IGRP and insulin vaccination induce CD8+ T cell-mediated autoimmune diabetes in the RIP-CD80GP mouse.

Autoimmune diabetes is characterized by autoantigen-specific T cell-mediated destruction of pancreatic islet beta cells, and CD8(+) T cells are key players during this process. We assessed whether the bitransgenic RIP-CD80 x RIP-LCMV-GP (RIP-CD80GP) mice may be a versatile antigen-specific model of inducible CD8(+) T cell-mediated autoimmune diabetes. Antigen-encoding DNA, peptide-loaded dendritic cells and antigen plus incomplete Freund's adjuvant were used for vaccination. Of 14 pancreatic proteins tested by DNA vaccination, murine pre-proinsulin 2 (100% of mice; median time after vaccination, 60 days) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (77%, 58 days) could induce diabetes. Vaccination with DNA encoding for zinc transporter 8, Ia-2, Ia-2ß, glutamic acid decarboxylase 67 (Gad67), chromogranin A, insulinoma amyloid polypeptide and homeobox protein Nkx-2.2 induced diabetes development in 25-33% of mice. Vaccination with DNA encoding for Gad65, secretogranin 5, pancreas/duodenum homeobox protein 1 (Pdx1), carboxyl ester lipase, glucagon and control hepatitis B surface antigen (HBsAg) induced diabetes in <20% of mice. Diabetes induction efficiency could be increased by DNA vaccination with a vector encoding a ubiquitin-antigen fusion construct. Diabetic mice had florid T cell islet infiltration. CD8(+) T cell targets of IGRP were identified with a peptide library-based enzyme-linked immunospot assay, and diabetes could also be induced by vaccination with major histocompatibility complex (MHC) class I-restricted IGRP peptides loaded on mature dendritic cells. Vaccination with antigen plus incomplete Freund's adjuvant, which can prevent diabetes in other models, led to rapid diabetes development in the RIP-CD80GP mouse. We conclude that RIP-CD80GP mice are a versatile model of antigen specific autoimmune diabetes and may complement existing mouse models of autoimmune diabetes for evaluating CD8(+) T cell-targeted prevention strategies.

[The care situation of patients with interstitial cystitis in Germany: results of a survey of 270 patients]. / Die Versorgungssituation von Patienten mit interstitieller Zystitis in Deutschland : Ergebnisse einer Umfrage unter 270 Betroffenen.

BACKGROUND: Using a comprehensive questionnaire the care situation of 270 patients with interstitial cystitis (IC) and bladder pain syndrome in Germany was recorded. Despite comprehensive literature on IC (62,000 citations in PubMed) almost nothing is known of the everyday care and quality of patient care in Germany. RESULTS: In total 94% of the patients were women and 6% men, the average age of women was 53.5 years and that of men 67 years and 47.77% of the patients felt that they were well or very well informed about the disease whereby the internet was the source of information in many cases. The exchange of information among patients will increase further through social networks. The diagnosis of IC was made most frequently (62.22%) by biopsy and histological examination followed by urodynamics, potassium test, hydrodistension and cystoscopy. The average duration of the diagnosis was 9 years, 46.67% of the patients consulted a doctor more than 20 times before the diagnosis was made and 51.84% had to pass water more than 14 times per day. Frequency, nocturia and pain were the leading symptoms and 25% of the patients complained of urge incontinence. Among oral medications, analgesics were taken most frequently (61.7%) followed by pentosan polysulphate, antidepressants, antiepileptic drugs, antispasmodics and remedies for urinary urgency. In the self-assessment of the success of treatment with oral medications (helped very well and well), pentosan polysulphate, analgesics, antidepressants and antiepileptic drugs were considered to be the best. Medications that restore the glucosamine lining of the bladder were used predominantly for instillation into the bladder included hyaluronic acid, chondroitin sulphate and a combination of both and pentosan polysulphate. In the self-assessment of the success of treatment with instillation therapy (helped very well or well) the order was: chondroitin sulphate (62.69%), hyaluronic acid (55.77%), a combination of both (53.66%) and pentosan polysulphate (46.30%). The electromotive drug administration (EMDA) procedure with the use of direct current to introduce medications into the bladder wall was mentioned surprisingly often, namely, in 119 patients. In the self-assessment success (helped very well or well) was considered the best for intravesical procedures with 61.34%. CONCLUSIONS: Compared with all drug procedures instillation of medications into the bladder was mentioned 368 times and was assessed by the patients as having helped very well and noticeably by 53.53%, followed by special invasive procedures at 50.56%/271 mentions, alternative therapies at 41.11%/287 mentions and oral medication at 39.75%/1,024 mentions. Hyaluronic acid and chondroitin sulphate products, the combination of both and pentosan polysulphate (oral and intravesical) are not reimbursed by the statutory health insurance. Over 40% of patients treated with these therefore discontinued the treatment for reasons of cost.

