary according to serum calcium (Ca) level. As a result, significant inhibition of the bone resorption markers, i.e., intact parathyroid hormone (i-PTH), pyridinoline (Pyr), and deoxypyridinoline (D-Pyr), was observed from the 8th week, and the bone formation markers, i.e., total activity and bone fraction of alkaline phosphatase, were also significantly inhibited from the 12th week. The bone mineral density (BMD) change rate in the bones of the whole body determined by dual-energy X-ray absorptiometry remained almost constant. When subjects were stratified according to the inhibition rate of bone metabolic parameters, BMD tended to increase in the group with strong inhibition and to decrease in the group with weak inhibition. Mean serum Ca level significantly increased from 9.5 mg/dl, but mean level was subsequently maintained at about 10 mg/dl until the end of administration by adjustment of the doses. These findings suggested that falecalcitriol may inhibit and normalize accelerated bone metabolic turnover without inducing excessive increases in serum Ca level in secondary hyperparathyroidism. With respect to safety, no specific adverse reactions associated with the prolonged administration period were observed. Received: July 1, 1997 / Accepted: Aug. 29, 1997     

Transient ischemia-induced changes of interleukin-2 and its receptor beta immunoreactivity and levels in the gerbil hippocampal CA1 region

Interlukin-2 (IL-2) is an important cytokine in the brain: IL-2 and its receptors are involved with inflammatory processes. Chronological changes in IL-2 level in serum, and IL-2 and its receptor (IL-2 receptor beta, IL-2Rbeta) immunoreactivities and levels were examined in the hippocampal CA1 region after transient forebrain ischemia in gerbils. IL-2 level in serum significantly decreased 12 h after ischemia/reperfusion. IL-2 immunoreactivity was detected in the somata of pyramidal cells in sham-operated group. At 15 min after ischemia, IL-2 immunoreactivity was shown in non-pyramidal cells as well as pyramidal cells. One day after ischemia, IL-2 immunoreactivity was lowest, and IL-2 immunoreactivity is shown in non-pyramidal cells from 2 days after ischemia. Four days after ischemia, IL-2 immunoreactivity was shown in dying pyramidal cells. IL-2Rbeta immunoreactivity in the sham-operated and 15 min-3 min post-ischemic groups is detected in the cell membrane of pyramidal cells. From 3 h after ischemia, IL-2Rbeta immunoreactivity is found in cytoplasm and nuclei, but not in cell membrane. IL-2Rbeta immunoreactivity decreases from 6 h after ischemia and is shown mainly in non-pyramidal cells from 3 days after ischemia. The data of Western blot analyses for IL-2 and IL-2Rbeta was similar to the immunohistochemical data. IL-2 infusion into cerebrospinal fluid did not protect hippocampal neurons from ischemic damage. These results suggest that IL-2 and IL-2Rbeta show malfunction from 3 h after ischemia, and exogenous IL-2 does not protect ischemic neuronal damage.     

Neuronal cytoskeleton and synaptic densities are altered after a chronic treatment with the cannabinoid receptor agonist WIN 55,212-2

