Optimisation of dose level and vaccination schedule for the VLA15 Lyme borreliosis vaccine candidate among healthy adults: two randomised, observer-blind, placebo-controlled, multicentre, phase 2 studies.

BACKGROUND: Rising Lyme borreliosis incidence rates, potential for severe outcomes, and limitations in accurate and timely diagnosis for treatment initiation suggest the need for a preventive vaccine; however, no vaccine is currently available for human use. We performed two studies in adults to optimise the dose level and vaccination schedule for VLA15, an investigational Lyme borreliosis vaccine targeting outer surface protein A (OspA) serotypes 1-6, which are associated with the most common pathogenic Borrelia species in Europe and North America. METHODS: Both randomised, observer-blind, placebo-controlled, multicentre phase 2 studies included participants aged 18-65 years without recent history of Lyme borreliosis or tick bites. Study one was conducted at nine clinical research and study centre sites in the USA (n=6), Germany (n=2), and Belgium (n=1); study two was conducted at five of the study one US sites. Based on a randomisation list created by an unmasked statistician for each study, participants were randomly assigned via an electronic case report form randomisation module to receive 90 µg (study one only), 135 µg, or 180 µg VLA15 or placebo by intramuscular injection at months 0, 1, and 2 (study one) or 0, 2, and 6 (study two). Study one began with a run-in phase to confirm safety, after which the Data Safety Monitoring Board recommended the removal of the 90 µg group and continuation of the study. In the study one run-in phase, randomisation was stratified by study site, whereas in the study one main phase and in study two, randomisation was stratified by study site, age group, and baseline B burgdorferi (sensu lato) serostatus. All individuals were masked, other than staff involved in randomisation, vaccine preparation or administration, or safety data monitoring. The primary endpoint for both studies was OspA-specific IgG geometric mean titres (GMTs) at 1 month after the third vaccination and was evaluated in the per-protocol population. Safety endpoints were evaluated in the safety population: all participants who received at least one vaccination. Both studies are registered at ClinicalTrials.gov (study one NCT03769194 and study two NCT03970733) and are completed. FINDINGS: For study one, 573 participants were screened and randomly assigned to treatment groups between Dec 21, 2018, and Sept, 26, 2019. For study two, 248 participants were screened and randomly assigned between June 26 and Sept 3, 2019. In study one, 29 participants were assigned to receive 90 µg VLA15, 215 to 135 µg, 205 to 180 µg, and 124 to placebo. In study two, 97 participants were assigned to receive 135 µg VLA15, 100 to 180 µg, and 51 to placebo. At 1 month after the third vaccination (ie, month 3), OspA-specific IgG GMTs in study one ranged from 74·3 (serotype 1; 95% CI 46·4-119·0) to 267·4 units per mL (serotype 3; 194·8-367·1) for 90 µg VLA15, 101·9 (serotype 1; 87·1-119·4) to 283·2 units per mL (serotype 3; 248·2-323·1) for 135 µg, and 115·8 (serotype 1; 98·8-135·7) to 308·6 units per mL (serotype 3; 266·8-356·8) for 180 µg. In study two, ranges at 1 month after the third vaccination (ie, month 7) were 278·5 (serotype 1; 214·9-361·0) to 545·2 units per mL (serotype 2; 431·8-688·4) for 135 µg VLA15 and 274·7 (serotype 1; 209·4-360·4) to 596·8 units per mL (serotype 3; 471·9-754·8) for 180 µg. Relative to placebo, the VLA15 groups had more frequent reports of solicited local adverse events (study one: 94%, 95% CI 91-96 vs 26%, 19-34; study two: 96%, 93-98 vs 35%, 24-49 after any vaccination) and solicited systemic adverse events (study one: 69%, 65-73 vs 43%, 34-52; study two: 74%, 67-80 vs 51%, 38-64); most were mild or moderate. In study one, unsolicited adverse events were reported by 52% (48-57) of participants in the VLA15 groups and 52% (43-60) of those in the placebo groups; for study two these were 65% (58-71) and 69% (55-80), respectively. Percentages of participants reporting serious unsolicited adverse events (study one: 2%, 1-4; study two: 4%, 2-7) and adverse events of special interest (study one: 1%, 0-2; study two: 1%, 0-3) were low across all groups. A single severe, possibly related unsolicited adverse event was reported (worsening of pre-existing ventricular extrasystoles, which resolved after change of relevant concomitant medication); no related serious adverse events or deaths were reported. INTERPRETATION: VLA15 was safe, well tolerated, and elicited robust antibody responses to all six OspA serotypes. These findings support further clinical development of VLA15 using the 180 µg dose and 0-2-6-month schedule, which was associated with the greatest immune responses. FUNDING: Valneva.