[Clinical trials with medical devices: lack of quality illustrated for faecal incontinence]. / Therapiestudien mit Medizinprodukten: Mangelnde Qualität illustriert anhand analer Inkontinenz.

BACKGROUND: Medical devices must be safe and functioning states the law. Treatments with medical devices need not be efficacious to be allowed. We investigated special requirements and problems arising from the law. METHODS: The market for medical devices is contrasted with that for drugs. The requirements of relevant laws are discussed. Finally, published clinical studies on anal incontinence are analysed with respect to their methodological quality. RESULTS: Clinical trials of medical devices for treat-ing anal incontinence are of poor methodological quality thus preventing evaluation of the devices' utility. CONCLUSION: Large, high quality clinical studies of the efficacy of medical devices for treating anal incontinence are urgently needed. Only such studies enable health technology assessment and comprehensible decisions on reimbursement by health insurance.

[3T-AI: a new treatment algorithm for anal incontinence with a higher evidence level]. / 3T-AI: Ein neuer, mit höherer Evidenz versehener Behandlungsalgorithmus bei analer Inkontinenz.

BACKGROUND: The evidence for conservative treatment of anal incontinence is poor. In our first publication [Schwandner et al. Dis Colon Rectum 2010; 53: 1007-1016] we demonstrated that a novel therapeutic concept, termed triple target treatment (3T), combining amplitude-modulated medium frequency stimulation and electromyography biofeedback (EMG-BF) was superior to EMG-BF alone. Questions about the required treatment duration and the relevant subgroups of patients with sphincter damage and damaged anal sensibility were not addressed. METHODS: We enrolled 158 patients with anal incontinence in this randomized study. Here, we -report on the important subgroup analyses of patients with and without sphincter damage and damaged anal sensibility for the endpoints Cleveland Clinic Score (CCS) and success record. Using the results of this study we propose a novel treatment algorithm which is open for discussion. RESULTS: In patients with sphincter damage, the median difference on the CCS from baseline to 9 months was 5 points higher for 3T than for EMG-BF (95 % confidence interval 0-8; p = 0.0168). While 47 % of the patients with sphincter damage became continent with 3T, only 18 % did with EMG-BF (p = 0.0036). Ten of 17 patients in the 3T group regained anal sensibility after 3 months stimulation. There was tendency towards improved continence in patients with neuropathy upon 3T treatment (p = 0.1219). CONCLUSIONS: 3T is superior to EMG-BF alone for patients with sphincter damage and neuropathic anal incontinence. It is a successful key element within our treatment algorithm, even in patients with sphincter damage and neuropathic anal incontinence.

Plasmodesmal targeting and intercellular movement of potato mop-top pomovirus is mediated by a membrane anchored tyrosine-based motif on the lumenal side of the endoplasmic reticulum and the C-terminal transmembrane domain in the TGB3 movement protein.

Live-cell fluorescence microscopy was used to investigate the third triple gene block protein (TGB3) of potato mop-top pomovirus and its role in assisted targeting of TGB2 to plasmodesmata (PD). Wild-type and mutant TGB3 proteins were expressed under the control of the 35S promoter or from a virus reporter clone. Assisted targeting of TGB2 to PD was optimal when the proteins were expressed from a bicistronic plasmid in the relative ratios expected in a virus infection, suggesting that excess TGB3 inhibited PD localisation. Contrary to the generally accepted view, bimolecular fluorescence complementation showed that the TGB3 N terminus is located in the cytosol. Mutational analysis to dissect TGB3 sub domain functions showed that PD targeting was mediated by a composite signal comprising an ER-lumenal tyrosine-based motif and the C-terminal transmembrane domain. Mutation of either of these domains also abolished cell-to-cell movement of the virus. The results are discussed in the context of TGB3 membrane topology.

