"For a man to go to hospital, then that would be his last option": A qualitative study exploring men's experiences, perceptions and healthcare needs in the implementation of Universal Health Coverage in Kenya.

The achievement of Universal Health Coverage (UHC) requires equitable access and utilization of healthcare services across all population groups, including men. However, men often face unique barriers that impede their engagement with health systems which are influenced by a myriad of socio-cultural, economic, and systemic factors. Therefore, understanding men's perspectives and experiences is crucial to identifying barriers and facilitators to their healthcare-seeking behaviour under UHC initiatives. This qualitative study sought to explore men's perceptions, experiences, healthcare needs and potential strategies to inform an impartial implementation of Universal Health Coverage (UHC) in Kenya. The study employed a qualitative research design to investigate men's healthcare experiences in 12 counties across Kenya. Thirty focus group discussions involving 296 male participants were conducted. Men were purposively selected and mobilized through the support of health facility-in-charges, public health officers, and community health extension workers. Data was coded according to emergent views and further categorized thematically into three main domains (1) Perspectives and experiences of healthcare access (2) Socio-cultural beliefs and societal expectations (3) Desires and expectations of health systems. Findings revealed complex sociocultural, economic, and health system factors that influenced men's healthcare experiences and needs which included: masculinity norms and gender roles, financial constraints and perceived unaffordability of services, lack of male-friendly and gender-responsive healthcare services, confidentiality concerns, and limited health literacy and awareness about available UHC services. Our study has revealed a disconnect between men's needs and the current healthcare system. The expectations concerning masculinity further exacerbate the problem and exclude men further hindering men's ability to receive appropriate care. This data provides important considerations for the development of comprehensive and gender-transformative approaches challenging harmful masculine norms, pushing for financial risk protection mechanisms and gender-responsive healthcare delivery attuned to the unique needs and preferences of men.

Adenosine receptor A2a blockade by caffeine increases IFN-gamma production in Th1 cells from patients with rheumatoid arthritis.

OBJECTIVE: Studies indicate that caffeine uptake may be a risk factor for rheumatoid arthritis (RA), but a definitive link between caffeine consumption and RA has not been established. This study aimed to investigate the interplay between caffeine, adenosine receptor A2a, and interferon-γ (IFN-γ) production in CD4+ T cells from RA patients. METHOD: Peripheral blood mononuclear cells were obtained from the peripheral blood of healthy individuals and patients with RA. CD4+ T cells were isolated using the magnetic activated cell sorting technique and cultured in vitro with caffeine or mock control. In addition, adenosine was used as a competitive inhibitor of caffeine. After 48 h, expression of IFN-γ and interleukin-17 (IL-17) was analysed by flow cytometry. Ex vivo expression levels of adenosine receptor A2a were also assessed. RESULTS: Caffeine promoted IFN-γ production in Th1 cells in vitro. Significantly higher concentrations of caffeine were required to increase IFN-γ levels in Th1 cells from healthy individuals compared to Th1 cells from patients with RA. Moreover, ex vivo levels of adenosine receptor A2a expression on CD4+ T cells were significantly higher in RA than in healthy individuals. Caffeine-driven IFN-γ production was completely reversed by adenosine, a competitive agonist of adenosine receptor A2a. In contrast to IFN-γ, production of IL-17 was not affected by caffeine. CONCLUSION: Caffeine promotes IFN-γ production in Th1 cells from RA patients in vitro by competitive inhibition of adenosine receptor A2a. Excessive coffee consumption could contribute to T-cell activation and inflammation in RA.

The best from East and West? Acupuncture and medical training therapy as monotherapies or in combination for adult patients with episodic and chronic tension-type headache: study protocol for a randomized controlled trial.