Safety and quality of cystectomy and pelvic lymph node dissection after neoadjuvant durvalumab and cisplatin/gemcitabine.

OBJECTIVE: To report on the surgical safety and quality of pelvic lymph node dissection (PLND) in patients treated with radical cystectomy (RC) and PLND for muscle-invasive bladder cancer (MIBC) after neoadjuvant chemo-immunotherapy. PATIENTS AND METHODS: The Swiss Group for Clinical Cancer Research (SAKK) 06/17 was an open-label single-arm phase II trial including 61 cisplatin-fit patients with clinical stage (c)T2-T4a cN0-1 operable urothelial MIBC or upper urinary tract cancer. Patients received neoadjuvant cisplatin/gemcitabine and durvalumab followed by surgery. Prospective quality assessment of surgeries was performed via central review of intraoperative photographs. Postoperative complications were assessed using the Clavien-Dindo Classification. Data were analysed descriptively. RESULTS: A total of 50 patients received RC and PLND. All patients received neoadjuvant chemo-immunotherapy. The median (interquartile range) number of lymph nodes removed was 29 (23-38). No intraoperative complications were registered. Grade ≥III postoperative complications were reported in 12 patients (24%). Complete nodal dissection (100%) was performed at the level of the obturator fossa (bilaterally) and of the left external iliac region; in 49 patients (98%) at the internal iliac region and at the right external iliac region; in 39 (78%) and 38 (76%) patients at the right and left presacral level, respectively. CONCLUSION: This study supports the surgical safety of RC and PLND following neoadjuvant chemo-immunotherapy in patients with MIBC. The extent and completeness of protocol-defined PLND varies between patients, highlighting the need to communicate and monitor the surgical template.

First-line durvalumab in patients with PD-L1 positive, advanced non-small cell lung cancer (NSCLC) with a performance status of 2 (PS2). Primary analysis of the multicenter, single-arm phase II trial SAKK 19/17.

INTRODUCTION: The safety and efficacy of first-line durvalumab in PS2 patients with advanced NSCLC is unknown. Here, we present the primary analysis of first-line durvalumab in PS2 patients, unsuitable for combination chemotherapy. METHODS: In this single-arm, multicenter, phase II trial patients with PD-L1 positive (tumor proportional score ≥25%), advanced NSCLC with PS2, received four-weekly durvalumab 1500 mg. The primary endpoint was overall survival (OS) at 6 months. RESULTS: Forty-eight patients were included. Median follow-up was 23.3 months (95% CI: 14.3-28.6). OS at 6 months was 60% (95% CI: 45-74%). Median OS was 8.5 months (95%CI: 4.4-16.7). Objective response rate and median progression free survival were 17% (95% CI: 8-30%) and 2.5 months (95% CI: 1.8-7.1), respectively. Thirty-three deaths were observed at the time point of the analysis. Seven early fatal events considered not treatment-related occurred during the first 5 weeks of treatment. Four out of the first 7 early fatal events (4/7; 57%) were respiratory failure in patients with advanced symptomatic primary lung tumors. Three more early fatal events occurred after exclusion of patients with grade ≥ 3 dyspnea. Treatment-related AEs ≥G3 were reported in 9 patients (19%) and included colonic perforation in one patient (grade 5), colitis in 4 patients (8%), increased lipase in 3 patients (6%), and hepatitis in 2 patients (4%). CONCLUSIONS: First-line durvalumab in PS2 patients with advanced PD-L1 positive NSCLC results in a high number of early fatal events. When patients with grade ≥ 3 dyspnea are excluded a promising 6-month OS with an acceptable toxicity profile can be observed. Durvalumab could be an option instead of single agent chemotherapy for PS2 patients who are not candidates for platinum doublet chemotherapy provided they are well selected.

