Use of platelet rich plasma in human infertility.

Successful embryo implantation requires good quality embryo but also needs a receptive endometrium site. In our clinical practice, we daily verify that an adequate endometrial growth is reached for successful implantation. To understand whether platelet rich plasma (PRP) can improve endometrium thickness and performance, PRP treatment was carried out after at least three of the classic medical protocols currently in use had been unsuccessfully adopted. Eight patients with more than 3 cryo-transfers cancelled because of failure of endometrial growth, defined as endometrium less than 6 mm, with negative hysteroscopic screening for endometrial pathology, and with negative bacteriologic screening, before present and all previous treatment, were selected to undergo PRP treatment. In 7 out of 8 treatments, an endometrial thickness greater than 6.5 mm (mean 6.9 mm) was reached, with endometrial three-layer pattern, before progesterone administration and embryo transfer was performed. In 6 out of 7 patients, who underwent embryo transfer, beta-HCG were positive, with 2 biochemical abortions, one miscarriage at 6-week pregnancy, two babies born and one drop-out. In this study, 8 patients had extraordinarily poor endometrial quality, and the endometrium was non-responsive to conventional estrogenic therapy, resulting in cycle cancellation. After application of PRP, the endometrial thickness was satisfactory in all the patients except one. Of these, beta-HCG was positive in 6 women, the pregnancy was progressing normally in 2 women, and one had an early miscarriage. We can suppose that the multiple implantation failures were caused by inefficient expression adhesion molecules, which can hypothetically be more represented after PRP application.

[Dermatomyositis with tonsillar carcinoma. Rare presentation as paraneoplastic syndrome]. / Dermatomyositis bei Tonsillenkarzinom. Seltener Zusammenhang als paraneoplastisches Syndrom.

BACKGROUND: Malignant tumors occur in up to 15 % of patients with paraneoplastic syndromes. The temporal association between malignancy and paraneoplasia is variable. Dermatomyositis belongs to the facultative cutaneous paraneoplasia. CASE REPORT: A patient presented with a cervical swelling and preexisting dermatomyositis. Staging revealed a tonsillar carcinoma with cervical, mediastinal and bone metastasis, and meningeal carcinomatosis. Systemic intrathecal chemotherapy was initiated. CONCLUSION: Dermatomyositis has only been described four times worldwide as a paraneoplastic disease with tonsillar carcinoma. Upon occurrence of a paraneoplastic syndrome, an intensive search for tumours is required at regular intervals until the primary tumor is diagnosed.

A framework for evaluation of on-farm mastitis diagnostics in Australia.

Numerous culture-based diagnostics are available on the Australian and international markets for on-farm detection of bacterial pathogens in milk. Use of such diagnostics may provide an opportunity to improve the prudent use of antimicrobials in udder health management. Farms are low-resource settings in terms of diagnostic microbiology capacity. The World Health Organisation has identified criteria for the evaluation of diagnostic tests in low resource settings based on Accuracy, Sensitivity, Specificity, User-friendliness, being Rapid or Robust, Equipment-free and being Deliverable (ASSURED). Here, we review how those criteria can be interpreted in the context of microbiological diagnosis of mastitis pathogens, and how on-farm diagnostics that are currently available in Australia perform relative to ASSURED criteria. This evaluation identifies multiple trade-offs, both with regard to scientific criteria and with regards to convenience criteria. More importantly, the purpose of testing may differ between farms, and test performance should be evaluated relative to its intended use. The ability of on-farm mastitis diagnostics to inform mastitis treatment decision-making in a timely and cost-effective manner depends not just on test characteristics but also on farm-specific pathogen prevalence, and on the farm enterprise's priorities and the farm manager's potential courses of action. With most assay evaluations to date conducted in professional laboratories, there is a surprising dearth of information on how well any of the diagnostic tests perform on-farm and, indeed, of the on-farm decision-making processes that they aim to inform.

Central role of PKCα in isoenzyme-selective regulation of cardiac transient outward current Ito and Kv4.3 channels.