BACKGROUND: This study aims to evaluate the feasibility and efficacy of a complex health intervention, based on the combination of conventional Western medicine and traditional Chinese medicine (TCM), in an outpatient department of a university hospital for patients with frequent episodic or chronic tension-type headaches. METHODS/DESIGN: This is a prospective randomized controlled pilot study with four balanced treatment arms (usual care, acupuncture, training, and training plus acupuncture). Each arm will have 24 patients. After the initial screening examination and randomization, a 6-week treatment period follows, with treatment frequencies decreasing at 2-week intervals. After completion of the intervention, two follow-up evaluations will be performed 3 and 6 months after the start of treatment. At predefined times, the various outcomes (pain intensity, health-related quality of life, pain duration, autonomic regulation, and heart rate variability) as well as the participants' acceptance of the complex treatment will be evaluated with valid assessment instruments (Migraine Disability Assessment, PHQ-D, GAD-7, and SF-12) and a headache diary. The acupuncture treatment will be based on the rules of TCM, comprising a standardized combination of acupuncture points and additional points selected according to individual pain localization. The training therapy comprises a combination of strength training, endurance training, and training to improve flexibility and coordination. Besides descriptive analyses of the samples, their comparability will be assessed using an analysis of variance (ANOVA) or chi-squared tests. Analyses will be performed on an intention-to-treat basis. Potential interaction effects will be calculated using a repeated-measures ANOVA to test the primary and secondary hypotheses. In supplementary analyses, the proportion of treatment responders (those with a 50% reduction in the frequency of pain episodes) will be determined for each treatment arm. DISCUSSION: This trial may provide evidence for the additive effects of acupuncture and medical training therapy as a combination treatment and may scientifically support the implementation of this complex health intervention. TRIAL REGISTRATION: Registered on 11 Feburary 2019. German Clinical Trials Register, DRKS00016723.

In vitro supplementation with unsaturated fatty acids improves boar sperm viability after storage at 6 °C.

Liquid preservation of the cold-sensitive boar sperm at a lesser temperature than the standard 17 °C would reduce bacterial growth and minimize the use of antibiotics. There was assessment, therefore, of the capacity of individual fatty acids bound to fatty acid free BSA to improve sperm survival at 6 °C because oxidative stress and lipid degradation are prominent detrimental factors. Different effects of the fatty acids were observed. Supplementation with naturally occurring fatty acids (linolenic, linoleic, oleic, palmitoleic acid), which may become metabolically incorporated into sperm lipids, increased the number of motile and progressively motile sperm after 2 days of storage during a thermo-resistance test (5 h at 38 °C) to that of control samples preserved at 17 °C in pure Beltsville Thawing Solution. With the exception of linolenic acid, all naturally occurring fatty acids enhanced the number of sperm with active mitochondria after 3 days of storage. Palmitoleic acid was the most effective supplement with effects already present when sperm were re-warmed for 30 min after 2 and 7 days of storage. The non-endogenous, non-integrated timnodonic acid (20:5) had no effect on sperm variables. Because the application of individual fatty acids attached to BSA had differing effects in preserving boar sperm at 6 °C, the use of combinations of fatty acids could be more efficacious than with use of natural lipid supplements for low temperature preservation of sperm.

Efficacy of a Carrageenan gel Against Transmission of Cervical HPV (CATCH): interim analysis of a randomized, double-blind, placebo-controlled, phase 2B trial.

OBJECTIVES: To evaluate the efficacy of a carrageenan-based lubricant gel in reducing the risk of genital human papillomavirus (HPV) infections in women. METHODS: We conducted a planned interim analysis of a randomized, double-blind, placebo-controlled, phase 2B trial. Women aged 18 years and older were randomly assigned (1:1) to a carrageenan-based gel or a placebo gel to be self-applied every other day for the first month and before and after each intercourse during follow-up. Assessments were performed at 0.5, 1, 3, 6, 9 and 12 months. The primary outcome was incidence of a new infection by an HPV type that was not present at baseline. Intention-to-treat analyses were performed. RESULTS: Between January 2013 and June 2017, a total of 280 participants were randomly assigned to the carrageenan (n = 141) or the placebo (n = 139) arm. All participants were included in safety analyses, but three (1%) were excluded from efficacy analyses (HPV results unavailable for two participants in the carrageenan and one participant in the placebo arm). The median follow-up time was 9.2 months (interquartile range, 1.9-13.2 months). A total of 59 (42%) of 139 participants in the carrageenan arm and 78 (57%) of 138 participants in the placebo arm became infected by at least one new HPV type (hazard ratio = 0.64, 95% confidence interval = 0.45-0.89, p 0.009). A total of 62 (44%) of 141 participants in the carrageenan arm versus 43 (31%) of 139 participants in the placebo arm reported an adverse event (p 0.02), none of which was deemed related to the gels. CONCLUSIONS: Our trial's interim analysis suggests that using a carrageenan-based lubricant gel can reduce the risk of genital HPV infections in women.