Cannabinoid CB1 receptors are the most abundant G-protein-coupled receptors in the brain. Its presynaptic location suggests a role for cannabinoids in modulating the release of neurotransmitters from axon terminals by retrograde signaling. The neuroprotective effects of cannabinoid agonists in animal models of ischemia, seizures, hypoxia, Multiple Sclerosis, Huntington and Parkinson disease have been demonstrated in several reports. The proposed mechanism for the neuroprotection ranges from antioxidant effects, reduction of microglial activation and anti-inflammatory reaction to receptor-mediated reduction of glutamate release. In the present work, we analyzed the morphological changes induced by a chronic treatment with the synthetic cannabinoid receptor agonist, WIN 55,212-2, in four brain regions where the CB1 cannabinoid receptor is present in high density: the CA1 hippocampal area, corpus striatum, cerebellum and frontal cortex. After a twice-daily treatment for 14 days with the cannabinoid receptor agonist (3 mg/kg sc, each dose) to male Wistar rats (150-170 g), the expression of neurofilaments (Nf-160 and Nf-200), microtubule-associated protein-2 (MAP-2), synaptophysin (Syn) and glial fibrillary acidic protein (GFAP) was studied by immunohistochemistry and digital image analysis. Ultrastructural study of the synapses was done using electron microscopy. After the treatment, a significant increase in the expression of neuronal cytoskeletal proteins (Nf-160, Nf-200, MAP-2) was observed, but we did not find changes in the expression of GFAP, the main astroglial cytoskeletal protein. In cerebellum, there was an increase in Syn expression and in the number of synaptic vesicles, while, in the hippocampus, an increase in the Syn expression and in the thickness of the postsynaptic densities was observed. The results obtained from these studies provide evidences on the absence of astroglial reaction and a sprouting phenomena induced by the WIN treatment that might be a key contributor to the long-term neuroprotective effects observed after cannabinoid treatments in different models of central nervous system (CNS) injury reported in the literature.     

The CSF concentration of ADMA, but not of ET-1, is correlated with the occurrence and severity of cerebral vasospasm after subarachnoid hemorrhage

Insulin-like growth factor-1 (IGF-1) has been demonstrated to have neuroprotective effects, but little is known concerning its role in vascular dementia (VaD). This study aimed to evaluate expression of IGF-1 signaling in hippocampus in rat model of VaD, and probe the underlying mechanisms. Permanent occlusion of bilateral common carotid arteries (2-VO) was used as VaD model. Learning and memory functions were declined significantly in 2-VO rats, and these impairments were further deteriorated with the prolongation of 2-VO treatment. IGF-1, IGF-1 receptor (IGF-1R), total Akt and phosphorylated Akt (p-Akt) were all measured at 1, 2 and 4 months following 2-VO injury. Compared with controls, IGF-1, IGF-1 mRNA and p-Akt expression were significantly decreased in hippocampus of 2-VO rats. However, changes of IGF-1R and total Akt levels were not significant. These results suggest that down-regulation of IGF-1 and p-Akt may contribute to the impairments of learning and memory functions after 2-VO. IGF-1/IGF-1R signaling system may involved in the onset and development of VaD.     

八例慢性肾衰患者的骨组织计量学研究

本文采用美国８７４００－０００型骨环钻（内径约７．５ｍｍ）作髂骨骨活检，不脱钙骨组织切片，作骨组织计量学参数测定．结果表明：氮质血症期骨组织形态参数已显示异常，主要表现骨形成增加，成骨细胞活性增高，骨吸收正常。尿毒症早期骨形成及骨吸收均增加，骨小梁体积减少，骨矿化障碍。尿毒症晚期主要表现为骨吸收明显增加，骨矿化障碍严重。维持血透患者除骨吸收增加及骨矿化障碍外，还存在骨组织纤维化形成。     

Individual trabecula segmentation (ITS)-based morphological analyses and microfinite element analysis of HR-pQCT images discriminate postmenopausal fragility fractures independent of DXA measurements