The transient outward current I(to) is an important determinant of the early repolarization phase. I(to) and its molecular basis Kv4.3 are regulated by adrenergic pathways including protein kinase C. However, the exact regulatory mechanisms have not been analyzed yet. We here analyzed isoenzyme specific regulation of Kv4.3 and I(to) by PKC. Kv4.3 channels were expressed in Xenopus oocytes and currents were measured with double electrode voltage clamp technique. Patch clamp experiments were performed in isolated rat cardiomyocytes. Unspecific PKC stimulation with PMA resulted in a reduction of Kv4.3 current. Similar effects could be observed after activation of conventional PKC isoforms by TMX. Both effects were reversible by pharmacological inhibition of the conventional PKC isoenzymes (Gö6976). In contrast, activation of the novel PKC isoforms (ingenol) did not significantly affect Kv4.3 current. Whereas TMX-induced PKC activation was not attenuated inhibition of PKCß, inhibition of PKCα with HBDDE prevented inhibitory effects of both PMA and TMX. Accordingly, stimulatory effects of PMA and TMX could be mimicked by the α-isoenzyme selective PKC activator iripallidal. Further evidence for the central role of PKCα was provided with the use of siRNAs. We found that PKCα siRNA but not PKCß siRNA abolished the TMX induced effect. In isolated rat cardiomyocytes, PMA dependent I(to) reduction could be completely abolished by pharmacologic inhibition of PKCα. In summary we show that PKCα plays a central role in protein kinase C dependent regulation of Kv4.3 current and native I(to). These results add to the current understanding of isoenzyme selective ion channel regulation by protein kinases.

Fine mapping of T-cell immunoglobulin mucin domain gene 1 failed to detect a significant association with multiple sclerosis.

The T-cell immunoglobulin mucin (TIM) gene family encodes receptors on T-cells that regulate Th1- and Th2-cell-mediated immunity. Recently published data implied differential expression of human TIM molecules by mononuclear cells in cerebrospinal fluid of patients with multiple sclerosis (MS) and might therefore be involved in different phases of the pathogenesis of MS. The purpose of this study was to investigate the association of TIM1 gene polymorphism with susceptibility to and clinical progression in MS. In total, 272 patients with MS and 272 sex- and age-matched healthy blood donors from Western Austria were genotyped for 10 single nucleotide polymorphisms (SNPs). Five SNPs were located in the promoter region of TIM1 (rs7702920, rs41297577, rs41297579, rs9313422 and rs34333511). Another five SNPs were selected in exon 4 (rs1553316 and rs12522248) and in the intronic regions 4 and 7 of TIM1 (rs1553318, rs2279804 and rs2277025), respectively. None of these SNPs showed a significant association with MS after correction for multiple comparisons. Haplotype analysis of our data resulted in 11 haplotypes and showed no significant differences between MS patients and controls. Our findings suggest that even fine mapping of TIM1 shows no significant association of this gene with multiple sclerosis.

Regulation of two-pore-domain (K2P) potassium leak channels by the tyrosine kinase inhibitor genistein.

BACKGROUND AND PURPOSE: Two-pore-domain potassium (K2P) channels mediate potassium background (or 'leak') currents, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Inhibition of K2P currents permits membrane potential depolarization and excitation. As expected for key regulators of excitability, leak channels are under tight control from a plethora of stimuli. Recently, signalling via protein tyrosine kinases (TKs) has been implicated in ion channel modulation. The objective of this study was to investigate TK regulation of K2P channels. EXPERIMENTAL APPROACH: The two-electrode voltage clamp technique was used to record K2P currents in Xenopus oocytes. In addition, K2P channels were studied in Chinese hamster ovary (CHO) cells using the whole-cell patch clamp technique. KEY RESULTS: Here, we report inhibition of human K2P3.1 (TASK-1) currents by the TK antagonist, genistein, in Xenopus oocytes (IC50=10.7 microM) and in CHO cells (IC50=12.3 microM). The underlying molecular mechanism was studied in detail. hK2P3.1 was not affected by genistin, an inactive analogue of genistein. Perorthovanadate, an inhibitor of tyrosine phosphatase activity, reduced the inhibitory effect of genistein. Current reduction was voltage independent and did not require channel protonation at position H98 or phosphorylation at the single TK phosphorylation site, Y323. Among functional hK2P family members, genistein also reduced K2P6.1 (TWIK-2), K2P9.1 (TASK-3) and K2P13.1 (THIK-1) currents, respectively. CONCLUSIONS AND IMPLICATIONS: Modulation of K2P channels by the TK inhibitor, genistein, represents a novel molecular mechanism to alter background K+ currents.

