RNA Polymerase Inhibitor Enisamium for Treatment of Moderate COVID-19 Patients: A Randomized, Placebo-Controlled, Multicenter, Double-Blind Phase 3 Clinical Trial.

Enisamium is an orally available therapeutic that inhibits influenza A virus and SARS-CoV-2 replication. We evaluated the clinical efficacy of enisamium treatment combined with standard care in adult, hospitalized patients with moderate COVID-19 requiring external oxygen. Hospitalized patients with laboratory-confirmed SARS-CoV-2 infection were randomly assigned to receive either enisamium (500 mg per dose, four times a day) or a placebo. The primary outcome was an improvement of at least two points on an eight-point severity rating (SR) scale within 29 days of randomization. We initially set out to study the effect of enisamium on patients with a baseline SR of 4 or 5. However, because the study was started early in the COVID-19 pandemic, and COVID-19 had been insufficiently studied at the start of our study, an interim analysis was performed alongside a conditional power analysis in order to ensure patient safety and assess whether the treatment was likely to be beneficial for one or both groups. Following this analysis, a beneficial effect was observed for patients with an SR of 4 only, i.e., patients with moderate COVID-19 requiring supplementary oxygen. The study was continued for these COVID-19 patients. Overall, a total of 592 patients were enrolled and randomized between May 2020 and March 2021. Patients with a baseline SR of 4 were divided into two groups: 142 (49.8%) were assigned to the enisamium group and 143 (50.2%) to the placebo group. An analysis of the population showed that if patients were treated within 4 days of the onset of COVID-19 symptoms (n = 33), the median time to improvement was 8 days for the enisamium group and 13 days for the placebo group (p = 0.005). For patients treated within 10 days of the onset of COVID-19 symptoms (n = 154), the median time to improvement was 10 days for the enisamium group and 12 days for the placebo group (p = 0.002). Our findings suggest that enisamium is safe to use with COVID-19 patients, and that the observed clinical benefit of enisamium is worth reporting and studying in detail.

Male Reproduction in Spinal Muscular Atrophy (SMA) and the Potential Impact of Oral Survival of Motor Neuron 2 (SMN2) Pre-mRNA Splicing Modifiers.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene resulting in reduced levels of SMN protein. SMN protein is produced by cells throughout the body, and evidence suggests that low SMN protein can have systemic implications, including in male reproductive organs. However, a paucity of research exists on this important topic. This article will discuss findings from non-clinical studies on the role of SMN in the male reproductive system; additionally, real-world observational reports of individuals with SMA will be examined. Furthermore, we will review the non-clinical reproductive findings of risdiplam, a small-molecule SMN2 splicing modifier approved for the treatment of SMA, which has widespread distribution in both the central nervous system and peripheral organs. Specifically, the available non-clinical evidence of the effect of risdiplam on male reproductive organs and spermatogenesis is examined. Lastly, the article will highlight available capabilities to assess male fertility as well as the advanced reproductive technologies utilized to treat male infertility. This article demonstrates the need for further research to better understand the impacts of SMA on male fertility and reproduction.

Luciferase Expressing Preclinical Model Systems Representing the Different Molecular Subtypes of Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease. More insight into the biological diversity of CRC is needed to improve therapeutic outcomes. Established CRC cell lines are frequently used and were shown to be representative models of the main subtypes of CRC at the genomic and transcriptomic level. In the present work, we established stable, luciferase expressing derivatives from 10 well-established CRC cell lines, generated spheroids and subcutaneous xenograft tumors in nude mice, and performed comparative characterization of these model systems. Transcriptomic analyses revealed the close relation of cell lines with their derived spheroids and xenograft tumors. The preclinical model systems clustered with patient tumor samples when compared to normal tissue thereby confirming that cell-line-based tumor models retain specific characteristics of primary tumors. Xenografts showed different differentiation patterns and bioluminescence imaging revealed metastatic spread to the lungs. In addition, the models were classified according to the CMS classification system, with further sub-classification according to the recently identified two intrinsic epithelial tumor cell states of CRC, iCMS2 and iCMS3. The combined data showed that regarding primary tumor characteristics, 3D-spheroid cultures resemble xenografts more closely than 2D-cultured cells do. Furthermore, we set up a bioluminescence-based spheroid cytotoxicity assay in order to be able to perform dose-response relationship studies in analogy to typical monolayer assays. Applying the established assay, we studied the efficacy of oxaliplatin. Seven of the ten used cell lines showed a significant reduction in the response to oxaliplatin in the 3D-spheroid model compared to the 2D-monolayer model. Therapy studies in selected xenograft models confirmed the response or lack of response to oxaliplatin treatment. Analyses of differentially expressed genes in these models identified CAV1 as a possible marker of oxaliplatin resistance. In conclusion, we established a combined 2D/3D, in vitro/in vivo model system representing the heterogeneity of CRC, which can be used in preclinical research applications.