A randomized, double-blinded Phase 3 study to demonstrate lot-to-lot consistency and to confirm immunogenicity and safety of the live-attenuated chikungunya virus vaccine candidate VLA1553 in healthy adults†.

BACKGROUND: The global spread of the chikungunya virus (CHIKV) increases the exposure risk for individuals travelling to or living in endemic areas. This Phase 3 study was designed to demonstrate manufacturing consistency between three lots of the single shot live-attenuated CHIKV vaccine VLA1553, and to confirm the promising immunogenicity and safety data obtained in previous trials. METHODS: This randomized, double-blinded, lot-to-lot consistency, Phase 3 study, assessed immunogenicity and safety of VLA1553 in 408 healthy adults (18-45 years) in 12 sites across the USA. The primary endpoint was a comparison of the geometric mean titre (GMT) ratios of CHIKV-specific neutralizing antibodies between three VLA1553 lots at 28 days post-vaccination. Secondary endpoints included immunogenicity and safety over 6 months post-vaccination. RESULTS: GMTs were comparable between the lots meeting the acceptance criteria for equivalence. The average GMT (measured by 50% CHIKV micro plaque neutralization test; µPRNT50) peaked with 2643 at 28 days post-vaccination and decreased to 709 at 6 months post-vaccination. An excellent seroresponse rate (defined as µPRNT50 titre ≥ 150 considered protective) was achieved in 97.8% of participants at 28 days post-vaccination and still persisted in 96% at 6 months after vaccination. Upon VLA1553 immunization, 72.5% of participants experienced adverse events (AEs), without significant differences between lots (related solicited systemic AE: 53.9% of participants; related solicited local AE: 19.4%). Overall, AEs were mostly mild or moderate and resolved without sequela, usually within 3 days. With 3.9% of participants experiencing severe AEs, 2.7% were classified as related, whereas none of the six reported serious adverse events was related to the administration of VLA1553. CONCLUSIONS: All three lots of VLA1553 recapitulated the safety and immunogenicity profiles of a preceding Phase 3 study, fulfilling pre-defined consistency requirements. These results highlight the manufacturability of VLA1553, a promising vaccine for the prevention of CHIKV disease for those living in or travelling to endemic areas.

[Individual Needs of the Patient in Breast Reconstruction - Are There Limits to Plastic Surgery?]

INTRODUCTION: The breast is essential to a woman's physical integrity. There are numerous techniques for breast reconstruction, so that the needs and limitations of each patient can be respected. The individual preferences of the patient play an important role in the decision of silicone implants vs. autologous tissue, size, and shape of the breast as well as the timing of the surgery. The only reasons not to perform a reconstruction are a locally incompletely removed tumor or the explicit wish of the patient against reconstruction. The costs for reconstruction are covered by the health insurance for all procedures, including symmetrizing the opposite breast, nipple reconstruction and autologous fat grafting.

Perioperative Chemoimmunotherapy With Durvalumab for Muscle-Invasive Urothelial Carcinoma: Primary Analysis of the Single-Arm Phase II Trial SAKK 06/17.

PURPOSE: The integration of immunotherapy in the perioperative setting of muscle-invasive urothelial carcinoma (MIUC) appears promising. SAKK 06/17 investigated the addition of neoadjuvant durvalumab to gemcitabine/cisplatin (GC) chemotherapy followed by radical surgery and adjuvant checkpoint inhibition with durvalumab. PATIENTS AND METHODS: SAKK 06/17 was an investigator-initiated, open-label, single-arm phase II study including cisplatin-fit patients with stage cT2-T4a cN0-1 operable MIUC. Four cycles of neoadjuvant GC in combination with four cycles of durvalumab (start with GC cycle 2) were administered, followed by radical surgery. Adjuvant durvalumab was given for 10 cycles. The primary end point was event-free survival (EFS) at 2 years. RESULTS: Sixty one patients were accrued at 12 sites. The full analysis set consisted of 57 patients, 54 (95%) had bladder cancer. Median follow-up was 40 months. The primary end point was met, with EFS at 2 years of 76% (one-sided 90% CI [lower bound], 67%; two-sided 95% CI, 62 to 85). EFS at 3 years was 73% (95% CI, 59 to 83). Complete pathologic response in resected patients (N = 52) was achieved in 17 patients (33%), and 31 (60%) had pathologic response