Increased number of Clostridium difficile infections and prevalence of Clostridium difficile PCR ribotype 001 in southern Germany.

In recent years, Clostridium difficile infection (CDI) has emerged as an increasing problem, both in in- and outpatients. In a rural region of southern Germany, the annual number of C. difficile toxin (Tcd)-positive patients has increased from 95 to 796 in the period from 2000 to 2007. Simultaneously, the proportion of positive tests among all Tcd examinations has risen from 7.0% to 12.8%, indicating that the higher number of affected patients was not solely due to an increase in the number of assays. Elevated numbers of CDI have recently been associated with outbreaks of the ribotype 027 strain, particularly in North America. This strain has also been isolated in Europe, including in Germany. Ribotyping and PCR testing for binary toxin genes of C. difficile strains isolated from in- and outpatients demonstrate a predominance (59%) of C. difficile ribotype 001, which exhibits antibiotic resistance to erythromycin, ciprofloxacin, and moxifloxacin, but lacks binary toxin genes. In summary, in our region of Germany, the number of patients affected by CDI has increased, probably due to spread of C. difficile ribotype 001.

[Molecular basis of primary electrical heart diseases]. / Molekulare Grundlagen primär elektrischer Herzerkrankungen.

The last decade has seen rapid progress in our understanding of the molecular basis of arrhythmias, particularly concerning hereditary arrhythmia syndromes. This has led to significant improvement regarding differentiation, risk stratification and therapy in these patients and their families. However, there is mounting evidence that the knowledge obtained by studying these rare monogenic disorders will also enable us to dissect the molecular mechanisms underlying polygenetic and multi-factorial arrhythmias that are by far more common in clinical practice. The goal of this review is to give a brief overview of current knowledge on the molecular basis of primary electrical heart diseases. A focus is on the long QT syndrome.

Urinary metabolites of amitriptylinoxide and amitriptyline in single-dose experiments and during continuous therapy.

In a cross-over design, six healthy volunteers received 50 mg amitriptylinoxide (AT-NO) IV and orally and 50 mg amitriptyline (AT) IV. Urine was collected completely for 8 h and occasionally up to 48 h. In addition, five patients each under treatment with AT-NO or AT for tension headache collected 24-h urine samples. The following compounds were analysed by HPLC: AT-NO, E- and Z-10-hydroxy-AT-NO (E- and Z-10-OH-AT-NO), free and conjugated AT, E- and Z-10-OH-AT and their mono- and didemethylated analogues, and 2-OH-nortriptyline (2-OH-NT). Unchanged AT-NO in urine accounted for an average of 34% and 22% of the single IV and oral doses, respectively, and for 28% in continuous therapy, with a further 8-9% being excreted as E- and Z-10-OH-AT-NO. The remaining part was converted to the same metabolites as was AT. In the steady state the measured compounds accounted for 74% and 77% of the daily AT-NO and AT doses, respectively. The renal plasma clearance of AT-NO varied between 75 and 265 ml/min in the six volunteers. Tubular secretion must play an important part in the renal excretion of AT-NO.

Transactivation of the early SV40 promoter by avian infectious laryngotracheitis virus in avian hepatoma cells.