Differential CMS-Related Expression of Cell Surface Carbonic Anhydrases IX and XII in Colorectal Cancer Models-Implications for Therapy.

Tumor-associated carbonic anhydrases IX (CAIX) and XII (CAXII) have long been in the spotlight as potential new targets for anti-cancer therapy. Recently, CAIX/CAXII specific inhibitor SLC-0111 has passed clinical phase I study and showed differential response among patients with colorectal cancer (CRC). CRC can be classified into four different consensus molecular subgroups (CMS) showing unique expression patterns and molecular traits. We questioned whether there is a CMS-related CAIX/CAXII expression pattern in CRC predicting response. As such, we analyzed transcriptomic data of tumor samples for CA9/CA12 expression using Cancertool. Protein expression pattern was examined in preclinical models comprising cell lines, spheroids and xenograft tumors representing the CMS groups. Impact of CAIX/CAXII knockdown and SLC-0111 treatment was investigated in 2D and 3D cell culture. The transcriptomic data revealed a characteristic CMS-related CA9/CA12 expression pattern with pronounced co-expression of both CAs as a typical feature of CMS3 tumors. Protein expression in spheroid- and xenograft tumor tissue clearly differed, ranging from close to none (CMS1) to strong CAIX/CAXII co-expression in CMS3 models (HT29, LS174T). Accordingly, response to SLC-0111 analyzed in the spheroid model ranged from no (CMS1) to clear (CMS3), with moderate in CMS2 and mixed in CMS4. Furthermore, SLC-0111 positively affected impact of single and combined chemotherapeutic treatment of CMS3 spheroids. In addition, combined CAIX/CAXII knockdown and more effective treatment with SLC-0111 reduced clonogenic survival of CMS3 modelling single cells. In conclusion, the preclinical data support the clinical approach of targeted CAIX/CAXII inhibition by showing linkage of expression with response and suggest that patients with CMS3-classified tumors would most benefit from such treatment.

Reproductive findings in male animals exposed to selective survival of motor neuron 2 (SMN2) gene splicing-modifying agents.

Risdiplam is a daily, orally dosed, survival of motor neuron 2 (SMN2) mRNA splicing-modifying agent approved for the treatment of spinal muscular atrophy (SMA). RG7800 is a closely related SMN2 mRNA-splicing compound. Effects on secondary mRNA splice targets such as Forkhead Box M1 (FOXM1) and MAP kinase-activating death domain protein (MADD), which have been implicated in cell-cycle regulation, were observed in non-clinical studies with both risdiplam and RG7800. Potential effects of risdiplam on male fertility via FOXM1 and MADD are important as these secondary splice targets exist in humans. This publication reports the findings from 14 in vivo studies that investigated the reproductive tissues of male animals in various stages of development. Exposure to risdiplam or RG7800 induced changes within the germ cells in the testes of male cynomolgus monkeys and rats. Germ-cell changes included both cell-cycle gene changes (alteration of mRNA-splicing variants) and seminiferous tubule degeneration. In monkeys treated with RG7800, there was no evidence of damage to spermatogonia. Observed testicular changes were stage-specific with spermatocytes in the pachytene stage of meiosis and were fully reversible in monkeys following a sufficient recovery period of eight weeks following cessation of RG7800. In rats, seminiferous tubule degeneration was present, and full reversibility of germ-cell degeneration in the testes was observed among half of the rats that were exposed to risdiplam or RG7800 and then allowed to recover. With these results, coupled with histopathological findings, the effects on the male reproductive system are expected to be reversible in humans for these types of SMN2 mRNA-splicing modifiers.

Arterial input function for quantitative dynamic contrast-enhanced MRI to diagnose prostate cancer.