Safety and immunogenicity of a single-shot live-attenuated chikungunya vaccine: a double-blind, multicentre, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: VLA1553 is a live-attenuated vaccine candidate for active immunisation and prevention of disease caused by chikungunya virus. We report safety and immunogenicity data up to day 180 after vaccination with VLA1553. METHODS: This double-blind, multicentre, randomised, phase 3 trial was done in 43 professional vaccine trial sites in the USA. Eligible participants were healthy volunteers aged 18 years and older. Patients were excluded if they had history of chikungunya virus infection or immune-mediated or chronic arthritis or arthralgia, known or suspected defect of the immune system, any inactivated vaccine received within 2 weeks before vaccination with VLA1553, or any live vaccine received within 4 weeks before vaccination with VLA1553. Participants were randomised (3:1) to receive VLA1553 or placebo. The primary endpoint was the proportion of baseline negative participants with a seroprotective chikungunya virus antibody level defined as 50% plaque reduction in a micro plaque reduction neutralisation test (µPRNT) with a µPRNT50 titre of at least 150, 28 days after vaccination. The safety analysis included all individuals who received vaccination. Immunogenicity analyses were done in a subset of participants at 12 pre-selected study sites. These participants were required to have no major protocol deviations to be included in the per-protocol population for immunogenicity analyses. This trial is registered at ClinicalTrials.gov, NCT04546724. FINDINGS: Between Sept 17, 2020 and April 10, 2021, 6100 people were screened for eligibility. 1972 people were excluded and 4128 participants were enrolled and randomised (3093 to VLA1553 and 1035 to placebo). 358 participants in the VLA1553 group and 133 participants in the placebo group discontinued before trial end. The per-protocol population for immunogenicity analysis comprised 362 participants (266 in the VLA1553 group and 96 in the placebo group). After a single vaccination, VLA1553 induced seroprotective chikungunya virus neutralising antibody levels in 263 (98·9%) of 266 participants in the VLA1553 group (95% CI 96·7-99·8; p<0·0001) 28 days post-vaccination, independent of age. VLA1553 was generally safe with an adverse event profile similar to other licensed vaccines and equally well tolerated in younger and older adults. Serious adverse events were reported in 46 (1·5%) of 3082 participants exposed to VLA1553 and eight (0·8%) of 1033 participants in the placebo arm. Only two serious adverse events were considered related to VLA1553 treatment (one mild myalgia and one syndrome of inappropriate antidiuretic hormone secretion). Both participants recovered fully. INTERPRETATION: The strong immune response and the generation of seroprotective titres in almost all vaccinated participants suggests that VLA1553 is an excellent candidate for the prevention of disease caused by chikungunya virus. FUNDING: Valneva, Coalition for Epidemic Preparedness Innovation, and EU Horizon 2020.

Novel sequential treatment strategy for patients with muscle-invasive bladder cancer (MIBC): intravesical recombinant BCG, followed by neoadjuvant chemoimmunotherapy, radical cystectomy plus pelvic lymphadenectomy and adjuvant immunotherapy - protocol of a multicentre, single arm phase 2 trial (SAKK 06/19).

INTRODUCTION: The combination of checkpoint inhibition and cisplatin-based chemotherapy is investigated in muscle invasive bladder cancer (MIBC) and results from phase 2 trials have been presented. Intravesical BCG has been used for non-MIBC (NMIBC) in patients with carcinoma in situ and high-grade Ta/T1 tumours. BCG induces innate and adapted immune response and upregulation of PD-L1 in preclinical models. The proposed trial is intended to implement a new immuno-immuno-chemotherapy induction therapy for MIBC. The combination of BCG and checkpoint inhibition with chemotherapy aims at higher intravesical responses and better local and systemic control of disease. METHODS AND ANALYSIS: SAKK 06/19 is an open-label single-arm phase II trial for patients with resectable MIBC T2-T4a cN0-1. Intravesical recombinant BCG (rBCG: VPM1002BC) is applied weekly for three instillations followed by four cycles of neoadjuvant cisplatin/gemcitabine every 3 weeks. Atezolizumab 1200 mg every 3 weeks is started together with rBCG and given for four cycles. All patients then undergo restaging and radical cystectomy and pelvic lymphadenectomy. Atezolizumab is continued as maintenance therapy after surgery every 3 weeks for 13 cycles. Pathological complete remission is the primary endpoint. Secondary endpoints include pathological response rate (

Continuous Production of Ethanol, 1-Butanol and 1-Hexanol from CO with a Synthetic Co-Culture of Clostridia Applying a Cascade of Stirred-Tank Bioreactors.