Phase II randomized trial of carboplatin, paclitaxel, bevacizumab with or without cixutumumab (IMC-A12) in patients with advanced non-squamous, non-small-cell lung cancer: a trial of the ECOG-ACRIN Cancer Research Group (E3508).

BACKGROUND: Cixutumumab is a fully human IgG1 monoclonal antibody to the insulin-like growth factor type I receptor that can potentially reverse resistance and enhance the efficacy of chemotherapy. METHODS: Bevacizumab-eligible patients with stage IV or recurrent non-squamous, non-small-cell lung cancer and good performance status were randomized to receive standard doses of paclitaxel, carboplatin, and bevacizumab to a maximum of six cycles followed by bevacizumab maintenance (CPB) until progression (arm A) or CPB plus cixutumumab 6 mg/kg i.v. weekly (arm B). RESULTS: Of 175 patients randomized, 153 were eligible and treated (78 in arm A; 75 in arm B). The median progression-free survival was 5.8 months (95% CI 5.4-7.1) in arm A versus 7 months (95% CI 5.7-7.6) in arm B (P = 0.33); hazard ratio 0.92 (95% CI 0.65-1.31). Objective response was 46.2% versus 58.7% in arm A versus arm B (P = 0.15). The median overall survival was 16.2 months in arm A versus 16.1 months in arm B (P = 0.95). Grade 3/4 neutropenia and febrile neutropenia, thrombocytopenia, fatigue, and hyperglycemia were increased with cixutumumab. CONCLUSIONS: The addition of cixutumumab to CPB increased toxicity without improving efficacy and is not recommended for further development in non-small-cell lung cancer. Both treatment groups had longer OS than historical controls which may be attributed to several factors, and emphasizes the value of a comparator arm in phase II trials. CLINICALTRIALS.GOV IDENTIFIER: NCT00955305.

Comprehensive evaluation of published gene expression prognostic signatures for biomarker-based lung cancer clinical studies.

Background: A more accurate prognosis for non-small-cell lung cancer (NSCLC) patients could aid in the identification of patients at high risk for recurrence. Many NSCLC mRNA expression signatures claiming to be prognostic have been reported in the literature. The goal of this study was to identify the most promising mRNA prognostic signatures in NSCLC for further prospective clinical validation. Experimental design: We carried out a systematic review and meta-analysis of published mRNA prognostic signatures for resected NSCLC. The prognostic performance of each signature was evaluated via a meta-analysis of 1927 early stage NSCLC patients collected from 15 studies using three evaluation metrics (hazard ratios, concordance scores, and time-dependent receiver-operating characteristic curves). The performance of each signature was then evaluated against 100 random signatures. The prognostic power independent of clinical risk factors was assessed by multivariate Cox models. Results: Through a literature search, we identified 42 lung cancer prognostic signatures derived from genome-wide expression profiling analysis. Based on meta-analysis, 25 signatures were prognostic for survival after adjusting for clinical risk factors and 18 signatures carried out significantly better than random signatures. When analyzing histology types separately, 17 signatures and 8 signatures are prognostic for adenocarcinoma and squamous cell lung cancer, respectively. Despite little overlap among published gene signatures, the top-performing signatures are highly concordant in predicted patient outcomes. Conclusions: Based on this large-scale meta-analysis, we identified a set of mRNA expression prognostic signatures appropriate for further validation in prospective clinical studies.

Semen cryopreservation and radical reduction capacity of seminal fluid in captive African lion (Panthera leo).