Early infant diet in children at high risk for type 1 diabetes.

Infant diet affects health and development. The aim of our study was to investigate WHO infant feeding compliance in children who have a first degree family history of type 1 diabetes (T1D). One hundred and fifty children who were first degree relatives of patients with T1D were intensively followed from birth in the BABYDIET intervention study. Infant feeding, infections, and medication were recorded daily by participating families. Weight and length of children were obtained from paediatric records. Only 5% of the families followed the WHO recommendations for infant feeding that include full breastfeeding for at least 6 months (18.8% of children) and introduction of complementary foods under continued breastfeeding thereafter (22.2% of children). Maternal age in the first quartile (<29 years; p<0.0001), and maternal smoking (p<0.0001) were associated with an earlier introduction of solid food and reduced breastfeeding. Full breastfeeding > or =6 months was associated with reduced frequency of gastrointestinal infections (12 vs. 38%, p=0.02) and antibiotic treatment (24 vs. 48%, p=0.04). Our findings indicate that WHO infant feeding recommendations were poorly followed by families with a family history of T1D. Action to improve levels of infant feeding behaviour is essential, especially among young mothers with T1D.

Mucosal insulin vaccination for type 1 diabetes prevention.

Mucosal administration of autoantigen, if efficacious, is an attractive therapeutic approach for prevention of type 1 diabetes. It can prevent or delay autoimmune diabetes in animal models, but although shown to be safe has not yet been proven effective in human studies. Efficacy may depend upon the dose and route at which antigen is administered, the stage in type 1 diabetes pathogenesis at which treatment is initiated, and the study cohort that is treated. Here we address these issues and propose a clinical translation possibility for mucosal insulin administration in genetically at risk individuals.

[Present state of radio frequency ablation of liver tumors in Germany]. / Aktueller Stand der Radiofrequenzablation von Lebertumoren. Eine deutschlandweite Umfrage.

INTRODUCTION: Radio frequency ablation (RFA) represents a new technique for local destruction of liver tumors. Indication and procedure are poorly validated at present. METHOD: To record the actual nationwide RFA application in Germany, a survey of 2026 hospitals with standardized questionnaires was conducted. RESULTS: With a respond rate of 17.5%, results from 58 hospitals covering 1700 ablated patients were analyzed. In 25.9% of them, RFA is already used in potentially curative resectable tumors, in 22.4% even when incomplete ablation for tumor mass reduction is expected. Of the 58 hospitals, 75% combine resection and RFA to reach a so-called R-0 situation. The maximal tumor sizes they quoted for achieving complete ablation ranged from <3 cm to 11 cm. In contraindications for RFA, the tumor size, number of tumors, critical localization of the tumor, and disorders of liver function were mentioned the most. CONCLUSION: This survey documents significant discrepancies in indication, application, procedure, and results in RFA for liver tumors. The lack of general standards and an overestimation of the method may lead to uncritical application, neglect of standard therapy, or unsatisfying results.

Subcellular localisation, protein interactions, and RNA binding of Potato mop-top virus triple gene block proteins.

Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied. The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata. In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata. However, the process is facilitated by RNA-protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2.

Real-time water quality monitoring and regression analysis to estimate nutrient and bacteria concentrations in Kansas streams.

An innovative approach currently is underway in Kansas to estimate and monitor constituent concentrations in streams. Continuous in-stream water-quality monitors are installed at selected U.S. Geological Survey stream-gaging stations to provide real-time measurement of specific conductance, pH, water temperature, dissolved oxygen, turbidity, and total chlorophyll. In addition, periodic water samples are collected manually and analyzed for nutrients, bacteria, and other constituents of concern. Regression equations then are developed from measurements made by the water-quality monitors and analytical results of manually collected samples. These regression equations are used to estimate nutrient, bacteria, and other constituent concentrations. Concentrations then are available to calculate loads and yields to further assess water quality in watersheds. The continuous and real-time nature of the data may be important when considering recreational use of a water body; developing and monitoring total maximum daily loads; adjusting water-treatment strategies; and determining high constituent concentrations in time to prevent adverse effects on fish or other aquatic life.

High glycolytic activity in rat glioma demonstrated in vivo by correlation peak 1H magnetic resonance imaging.