Syngas fermentation with clostridial co-cultures is promising for the conversion of CO to alcohols. A CO sensitivity study with Clostridium kluyveri monocultures in batch operated stirred-tank bioreactors revealed total growth inhibition of C. kluyveri already at 100 mbar CO, but stable biomass concentrations and ongoing chain elongation at 800 mbar CO. On/off-gassing with CO indicated a reversible inhibition of C. kluyveri. A continuous supply of sulfide led to increased autotrophic growth and ethanol formation by Clostridium carboxidivorans even at unfavorable low CO concentrations. Based on these results, a continuously operated cascade of two stirred-tank reactors was established with a synthetic co-culture of both Clostridia. An amount of 100 mbar CO and additional sulfide supply enabled growth and chain elongation in the first bioreactor, whereas 800 mbar CO resulted in an efficient reduction of organic acids and de-novo synthesis of C2-C6 alcohols in the second reactor. High alcohol/acid ratios of 4.5-9.1 (w/w) were achieved in the steady state of the cascade process, and the space-time yields of the alcohols produced were improved by factors of 1.9-5.3 compared to a batch process. Further improvement of continuous production of medium chain alcohols from CO may be possible by applying less CO-sensitive chain-elongating bacteria in co-cultures.

Substances from unregulated drug markets - A retrospective data analysis of customer-provided samples from a decade of drug checking service in Zurich (Switzerland).

BACKGROUND: Drug checking services (DCS) are harm reduction interventions for people who consume illicit substances. Unregulated drug markets lead to samples with unexpected and variable contents. A retrospective data analysis of Zurich's DCS was performed to determine the nature of these samples. METHODS: This study aims to investigate the qualitative and quantitative properties of 16,815 customer-provided psychoactive drug samples analyzed chemically through the DCS in Zurich from 1st January 2011 to 31st December 2021. The main analytical method utilized for characterizing these substances was high-performance liquid chromatography and gas chromatography-mass spectrometry. Data sets are summarized using descriptive statistics. RESULTS: There was a 2.5-fold increase in the number of tested samples over the past decade. An overall proportion of 57.9% (weighted mean) of samples within our database demonstrates unexpected analytical findings and additional low sample contents during the observation period. Substantial differences in quality and quantity between substance groups were detected and an increase of sample quality and content over time was demonstrated. CONCLUSIONS: Chemical analysis reveals that over half of substances acquired from unregulated drug markets analyzed through DCS in Zurich are with low qualitative and quantitative properties, which may expose users to risks. Based on longitudinal analyses over a decade, this study contributes to the body of evidence that DCS may potentially manipulate unregulated drug markets towards providing better quality substances, as well as may stabilize these markets over time. The necessity for drug policy changes to make this service accessible in further settings was highlighted, as DCS still often take place in legal grey zones.

A randomized, placebo-controlled, blinded phase 1 study investigating a novel inactivated, Vero cell-culture derived Zika virus vaccine.