Optimizing cryopreservation protocols for nondomestic felids contributes to the successful development of assisted reproduction techniques and genetic resource banking. In this study, we describe a simple cryopreservation procedure for African lion (Panthera leo) ejaculates, which was tested with different packaging options and different sperm numbers per dose. By applying urethral catheterization and electroejaculation, 17 ejaculates with greater than 20% motile and greater than 5% progressively motile sperm were collected. A lyophilized extender (a modified egg yolk-Tes-Tris-fructose-glycerol medium) was rehydrated and added in one step at ambient temperature (∼25 °C) to semen, which was prediluted in cell culture medium M199. After slow cooling of insulated samples to 15 °C in a refrigerator (4 °C), the samples were fast frozen over the surface of liquid nitrogen or in a dry shipper. Aliquots of 300 µL containing 20 × 106 sperm were frozen in cryovials and in 0.5-mL straws. Differences were observed in the total motility after thawing between vial (31.5 ± 14.1%) and straw freezing (20.1 ± 8.6%). However, the subpopulations of vital (22.7 ± 7.8% for vial and 19.8 ± 8.5% for straw) and progressively motile (10.0 ± 7.9% for vial and 10.0 ± 6.4% for straw) sperm after washing and 1 hour incubation at 38 °C were of similar magnitude, velocity, and linearity for both packaging options. After freezing of five ejaculates with 20, 60, and 100 × 106 sperm per dose, best results were achieved at the lowest concentration. In general, post-thaw results were highly variable (2.2% and 56.5% total motility) and not correlated to motility or morphology of the fresh semen. To further characterize semen quality, we assessed the protective potential of seminal fluid against oxidative stress, which might be challenged on freeze thawing. The capacity of seminal fluid to reduce radicals was measured in 10 semen samples by electron spin resonance spectroscopy and a spin-labeled fatty acid as a radical probe. Moreover, we determined the lysophosphatidylcholines (LPC) as potential lipid oxidation products in the sperm and erythrocytes of the males. Individuals with a high radical reduction capacity in the seminal fluid and a low LPC content in their erythrocytes showed a better cryosurvival of sperm. This is a first indication that seminal fluid may affect the freezing potential of African lion ejaculates.

Renal Transplantation With Final Allocation Based on the Virtual Crossmatch.

Solid phase immunoassays (SPI) are now routinely used to detect HLA antibodies. However, the flow cytometric crossmatch (FCXM) remains the established method for assessing final donor-recipient compatibility. Since 2005 we have followed a protocol whereby the final allocation decision for renal transplantation is based on SPI (not the FCXM). Here we report long-term graft outcomes for 508 consecutive kidney transplants using this protocol. All recipients were negative for donor-specific antibody by SPI. Primary outcomes are graft survival and incidence of acute rejection within 1 year (AR <1 year) for FCXM+ (n = 54) and FCXM- (n = 454) recipients. Median follow-up is 7.1 years. FCXM+ recipients were significantly different from FCXM- recipients for the following risk factors: living donor (24% vs. 39%, p = 0.03), duration of dialysis (31.0 months vs. 13.5 months, p = 0.008), retransplants (17% vs. 7.3%, p = 0.04), % sensitized (63% vs. 19%, p = 0.001), and PRA >80% (20% vs. 4.8%, p = 0.001). Despite these differences, 5-year actual graft survival rates are 87% and 84%, respectively. AR <1 year occurred in 13% FCXM+ and 12% FCXM- recipients. Crossmatch status was not associated with graft outcomes in any univariate or multivariate model. Renal transplantation can be performed successfully, using SPI as the definitive test for donor-recipient compatibility.

Nonmyeloablative allogeneic stem cell transplantation for chronic lymphocytic leukaemia offers the possibility of disease control with minimal morbidity and mortality--a single institution experience.

Allogeneic stem cell transplantation is a treatment option for patients with poor risk CLL. We conducted a retrospective analysis of all CLL patients allografted at our institution, the University Hospital of Cologne, Germany. Data was collected on 40 patients from 2004 to 2012. The mean age was 54, and the majority were male (75 %). On average, the patients were diagnosed 6 years (range 2-12) prior to transplant with an average of 4 years (range 1-8) from time of first-line therapy to transplant. The remission states at the time of transplant were complete remission (CR) (n = 4), stable disease (n = 10), partial remission (n = 20) and progressive disease (n = 6). Only reduced intensity conditioning regimens were employed. The average CD34(+) cell dose was 4.16 × 10(6)/kg. Neutrophil engraftment was seen by day +17 (range 10-23) post-transplant, and 88 % achieved 95-100 % donor chimerism by day 100. Overall survival, progression-free survival and non-relapse mortality at 2 years post-transplant were 65, 52.5 and 27.5 %, respectively. A total of 51 % of patients were found to be minimal residual disease (MRD)-negative at 1 year post-transplant. Our single-centre experience confirms the valuable role of allogeneic stem cell transplantation (allo-SCT) in the treatment of poor risk CLL patients with promising long-term survival and acceptable transplant-related mortality. The advent of newer therapeutic agents should not hinder the consideration of allo-SCT for this patient cohort as it remains the only curative option for these patients.

A randomized phase II study of the telomerase inhibitor imetelstat as maintenance therapy for advanced non-small-cell lung cancer.