An avian hepatoma cell line has been reported to be suitable for the cultivation of avian laryngotracheitis virus (ILTV) (Scholz et al. (1993) J. Virol. Methods, 273-286; Guo et al. (1993) Am. J. Vet. Res., in press). To provide information for the establishment of avian expression systems and for the construction of avian recombinant viruses, five expression plasmids were constructed to test two avian viral and two mammalian viral promotors for their suitability and strength for gene expression in this cell line. Chicken hepatoma cells were transfected with plasmids carrying the bacterial beta-galactosidase (beta-gal) gene as a reporter gene. The beta-gal gene of three plasmid constructs expressed in both E. coli and avian hepatoma cells, while the beta-gal gene of two other constructs expressed only in avian hepatoma cells. The beta-gal gene expressed independently of any viral infection when under the control of the early Rous sarcoma virus (RSV) promoter or the immediate-early cytomegalovirus (CMV) promoter. However, expression of beta-gal gene under the control of the SV40 early promoter/enhancer and the ILTV TK promoter was greatly potentiated when the transfected cells were co-infected with ILTV. This finding provides a system for the enhancement of gene expression in avian cells, especially when ILTV is used as vector.

Differential diagnosis of infectious laryngotracheitis from other avian respiratory diseases by a simplified PCR procedure.

A simple polymerase chain reaction (PCR)-based procedure was developed for the detection of avian infectious laryngotracheitis virus (ILTV) in chicken trachea, chorio-allantoic membrane (CAM), infected hepatoma cells and infectious cell culture supernatant. Samples were prepared by dilution in distilled water. After boiling and low speed centrifugation, samples were used for PCR analysis with two primers without special labeling. The PCR analysis for ILT virus could be completed in less than 8 h. Standard agarose gel electrophoretic analysis of the PCR products revealed a prominent band of 300 base-pairs in samples from ILTV-infected specimens, but not from specimens containing Newcastle disease virus, infectious bronchitis virus, avian adenovirus, fowlpox virus, Pachecoz or Marek's disease virus. One single ILTV infected cell or 10 plaque forming units of ILTV could be detected with this procedure. The procedure can be used for the identification of ILTV and the differentiation of ILTV from other avian respiratory tract infectants.

An avian hepatoma cell line for the cultivation of infectious laryngotracheitis virus and for the expression of foreign genes with a mammalian promoter.

Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly infectious upper respiratory tract disease in chickens. Vaccine development and basic studies on ILTV have been hampered by the lack of a cell line for the cultivation of this herpesvirus which was identified in 1930. Four different avian cell lines were tested for their suitability to propagate ILTV. Here we report the successful growth of ILTV with a chemically-induced avian hepatoma cell line, while retrovirus transformed cell lines derived from permissive primary cells, were found to be non-permissive for ILTV. After multiple passaging of ILTV in the hepatoma cells, the virus could be grown up to a titre of 1 x 10(7) EID50 per ml with a replication cycle comparable to that in primary hepatocytes. Methods of plaque assay, DNA-transfection, and expression of a reporter gene were established. The gene coding for the bacterial beta-galactosidase gene under the control of the cytomegalovirus (CMV) immediate-early promotor was transiently expressed, indicating that a mammalian herpesvirus promotor was recognized by this avian cell line. Infectious ILTV virions were produced after transfecting this cell-line with purified ILTV DNA. The results indicated that the cell line is suitable for the construction of recombinant ILTV and for the molecular biological study of this important avian pathogen.

Twenty-four-hour tremor recordings in the evaluation of the treatment of Parkinson's disease.

A new method of prolonged recording of EMG provides a good estimate of spontaneous and induced diurnal variations in resting tremor in Parkinson's disease. It provides a record and a measure of the effects of treatment. Tremor intensity shows considerable variations even over short periods of time. Therefore short-term measurements of tremor are unhelpful. Long-term recordings agree better with the patient's assessment than with the clinical rating score. Repeated recordings over a similar 10-h period on 3 consecutive days in one patients showed fairly constant measures of occurrence and intensity of tremor. In contrast to accelerometer measurements of tremor, artefacts caused by movements and general activity of the patient do not materially interfere with tremor evaluation using surface EMG.

Incidence of peripheral neuropathy and cerebellar ataxia in chronic alcoholics.

A total of 78 chronic alcoholics were examined neurologically as well as by electroneurography, myography and posturography. Clinical signs of peripheral neuropathy were detected in 45% of these patients, with electromyographic and neurographic abnormality in 67% and 55% respectively. Clinical signs of cerebellar ataxia were found in 33% of our patients, whereas posturographic measurements of increased sway were recorded in 69%. The posturographic characteristics of cerebellar anterior lobe atrophy were observed in two-thirds of the latter patients. The severity of cerebellar-ataxia did not correlate with the degree of neuropathy. This lack of correlation is interpreted as an indication of different pathogenetic mechanisms acting on peripheral nerves and cerebellum.