Osteoporosis is typically diagnosed by dual-energy X-ray absorptiometry (DXA) measurements of areal bone mineral density (aBMD). Emerging technologies, such as high-resolution peripheral quantitative computed tomography (HR-pQCT), may increase the diagnostic accuracy of DXA and enhance our mechanistic understanding of decreased bone strength in osteoporosis. Women with (n = 68) and without (n = 101) a history of postmenopausal fragility fracture had aBMD measured by DXA, trabecular plate and rod microarchitecture measured by HR-pQCT image-based individual trabecula segmentation (ITS) analysis, and whole bone and trabecular bone stiffness by microfinite element analysis (μFEA) of HR-pQCT images at the radius and tibia. DXA T-scores were similar in women with and without fractures at the spine, hip, and 1/3 radius, but lower in fracture subjects at the ultradistal radius. Trabecular microarchitecture of fracture subjects was characterized by preferential reductions in trabecular plate bone volume, number, and connectivity over rod trabecular parameters, loss of axially aligned trabeculae, and a more rod-like trabecular network. In addition, decreased thickness and size of trabecular plates were observed at the tibia. The differences between groups were greater at the radius than the tibia for plate number, rod bone volume fraction and number, and plate-rod and rod-rod junction densities. Most differences between groups remained after adjustment for T-score by DXA. At a fixed bone volume fraction, trabecular plate volume, number, and connectivity were directly associated with bone stiffness. In contrast, rod volume, number, and connectivity were inversely associated with bone stiffness. In summary, HR-pQCT-based ITS and μFEA measurements discriminate fracture status in postmenopausal women independent of DXA measurements. Moreover, these results suggest that preferential loss of plate-like trabeculae contribute to lower trabecular bone and whole bone stiffness in women with fractures. We conclude that HR-pQCT-based ITS and μFEA measurements increase our understanding of the microstructural pathogenesis of fragility fracture in postmenopausal women.     

Antigenicity of linear and cyclic peptides mimicking the disulfide loops in HIV-2 envelope glycoprotein: synthesis,reoxidation and purification

The external envelope glycoprotein (gp125) of human immunodeficiency virus type 2 (HIV-2) contains 22 cysteine residues. The positions of the 11 disulfide bridges in HIV-2 gp125 were determined by analogy with the experimental position of the disulfide bonds found in the gp120 of HIV-I. Peptides expected to mimic all 11 disulfide-bonded domains containing from 13 to 47 amino acids were synthesized by the solid-phase method according to 9-fluorenylmethoxycarbonyl strategy, except for peptide 5, which was assembled according to t-butoxycarbonyl (Boc) strategy. Analysis of all the crude peptides showed that the expected peptides were obtained with good yields, between 75% and 85%. Peptides were purified further by high-performance liquid chromatography (HPLC) on an Aquapore RPC30 C8 column. Peptide homogeneity was more than 90%. For each peptide, linear peptides (L) were SH-iodoacetamidated, whereas cyclization of peptides (C) was performed by air oxidation. Oxidation kinetics was followed with the Ellman test and HPLC. Cyclic peptides were purified by HPLC and characterized by fast atom bombardment mass spectrometry. This analysis showed that a small quantity (?10%) of dimeric peptides (2 and 8) and cyclic peptides containing oxidized methionine or tryptophan residues (4, 9 and 10) were formed. To assess the relevance of conformation for the antigenicity of disulfide-bonded loops of HIV-2 gp125, the antigenicity of linear and cyclic peptides was tested against a set of 76 HIV-2 positive human sera by enzyme-linked immunosorbent assay. Peptides 2, 4 and 9, mimicking the VI, V2 and V3 regions of the external envelope glycoprotein (gp 125) of HIV-2, were the most highly reactive with HIV-2 positive human sera tested at the dilution of 150. Cyclic peptides generally were recognized more than linear peptides, as shown by their greater inhibition (2 to 10 times more) of antigen-antibody complexes. Structure-antigenicity of peptide V3, the most reactive peptide (75% of the HIV-2 positive sera tested), was analyzed further. Cyclic peptide 9C had a higher affinity for anti-gpl25 antibodies than linear peptide 9L. In addition, circular dichroism showed that linear and cyclic peptides 9 had a similar structure, but when analyzed in aqueous solution or in trifluoroethanol (TFE), the structural difference shown with antibodies was not confirmed. No significant difference was observed between the antigenicity of linear and cyclic peptides 1, 8 and 11, mimicking the C1, C2 and C4 regions of HIV-l gp125. These peptides were weakly reactive with HIV-2 positive sera. This result agrees with the low immunogenicity of conserved regions. © Munksgaard 1998.