BACKGROUND: Zika virus (ZIKV) is an emerging public health threat, rendering development of a safe and effective vaccine against the virus a high priority to face this unmet medical need. Our vaccine candidate has been developed on the same platform used for the licensed vaccine IXIARO®, a vaccine against Japanese Encephalitis virus, another closely related member of the Flaviviridae family. METHODS: Between February 24, 2018 and November 16, 2018, we conducted a randomized, observer-blinded, placebo controlled, single center phase 1 study to assess the safety and immunogenicity of an adjuvanted, inactivated, purified whole-virus Zika vaccine candidate in the U.S. A total of 67 healthy flavivirus-naïve adults aged 18 to 49 years were randomly assigned to one of five study arms to receive two immunizations of either high dose or low dose (6 antigen units or 3 antigen units) with both dose levels applied in two different immunization regimens or placebo as control. RESULTS: Our vaccine candidate showed an excellent safety profile independent of dose and vaccination regimen with predominantly mild adverse events. No serious adverse event has been reported. The ZIKV vaccine induced neutralizing antibodies in all tested doses and regimens with seroconversion rates up to 85.7% (high dose), which remained up to 40% (high dose) at 6 months follow-up. Of note, the rapid regimen triggered a substantial immune response within days. CONCLUSIONS: The rapid development and production of a ZIKV vaccine candidate building on a commercial Vero-cell manufacturing platform resulted in a safe and immunogenic vaccine suitable for further clinical development. To optimize antibody persistence, higher doses and a booster administration might be considered.

Fibroblast growth factor receptor (FGFR) inhibitor rogaratinib in patients with advanced pretreated squamous-cell non-small cell lung cancer over-expressing FGFR mRNA: The SAKK 19/18 phase II study.

BACKGROUND: Patients with advanced squamous-cell lung cancer (SQCLC) frequently (46%) exhibit tumor overexpression of fibroblast growth factor receptor (FGFR) messenger ribonucleic acid (mRNA). Rogaratinib is a novel oral pan-FGFR inhibitor with a good safety profile and anti-tumor activity in early clinical trials as a single agent in FGFR pathway-addicted tumors. SAKK 19/18 determined clinical activity of rogaratinib in patients with advanced SQCLC overexpressing FGFR1-3 mRNA. METHODS: Patients with advanced SQCLC failing standard systemic treatment and with FGFR1-3 mRNA tumor overexpression as defined in the protocol received rogaratinib 600 mg BID until disease progression or intolerable toxicity. A 6-months progression-free survival rate (6mPFS) ≤15 % was considered uninteresting (H0), whereas a 6mPFS ≥38 % was considered promising (H1). According to a Simon 2-stage design, 2 out of 10 patients of the first stage were required to be progression-free at 6 months. Comprehensive Genomic Profiling was performedusing the Oncomine Comprehensive Assay Plus (Thermo Fisher Scientific). RESULTS: Between July 2019 and November 2020, 49 patients were screened and 20 were classified FGFR-positive. Among a total of 15 patients, 6mPFS was reached in 1 patient (6.7 %), resulting in trial closure for futility after the first stage. There were 7 (46.7 %) patients with stable disease and 5 (33.3 %) patients with progressive disease. Median PFS was 1.6 (95 % CI 0.9-3.5) months and median overall survival (OS) 3.5 (95 % CI 1.0-5.9) months. Most frequent treatment-related adverse events (TRAEs) included hyperphosphatemia in 8 (53 %), diarrhea in 5 (33 %), stomatitis in 3 (20 %) and nail changes in 3 (20 %) patients. Grade ≥3 TRAEs occurred in 6 (40 %) patients. No associations between mutational profile and treatment outcome were observed. CONCLUSION: Despite preliminary signals of activity, rogaratinib failed to improve PFS in patients with advanced SQCLC overexpressing FGFR mRNA. FGFR inhibitors in SQCLC remain a challenging field, and more in-depth understanding of pathway crosstalks may lead to the development of drug combinations with FGFR inhibitors resulting in improved outcomes.

Adaptive wireless millirobotic locomotion into distal vasculature.

Microcatheters have enabled diverse minimally invasive endovascular operations and notable health benefits compared with open surgeries. However, with tortuous routes far from the arterial puncture site, the distal vascular regions remain challenging for safe catheter access. Therefore, we propose a wireless stent-shaped magnetic soft robot to be deployed, actively navigated, used for medical functions, and retrieved in the example M4 segment of the middle cerebral artery. We investigate shape-adaptively controlled locomotion in phantoms emulating the physiological conditions here, where the lumen diameter shrinks from 1.5 mm to 1 mm, the radius of curvature of the tortuous lumen gets as small as 3 mm, the lumen bifurcation angle goes up to 120°, and the pulsatile flow speed reaches up to 26 cm/s. The robot can also withstand the flow when the magnetic actuation is turned off. These locomotion capabilities are confirmed in porcine arteries ex vivo. Furthermore, variants of the robot could release the tissue plasminogen activator on-demand locally for thrombolysis and function as flow diverters, initiating promising therapies towards acute ischemic stroke, aneurysm, arteriovenous malformation, dural arteriovenous fistulas, and brain tumors. These functions should facilitate the robot's usage in new distal endovascular operations.