High-grade brain tumors are known to have a high rate of glucose (Glc) consumption. Postmortem measurements have suggested that Glc content in experimental brain tumors is relatively low. We used magnetic resonance spectroscopy to investigate this, in vivo, in the brains of seven rats bearing intracerebral C6 gliomas. We combined the high spectral resolution allowed by two-dimensional proton nuclear magnetic resonance with spatial encoding by magnetic field gradient pulses to obtain in vivo maps of Glc, alanine, hypotaurine, aspartate, phosphoethanolamine, Glu/Gln, N-acetylaspartate (NAA), phosphocreatine/creatine (PCr/Cr), choline-containing compounds, and lactate (Lac) (some of which are involved in energy metabolism). Compared with normal brain tissue, the main differences found in the gliomas were that Glc, NAA, PCr/Cr, and aspartate concentrations were much lower, whereas concentrations of alanine, hypotaurine, phosphoethanolamine, and Lac were higher, whatever the extent of necrosis. A striking observation is the similarity of the NAA and Glc images: the concentrations of both metabolites are lower in the tumor than they are in the contralateral brain. If Glc was completely absent from the tumor tissue, and if the residual Glc level was due only to a partial volume effect like that for NAA, a neuronal marker, the ratio [Glc]tumor/[Glc]contralateral tissue, should be similar to that found for NAA. The ratio for Glc was 0.48 +/- 0.22 (+/- SD; n = 6), a ratio similar to that found for PCr/Cr (0.50 +/- 0.19) but significantly higher than that obtained for NAA (0.29 +/- 0.07). This observation indicates that a measurable Glc concentration is still present in the tumor tissue. Intense glycolysis in tumor cells may explain the increased production of Lac and alanine and decreased amount of Glc. These nuclear magnetic resonance measurements of metabolite concentrations are complementary to positron emission tomography, which measures Glc consumption.

Pharmacological investigation on nigrescigenin-a cardenolide from Parquetina nigrescens (Afzel.) Bullock: comparative studies on cardiotonic effects of Parquetina nigrescens, g-strophanthin and noradrenaline in guinea-pig isolated atria.

The cardiotonic and catecholamine-like effects of Parquetina nigrescens extract-induced contractile force of guinea-pig left and right atria were investigated in-vitro. Isometric contractions were recorded. P. nigrescens extract, 5-150 microg mL(-1), increased the force of contraction dose dependently in electrically driven left atria. The concentration of P. nigrescens extract producing 50% of the maximal effect (EC50 value) was 7.5 microg mL(-1). The positive inotropic response differed from that of g-strophanthin by its high rate of onset and its complete reversibility upon removal of the extract from the incubation medium. In spontaneously beating right atrial muscle, P. nigrescens extract increased the rate of contractions. Its positive chronotropic and inotropic effects were partly antagonized by propranolol and atenolol indicating the presence of an adrenergic acting principle in P. nigrescens extract. In contrast, the inotropic response to P. nigrescens extract could not be completely suppressed by beta-blocking agents, suggesting that the force of contraction is not only increased by a sympathomimetic ingredient of P. nigrescens extract but also by the cardenolides known to be present in P. nigrescens.

Sympathomimetic effects of Parquetina nigrescens (Periplocaceae) extract in isolated portal vein smooth muscle.

The purpose of this study was to examine the mechanisms underlying the pharmacological effects of the extract of Parquetina nigrescens (Expar) on vascular smooth muscle contractility. To evaluate the Expar effect, the contractile activity of portal veins isolated from Wistar Kyoto rats was isometrically recorded. Isolated portal vein preparations developed rhythmic and spontaneous contractile activity. Expar increased the contractile response of the portal vein preparations in a dose-dependent manner. The maximal effect of the dose-response curve for Expar was prevented by the alpha1-adrenergic blocking agent prazosin at 10 nM and 30 nM concentration dependently. The contractile responses of the muscle to Expar were partly blocked after chemical sympathectomy of the preparations with 6-hydroxydopamine, and those obtained in the same conditions with tyramine were completely abolished, whereas responses to noradrenaline were unaffected by the 6-hydroxydopamine pretreatment. It is concluded that Expar contains principles, which can be characterized as direct and indirect sympathomimetic.