BACKGROUND: Continuation or 'switch' maintenance therapy is commonly used in patients with advancd non-small-cell lung cancer (NSCLC). Here, we evaluated the efficacy of the telomerase inhibitor, imetelstat, as switch maintenance therapy in patients with advanced NSCLC. PATIENTS AND METHODS: The primary end point of this open-label, randomized phase II study was progression-free survival (PFS). Patients with non-progressive, advanced NSCLC after platinum-based doublet (first-line) chemotherapy (with or without bevacizumab), any histology, with Eastern Cooperative Oncology Group performance status 0-1 were eligible. Randomization was 2 : 1 in favor of imetelstat, administered at 9.4 mg/kg on days 1 and 8 of a 21-day cycle, or observation. Telomere length (TL) biomarker exploratory analysis was carried out in tumor tissue by quantitative PCR (qPCR) and telomerase fluorescence in situ hybridization. RESULTS: Of 116 patients enrolled, 114 were evaluable. Grade 3/4 neutropenia and thrombocytopenia were more frequent with imetelstat. Median PFS was 2.8 and 2.6 months for imetelstat-treated versus control [hazard ratio (HR) = 0.844; 95% CI 0.54-1.31; P = 0.446]. Median survival time favored imetelstat (14.3 versus 11.5 months), although not significantly (HR = 0.68; 95% CI 0.41-1.12; P = 0.129). Exploratory analysis demonstrated a trend toward longer median PFS (HR = 0.43; 95% CI 0.14-1.3; P = 0.124) and overall survival (OS; HR = 0.41; 95% CI 0.11-1.46; P = 0.155) in imetelstat-treated patients with short TL, but no improvement in median PFS and OS in patients with long TL (HR = 0.86; 95% CI 0.39-1.88; and HR = 0.51; 95% CI 0.2-1.28; P = 0.145). CONCLUSIONS: Maintenance imetelstat failed to improve PFS in advanced NSCLC patients responding to first-line therapy. There was a trend toward a improvement in median PFS and OS in patients with short TL. Short TL as a predictive biomarker will require further investigation for the clinical development of imetelstat.

Changes of murine sperm phospholipid composition during epididymal maturation determined by MALDI-TOF mass spectrometry.

After leaving the testis, spermatozoa undergo several important steps of biochemical maturation during the passage through the epididymis, increasing their motility and fertilizing ability. These changes comprise (among others) the modification of the phospholipid composition of the sperm membrane. This process is thought to be important for the achievement of motility and fertilizing capacity. The lipids of the sperm membrane are characterized by a significant content of unsaturated fatty acyl residues, resulting in a high sensitivity against oxidative stress. This is evidenced by the appearance of lysolipids, for example, lysophosphatidylcholine, which acts like a detergent and is normally present in only very small amounts in biological membranes. The epididymis represents a tubular system comprising three main parts (caput, corpus, and cauda), through which the spermatozoa are consecutively transported undergoing distinct maturation stages. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we established three striking differences in the lipid composition of murine spermatozoa from the different epididymal regions: in comparison to the caput sperm, sperm from the cauda are characterized by (1) a higher degree of unsaturation (PC 18:0/22:5 and 18:0/22:6 vs. 18:0/20:4 and 18:0/18:1), (2) an enhanced plasmalogen content, and (3) an enhanced content of lysolipids. These changes are likely to be of physiological relevance and potentially useful as diagnostic markers of sperm maturation and acquisition of motility.

Dosing to rash: a phase II trial of the first-line erlotinib for patients with advanced non-small-cell lung cancer an Eastern Cooperative Oncology Group Study (E3503).

BACKGROUND: The development of a rash has been retrospectively associated with increased response and improved survival when treated with erlotinib at the standard dose of 150 mg per day. The objective of this trial was to evaluate the association of the activity of erlotinib in the first-line setting in patients with advanced non-small-cell lung cancer (NSCLC) with the development of a tolerable rash via dose escalation of erlotinib or tumour characteristics. METHODS: Patients, with advanced NSCLC without prior systemic therapy, were treated with erlotinib 150 mg orally per day. The dose was increased by 25mg every two weeks until the development of grade 2/tolerable rash or other dose limiting toxicity. Tumour biopsy specimens were required for inclusion. RESULTS: The study enrolled 137 patients, 135 were evaluable for safety and 124 were eligible and evaluable for response. Only 73 tumour samples were available for analysis. Erlotinib dose escalation occurred in 69/124 patients. Erlotinib was well tolerated with 70% of patients developing a grade 1/2 rash and 10% developing grade 3 rash. Response rate and disease control rate were 6.5% and 41.1% respectively. Median overall survival was 7.7 months. Toxicity and tumour markers were not associated with response. Grade 2 or greater skin rash and low phosphorylated mitogen-activated protein kinase (pMAPK) were associated with improved survival. CONCLUSIONS: Overall survival was similar in this trial compared to first-line chemotherapy in this unselected patient population. Dose escalation to the development of grade 2 skin rash was associated with improved survival in this patient population.