Analgesic-induced chronic headache: long-term results of withdrawal therapy.

Headache characteristics are described in 139 patients with chronic daily or almost daily headaches due to regular intake of analgesics and the short- and long-term results of drug withdrawal. Drug-induced headache was described as dull, diffuse, and band-like, and usually started in the early morning. The mean duration of the original headache (migraine or tension headache) was 25 years; regular intake of drugs and chronic daily headache had started 10 and 6 years prior to withdrawal therapy, respectively. Patients took an average of 34.6 tablets or analgesic suppositories or antimigraine drugs per week containing 5.8 different substances. The drugs most often used were caffeine (95%), ergotalkaloids (89%), barbiturates (64%), and spasmolytics, paracetamol, and pyrazolone derivates (45%-46%). A total of 103 patients (68 migraine, 35 tension or combination headache) were available for interviews at a mean time interval of 2.9 years after an inpatient drug withdrawal programme. Chronic headache had disappeared or was reduced by more than 50% in two-thirds of the patients. Positive predictors for successful treatment were migraine as primary headache, chronic headache lasting less than 10 years, and regular intake of ergotamine. Drug intake was significantly reduced and patients used single substances more often. Patients who originally suffered from migraine, superimposed on the daily headache, also experienced a significant improvement in the frequency of the migraines and their intensity. Migraine prophylaxis through beta-blocking agents and calcium channel antagonists was more efficient after drug-withdrawal therapy.

The economic impact of Parkinson's disease. An estimation based on a 3-month prospective analysis.

OBJECTIVE: This study prospectively assesses the medical costs of Parkinson's disease (PD). DESIGN: Over a period of 3 months (from July to September 1995), patients with PD documented all items of healthcare provision. These data were then used to calculate medical costs for an individual patient as well as the costs of PD. PATIENTS AND SETTING: We included 20 outpatients with idiopathic PD from the neurological outpatient clinic, Klinikum Grosshadern, Munich, and 20 patients from two office-based neurologists in South-West Germany. MAIN RESULTS: The mean 3-month medical cost of PD in 1995 deutschmarks (DM) was 5210 ($US3390, 2240 Pounds) consisting of DM1410 ($US920, 610 Pounds) for care and nursing, DM1580 ($US1030, 680 Pounds) for drug therapy, DM1320 ($US860, 570 Pounds) for inpatient hospital care, DM40 ($US26, 17 Pounds) for outpatient care and DM860 for other expenses ($US560, 370 Pounds). The expenditure was related to the disease evolution. Patients complaining of one-sided symptoms [Hoehn and Yahr stage I; (HY I)] were less expensive to treat (DM1930, $US1250, 830 Pounds) than patients who were severely incapacitated (HY V) [DM9740, $US6330, 4200 Pounds; HY V]. After 3 to 5 years of levodopa treatment approximately 50% of patients start to experience fluctuations in motor ability and dyskinesias [Unified Parkinson's disease rating scale, part IV (UPDRS IV)]. This onset of motor complications parallels an increase in costs. For patients who experienced motor fluctuations, annual costs were DM6550 ($US4260, 2820 Pounds) compared with DM3030 ($US1960, 1300 Pounds) for patients lacking this problem. Indirect non-medical costs were not calculated due to the limited number of patients. The impact of the disease on work, however, is clearly apparent from the patients' history: 19 out of 34 patients who had already stopped working attributed this to the disease, and only 6 patients were still working at the time of the survey. CONCLUSION: PD poses a major financial impact to society which is expected to increase in future years as the age distribution shifts to older age groups. On the basis of a prevalence of PD of 183 per 100,000, we calculated an annual expenditure of DM3.0 billion for the direct medical costs of PD in Germany.

Structure of a persistent heptachlorobornane in toxaphene (b7-1000) agrees with molecular model predictions.