Heterosubtypic, cross-reactive immunity to human Cytomegalovirus glycoprotein B.

Cytomegalovirus (CMV) genome is highly variable and heterosubtypic immunity should be considered in vaccine development since it can enhance protection in a cross-reactive manner. Here, we developed a protein array to evaluate heterosubtypic immunity to CMV glycoprotein B (gB) in natural infection and vaccination. DNA sequences of four antigenic domains (AD1, AD2, AD4/5, and AD5) of gB were amplified from six reference and 12 clinical CMV strains, and the most divergent genotypes were determined by phylogenetic analysis. Assigned genotypes were in vitro translated and immobilized on protein array. Then, we tested immune response of variable serum groups (primarily infected patients, reactivated CMV infections and healthy individuals with latent CMV infection, as well gB-vaccinated rabbits) with protein in situ array (PISA). Serum antibodies of all patient cohorts and gB-vaccinated rabbits recognized many genetic variants of ADs on protein array, including but not limited to the subtype of infecting strain. High-grade cross-reactivity was observed. In several patients, we observed none or neglectable immune response to AD1 and AD2, while the same patients showed high antibody response to AD4/5 and AD5. Among the primary infected patients, AD5 was the predominant AD, in antibody response. The most successful CMV vaccine to date contains gB and demonstrates only 50% efficacy. In this study, we showed that heterosubtypic and cross-reactive immunity to CMV gB is extensive. Therefore, the failure of CMV gB vaccines cannot be explained by a highly, strain-specific immunity. Our observations suggest that other CMV antigens should be addressed in vaccine design.

A Tissue Adhesion-Controllable and Biocompatible Small-Scale Hydrogel Adhesive Robot.

Recently, the realization of minimally invasive medical interventions on targeted tissues using wireless small-scale medical robots has received an increasing attention. For effective implementation, such robots should have a strong adhesion capability to biological tissues and at the same time easy controlled detachment should be possible, which has been challenging. To address such issue, a small-scale soft robot with octopus-inspired hydrogel adhesive (OHA) is proposed. Hydrogels of different Young's moduli are adapted to achieve a biocompatible adhesive with strong wet adhesion by preventing the collapse of the octopus-inspired patterns during preloading. Introduction of poly(N-isopropylacrylamide) hydrogel for dome-like protuberance structure inside the sucker wall of polyethylene glycol diacrylate hydrogel provides a strong tissue attachment in underwater and at the same time enables easy detachment by temperature changes due to its temperature-dependent volume change property. It is finally demonstrated that the small-scale soft OHA robot can efficiently implement biomedical functions owing to strong adhesion and controllable detachment on biological tissues while operating inside the body. Such robots with repeatable tissue attachment and detachment possibility pave the way for future wireless soft miniature robots with minimally invasive medical interventions.

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry.

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO2 -gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.

Monitoring co-cultures of Clostridium carboxidivorans and Clostridium kluyveri by fluorescence in situ hybridization with specific 23S rRNA oligonucleotide probes.

The development of co-cultures of clostridial strains which combine different physiological traits represents a promising strategy to achieve the environmentally friendly production of biofuels and chemicals. For the optimization of such co-cultures it is essential to monitor their composition and stability throughout fermentation. FISH is a quick and sensitive method for the specific labeling and quantification of cells within microbial communities. This technique is neither limited by the anaerobic fermenter environment nor by the need of prior genetic modification of strains. In this study, two specific 23S rRNA oligonucleotide probes, ClosKluy and ClosCarb, were designed for the monitoring of C. kluyveri and C. carboxidivorans, respectively. After the optimization of hybridization conditions for both probes, which was achieved at 30% (v/v) formamide, a high specificity was observed with epifluorescence microscopy using cells from different pure reference strains. The discriminating properties of the ClosKluy and ClosCarb probes was verified with samples from heterotrophic co-cultures in anaerobic flasks as well as autotrophic stirred-tank bioreactor co-cultures of C. kluyveri and C. carboxidivorans. Besides being suited to monitor defined co-cultures of these two species, the new specific FISH oligonucleotide probes for C. kluyveri and C. carboxidivorans additionally have potential to be applied in environmental studies.