Randomised phase II study of axitinib or bevacizumab combined with paclitaxel/carboplatin as first-line therapy for patients with advanced non-small-cell lung cancer.

BACKGROUND: Efficacy and safety of first-line axitinib/paclitaxel/carboplatin versus bevacizumab/paclitaxel/carboplatin in advanced non-squamous non-small-cell lung cancer (NSCLC) was evaluated. PATIENTS AND METHODS: Patients with stage IIIB/IV disease stratified by adjuvant therapy and gender were randomised 1 : 1 to axitinib (5 mg twice daily) or bevacizumab [15 mg/kg every 3 weeks (Q3W)], both with paclitaxel (200 mg/m(2) Q3W)/carboplatin (AUC 6 mg min/ml Q3W). RESULTS: The trial was discontinued after preliminary analysis. Median progression-free survival (primary end point) for axitinib (N = 58) and bevacizumab (N = 60), respectively, was 5.7 and 6.1 months [hazard ratio (HR) 1.09, 95% confidence interval (CI) 0.68-1.76; one-sided stratified P = 0.64]; median overall survival was 10.6 and 13.3 months (HR 1.12, 95% CI 0.74-1.69; one-sided stratified P = 0.70). Objective response rates (95% CI) were 29.3% (18.1-42.7) and 43.3% (30.6-56.8), respectively; risk ratio 0.676 (95% CI 0.41-1.11; one-sided stratified P = 0.94). The most common grade 3/4 adverse events included neutropenia (28% versus 20%), fatigue (14% versus 7%), and hypertension (14% versus 5%). Patient-reported outcomes based on the EORTC QLQ-C30 were similar between arms. CONCLUSIONS: In patients with advanced non-squamous NSCLC, axitinib/paclitaxel/carboplatin did not improve efficacy versus bevacizumab/paclitaxel/carboplatin, and was less well tolerated.

Baseline tumour measurements predict survival in advanced non-small cell lung cancer.

BACKGROUND: The association between tumour measurements and survival has been studied extensively in early-stage and locally advanced non-small cell lung cancer (NSCLC). We analysed these factors in patients with advanced NSCLC. METHODS: Data were derived from the E4599 trial of paclitaxel-carboplatin±bevacizumab. Associations between the Response Evaluation Criteria in Solid Tumors (RECIST) baseline sum longest diameter (BSLD), response rate, progression-free survival (PFS) and overall survival (OS) were evaluated using univariate and multivariable Cox regression models. RESULTS: A total of 759 of the 850 patients (89%) in the E4599 trial had measurable diseases and were included in this analysis. The median BSLD was 7.5 cm. BSLD predicted OS (hazard ratio (HR) 1.41; P<0.001) and had a trend towards association with PFS (HR 1.14; P=0.08). The median OS was 12.6 months for patients with BSLD <7.5 cm compared with 9.5 months for BSLD ≥ 7.5 cm. This association persisted in a multivariable model controlling multiple prognostic factors, including the presence and sites of extrathoracic disease (HR 1.24; P=0.01). There was no association between BSLD and response rate. CONCLUSION: Tumour measurements are associated with survival in the E4599 trial. If validated in other populations, this parameter may provide important prognostic information to patients and clinicians.

Sulfated glycosaminoglycans as promising artificial extracellular matrix components to improve the regeneration of tissues.