A Cl(7) component of technical toxaphene (CTT), previously detected in marine mammals and fish and referred to as "7-1", was isolated from contaminated estuarine sediment using preparative solid-liquid chromatography followed by reversed-phase HPLC. The structure of this compound, elucidated by GC/MS and (1)H NMR, was 2-endo,3-exo,5-endo,6-exo,8,8,10-heptachlorobornane (hereafter referred to as B7-1000). This newly identified CTT eluted in the nonpolar fraction from silica and shares the alternating endo-exo chlorine substitution pattern with other relatively nonpolar, persistent congeners (e.g., B8-1413 and B9-1679). Based on ECNI-MS response, levels of B7-1000 in tissue samples of various higher organisms including humans were as high as 16% of B8-1413. Enantioselective determination of B7-1000 using a modified cyclodextrin chiral stationary phase (beta-BSCD) resulted in enantiomer ratios that were depleted in adipose tissue of a marine bird (skua) and Weddell seal blubber (0.3 and 0.5, respectively), but not in elephant seal blubber (1.1). Elucidation of the structure of B7-1000 thus validates previous predictions of persistence based on structure-activity relationships, chromatographic properties, and molecular modeling.

Systemic Candida albicans infection with cerebral abscess and granulomas.

This study reports the case of a 19-year-old Turkish woman who was suffering from a cerebral abscess and granuloma formation caused by Candida albicans. The diagnosis of her illness was established by cytologic analysis of the cerebrospinal fluid. Combined application of amphotericin B and 5-fluorocytosine cured the patient's disorder. Its remission was documented over a period of 2 years by the use of computed tomography.

Assembly pathway of avian infectious laryngotracheitis virus.

Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly contagious upper respiratory tract infection in chickens. At present, ILTV vaccines are not satisfactory because of development of a latent carrier status in vaccinated birds. Development of recombinant virus vaccines has been hampered by the limited information available on the molecular level and organization of this virus. We isolated 3 assembly intermediates, designated A, B, and C from ILTV-infected cells. Analysis of [3H]thymidine-and [35S]methionine-labeled particles, and electron microscopic studies indicated that particle A was the empty capsid, particle B was the procapsid containing scaffolding protein, and particle C was the DNA-filled capsid. The ILTV procapsids could only be found in the nucleus, which indicated that procapsids could not translocate through the nuclear membrane until they packaged the DNA. The DNA-filled capsids migrated through the nuclear membrane and obtained an envelope from the inner membrane of the nucleus. The enveloped particles then migrated through the lumen of the endoplasmic reticulum into vacuoles in the cytoplasm. Infective virions were isolated from within the infected cells, indicating that budding through the cytoplasmic membrane is not a necessary step in ILTV maturation. Abundant arrays composed of tubules about 45 to 50 nm wide were found in the cytoplasm of chicken embryonic liver cells about 30 to 38 hours after infection. Comparison of the assembly intermediates and the DNA packaging pathway of ILTV with that of bacteriophage pi 29 indicates that similarity exists.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a basement membrane protein (BM180) using capillary electrophoresis.

Coeliac disease (gluten intolerance) is a genetically based autoimmune disease that becomes evident following ingestion of cereal prolamins such as the wheat gliadins. The process of this disease is not yet thoroughly understood. Purification of basement membrane protein-180 (BM180) (a basement membrane protein with a potential autoantigen role) and multiple analysis of the purified protein can lead to a better understanding of this disorder, which affects millions of people worldwide. Our preliminary work on the purification and characterization of this protein is presented in this paper. BM180 proteins were expressed in mouse EHS (Engelberth-Holm-Swarm) tumor cells. A crude purification process (see "Methods") was performed and the purified fractions were analyzed by capillary gel electrophoresis (CGE) for determination of the apparent molecular weight of the protein components in each fraction. The fractions, which contained compounds of the expected molecular weight, were further analyzed using a capillary zone electrophoresis (CZE) method developed for the routine analysis of wheat gliadins. Using the two CE methods, we were able to compare BM180 with certain gliadin fractions. Additional information on the protein stability was also obtained.