Protective role of Gremlin-1 in myocardial function.

BACKGROUND: Gremlin-1 is a cystine knot protein and is expressed in organs developing fibrosis. Transient ischaemia leads to myocardial fibrosis, a major determinant of impaired myocardial function. MATERIALS AND METHODS: Expression of Gremlin-1 was investigated in infarcted myocardium by real-time PCR, Western blot analysis, histological and immunohistochemistry staining. We further elaborated the colocalization of Gremlin-1 and TGF-ß proteins by confocal microscopy and co-immunoprecipitation experiments. The interaction between Gremlin-1 and TGF-ß was analysed by photon correlation spectroscopy. Gremlin-1 modulation of the TGF-ß-dependent collagen I synthesis in fibroblasts was investigated using ELISA and immunohistochemistry experiments. The effect of prolonged administration of recombinant Gremlin-1 on myocardial function following ischaemia/reperfusion was accessed by echocardiography and immunohistochemistry. RESULTS: Gremlin-1 is expressed in myocardial tissue and infiltrating cells after transient myocardial ischaemia (P < .05). Gremlin-1 colocalizes with the pro-fibrotic cytokine transforming growth factor-ß (TGF-ß) expressed in fibroblasts and inflammatory cell infiltrates (P < .05). Gremlin-1 reduces TGF-ß-induced collagen production of myocardial fibroblasts by approximately 20% (P < .05). We found that Gremlin-1 binds with high affinity to TGF-ß (KD  = 54 nmol/L) as evidenced by photon correlation spectroscopy and co-immunoprecipitation. intravenous administration of m Gremlin-1-Fc, but not of equivalent amount of Fc control, significantly reduced infarct size by approximately 20%. In the m Gremlin-1-Fc group, infarct area was reduced by up to 30% in comparison with mice treated with Fc control (I/LV: 4.8 ± 1.2% vs 6.0 ± 1.2% P < .05; I/AaR: 15.2 ± 1.5% vs 21.1 ± 5%, P < .05). CONCLUSIONS: The present data disclose Gremlin-1 as an antagonist of TGF-ß and presume a role for Gremlin-1/TGF-ß interaction in myocardial remodelling following myocardial ischaemia.

Cytomegalovirus Infection Downregulates Vitamin D Receptor in Patients Undergoing Hematopoietic Stem Cell Transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative option for patients with hematologic diseases but is associated with high mortality and morbidity. Cytomegalovirus (CMV) infection is common in HSCT patients and modulates vitamin D metabolism in vitro. We aimed at validating CMV-associated vitamin D metabolism in vivo in HSCT. METHODS: Patients treated for significant CMV viremia after HSCT were evaluated for CMV load before, during, and after antiviral treatment. RNA was isolated from whole-blood samples to test for regulation of key components of the vitamin D receptor (VDR) pathway during different phases of CMV viremia. RESULTS: CMV viremia developed a mean time of 102 (±34) d post-HSCT. Maximum levels of CMV-DNA reached a mean of 5668 (±7257) copies/mL. VDR expression was downregulated to a mean of 64.3% (±42.5%) relative to the VDR expression pre-CMV viremia (P = 0.035) and lagged in recovery following antiviral treatment. Toll-like receptor (TLR) 2 mRNA was upregulated to 225.4% during CMV viremia relative to the expression pre-CMV viremia (P = 0.012) but not TLR6/7/8 and the TLR-adaptor protein MyD88. Levels of 25-OH vitamin D were reduced in all viremic patients (48.0 ± 4.8 versus 25.1 ± 3.7 ng/mL) and were even lower after periods of CMV viremia compared with the control group (48.3 ± 3.5 versus 17.8 ± 1.8 ng/mL; P = 0.008). CONCLUSIONS: CMV viremia is associated with significant dysregulation of vitamin D metabolism in HSCT patients.