Glycosaminoglycans (GAG) such as hyaluronan (HA) or chondroitin/dermatan sulfate (CS/DS) occur in many connective tissues, for instance, in bone, cartilage and skin. Due to their significant water-binding capacity, GAG are essential for the biomechanical properties of these tissues. However, there is also increasing evidence that the sulfation of GAG does not occur at random, but a "sulfation code" exists that mediates the physiological functions of GAG. Thus, the biological properties of these biomacromolecules are strongly influenced by the degree of sulfation (ds) and the sulfate group distribution along the polymer. Therefore, certain GAG might also have interesting pharmacological properties. It is, thus, commonly accepted that GAG represent promising biomaterials in the field of tissue engineering as well as to design new bioactive materials for tissue repair and reconstruction. In this review we will focus on chemically sulfated GAG and provide a survey of these compounds on four different levels. First, we will provide an overview on chemical functionalization strategies of naturally occurring HA and CS/DS with special emphasis on regioselective methods to introduce a defined number of sulfate residues into the carbohydrate backbone. Second, chemical and biochemical methods to characterize the synthesized compounds will be introduced with the focus on methods based on nuclear magnetic resonance (NMR) and mass spectrometry (MS). In the third part, we will discuss the interaction of natural and chemically sulfated GAG with proteins and other biomolecules with regulatory functions. Additionally, biological consequences of these interactions regarding healing processes of skin and bone will be presented by discussing selected cell culture experiments. Finally, in vivo effects of GAG as components of artificial extracellular matrices will be discussed.

Intravaginal TLR agonists increase local vaccine-specific CD8 T cells and human papillomavirus-associated genital-tumor regression in mice.

Human papillomaviruses (HPV)-related cervical cancer is the second leading cause of cancer death in women worldwide. Despite active development, HPV E6/E7 oncogene-specific therapeutic vaccines have had limited clinical efficacy to date. Here, we report that intravaginal (IVAG) instillation of CpG-ODN (TLR9 agonist) or poly-(I:C) (TLR3 agonist) after subcutaneous E7 vaccination increased ~fivefold the number of vaccine-specific interferon-γ-secreting CD8 T cells in the genital mucosa (GM) of mice, without affecting the E7-specific systemic response. The IVAG treatment locally increased both E7-specific and total CD8 T cells, but not CD4 T cells. This previously unreported selective recruitment of CD8 T cells from the periphery by IVAG CpG-ODN or poly-(I:C) was mediated by TLR9 and TLR3/melanoma differentiation-associated gene 5 signaling pathways, respectively. For CpG, this recruitment was associated with a higher proportion of GM-localized CD8 T cells expressing both CCR5 and CXCR3 chemokine receptors and E-selectin ligands. Most interestingly, IVAG CpG-ODN following vaccination led to complete regression of large genital HPV tumors in 75% of mice, instead of 20% with vaccination alone. These findings suggest that mucosal application of immunostimulatory molecules might substantially increase the effectiveness of parenterally administered vaccines.

Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure.

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

[Efficacy of a manual treatment method according to the fascial distortion model in the management of contracted ("frozen") shoulder]. / Wirksamkeit einer manuellen Behandlungstechnik nach dem Fasziendistorsionsmodell bei schmerzhaft eingeschränkter Schulterbeweglichkeit (Frozen Shoulder).

BACKGROUND: Frozen shoulder is a common problem and difficult to treat. The present prospective randomised single-blind controlled trial evaluates the efficacy of the 'fascial distortion model' according to Typaldos as a remedy for the 'frozen shoulder'. MATERIALS AND METHODS: A total of 60 patients were randomised to receive either the FDM-guided treatment (FDM, n = 30) or a 'conventional' manual therapy (MT, n = 30). The primary endpoint for the treatment effect was the shoulder mobility, and secondary endpoints were pain (measured on a VAS), raw force and function as expressed by the Constant-Murley and DASH scores. RESULTS: Before therapy, groups were well comparable in terms of all outcome parameters. All endpoints showed a substantial and significant improvement in both treatment groups. Improvement was significantly more marked in the FDM group as compared to the MT group, and the effect occurred significantly faster. During post-treatment observation, there was no further improvement and the achieved benefit in mobility in the FDM group decreased. However, the abduction ability of 150.2 ± 37.2° continued to be substantially better than in control patients (124.1 ± 38.6°, p < 0.01), and the ultimate improvement in abduction was 59.4° (64 % more than baseline) as opposed to 25.9° (27 %) in controls. Secondary outcome parameters (raw force, functional handicap, and pain) showed a significant improvement in both groups but a significantly better result in patients treated according to FDM guidelines. However, patients in this group experienced pain during the treatment more frequently (21/27 vs. 10/27, p < 0.01). CONCLUSION: Frozen shoulder treatment according to the FDM is an effective modality with swift onset of action and acceptable side effects that is superior to conventional manual therapy. Long-term effects and modes of action need to be investigated.