Genomic organization of the HSET locus and the possible association of HLA-linked genes with immotile cilia syndrome (ICS).

The kinesin-related protein (HSET) gene belongs to the kinesin superfamily, the members of which are involved in cellular transport processes. The HSET gene product was previously characterized by partial cDNA sequencing. The gene is located on the short arm of human Chromosome 6 (6p21.3), at the centromeric end of the major histocompatibility complex. Here, we report the genomic structure of the complete HSET gene together with its flanking loci. Sequence analysis of the 40 kilobase (kb) cosmid clone containing the HSET gene also revealed the presence of several new genes not related to the kinesin superfamily. These include a 60S ribosomal protein L35A-like pseudogene (rPL35A-like) on the telomeric side and a polycomb-like gene (PHF1), a copper tolerance-like gene (CUTA1) and the 5' part of the synaptic ras-GTPase-activating protein (SynGAP) gene centromeric of HSET. In addition, a complete 60S ribosomal protein L12-like (rPL12L) gene in intron 3 of the HSET gene was identified which appears to have an open reading frame. The possible involvement of the HSET gene and a beta-tubulin gene (TUBB) in the pathogenesis of immotile cilia syndrome (ICS) was studied by screening two unrelated ICS families with microtubular defects and suspected HLA linkage for mutations within the HSET gene and the TUBB gene. Four single base substitutions were detected in the HSET gene, and none in the TUBB gene. On the basis of these data, a role of the HSET and TUBB products in the pathogenesis of ICS in the two families is unlikely.

Evaluation of the MTHFR C677T allele and the MTHFR gene locus in a German spina bifida population.

UNLABELLED: A number of recent studies have demonstrated that the occurrence and recurrence risk of neural tube defects (NTD) is reduced by folic acid supplementation before and during pregnancy. Epidemiological studies have shown low plasma folate and raised plasma homocysteine in women with spina bifida aperta (SB) children suggesting an abnormal folate metabolism. The 5,10-methylenetetrahydrofolate reductase (MTHFR) variant C677T, resulting in a decreased activity of the enzyme, has been associated with the development of NTD. Several studies demonstrated that homozygosity for the C677T mutation occurs at a higher frequency in patients with SB phenotype than in control individuals. The SB risk is strongest if both the mother and her child have the mutation in the homozygous state. In the present study we compared the frequency of the C- and T-alleles in healthy German individuals (n = 153) with German SB patients (n = 137). Our study groups reveal no significant difference in C/T-allele frequencies and genotype distributions. A family based association study, the transmission disequilibrium test, confirms the absence of an association between T-allele and SB. In 9 of 40 families we were able to exclude linkage to the MTHFR locus (1p36.3) employing different inheritance models. CONCLUSION: Our data show no evidence for an association between the C677T mutation and the occurrence of the SB phenotype. Therefore we cannot support the hypothesis that the MTHFR variant does account for a significant genetic predisposition to the SB phenotype in the studied German patients.

Effects of leaf extract of Caesalpinia bonduc (Caesalpiniaceae) on the contractile activity of uterine smooth muscle of pregnant rats.

The calcium dependency and the cholinergic effect of the leaf extract of Caesalpinia bonduc Roxb. (Caesalpiniaceae) was studied in isolated pregnant rat myometrium preparations. Isometric contractions were recorded. The extract (Cebo) increased the contractile force in the isolated strips in a concentration-dependent manner. The effects were comparable to those obtained with acetylcholine. Contractions induced by Cebo or acetylcholine were inhibited in the presence of atropine. The stimulating action of Cebo on the contractile responses of isolated myometrium preparations inhibited by atropine may be mediated by cholinergic receptors. In calcium-free solution Cebo induced a tonic contraction (contracture) of the muscle. Moreover, in high-potassium calcium-free solution Cebo caused contracture of the uterine smooth muscle. Cebo was still able to elicit contractions in calcium-free solution containing EDTA or EGTA. These findings suggest the existence of cholinergic receptors sensitive to Cebo which could influence the influx of calcium (phasic contraction) and mobilization of calcium from cellular stores (tonic contraction), both of which are responsible for the increase of contractile activity and development of the contracture of uterine smooth muscle.

Selection of single-chain variable fragment antibodies to black currant reversion associated virus from a synthetic phage display library.

ABSTRACT Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.

A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA.

A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.