PURPOSE This study aims to analyze the ability of quantitative dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to distinguish between prostate cancer (PCa) and benign lesions in transition zone (TZ) and peripheral zone (PZ) using different methods for arterial input function (AIF) determination. Study endpoints are identification of a standard AIF method and optimal quantitative perfusion parameters for PCa detection. METHODS DCE image data of 50 consecutive patients with PCa who underwent multiparametric MRI were analyzed retrospectively with three different methods of AIF acquisition. First, a region of interest was manually defined in an artery (AIFm); second, an automated algorithm was used (AIFa); and third, a population-based AIF (AIFp) was applied. Values of quantitative parameters after Tofts (Ktrans, ve, and kep) in PCa, PZ, and TZ in the three different AIFs were analyzed. RESULTS Ktrans and kep were significantly higher in PCa than in benign tissue independent from the AIF method. Whereas in PZ, Ktrans and kep could differentiate PCa (P < .001), in TZ only kep using AIFpdemonstrated a significant difference (P = .039). The correlations of the perfusion parameters that resulted from AIFm and AIFa were higher than those that resulted from AIFp, and the absolute values of Ktrans, kep, and ve were significantly lower when using AIFp. The values of quantitative perfusion parameters for PCa were similar regardless of whether PCa was located in PZ or TZ. CONCLUSION Ktrans and kep were able to differentiate PCa from benign PZ independent of the AIF method. AIFaseems to be the most feasible method of AIF determination in clinical routine. For TZ, none of the quantitative perfusion parameters provided satisfying results.

Addressing Today's Absorption, Distribution, Metabolism, and Excretion (ADME) Challenges in the Translation of In Vitro ADME Characteristics to Humans: A Case Study of the SMN2 mRNA Splicing Modifier Risdiplam.

Small molecules that present complex absorption, distribution, metabolism, and elimination (ADME) properties can be challenging to investigate as potential therapeutics. Acquiring data through standard methods can yield results that are insufficient to describe the in vivo situation, which can affect downstream development decisions. Implementing in vitro-in vivo-in silico strategies throughout the drug development process is effective in identifying and mitigating risks while speeding up their development. Risdiplam (Evrysdi)-an orally bioavailable, small molecule approved by the US Food and Drug Administration and more recently by the European Medicines Agency for the treatment of patients ≥2 months of age with spinal muscular atrophy-is presented here as a case study. Risdiplam is a low-turnover compound whose metabolism is mediated through a non-cytochrome P450 enzymatic pathway. Four main challenges of risdiplam are discussed: predicting in vivo hepatic clearance, determining in vitro metabolites with regard to metabolites in safety testing guidelines, elucidating enzymes responsible for clearance, and estimating potential drug-drug interactions. A combination of in vitro and in vivo results was successfully extrapolated and used to develop a robust physiologically based pharmacokinetic model of risdiplam. These results were verified through early clinical studies, further strengthening the understanding of the ADME properties of risdiplam in humans. These approaches can be applied to other compounds with similar ADME profiles, which may be difficult to investigate using standard methods. SIGNIFICANCE STATEMENT: Risdiplam is the first approved, small-molecule, survival of motor neuron 2 mRNA splicing modifier for the treatment of spinal muscular atrophy. The approach taken to characterize the absorption, distribution, metabolism, and excretion (ADME) properties of risdiplam during clinical development incorporated in vitro-in vivo-in silico techniques, which may be applicable to other small molecules with challenging ADME. These strategies may be useful in improving the speed at which future drug molecules can be developed.

Enisamium is an inhibitor of the SARS-CoV-2 RNA polymerase and shows improvement of recovery in COVID-19 patients in an interim analysis of a clinical trial.

Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called Coronavirus Disease 2019 (COVID-19). Control of SARS-CoV-2 spread will depend on vaccine-induced or naturally acquired protective herd immunity. Until then, antiviral strategies are needed to manage COVID-19, but approved antiviral treatments, such as remdesivir, can only be delivered intravenously. Enisamium (laboratory code FAV00A, trade name Amizon®) is an orally active inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. Here we show that enisamium can inhibit SARS-CoV-2 infections in NHBE and Caco-2 cells. In vitro, the previously identified enisamium metabolite VR17-04 directly inhibits the activity of the SARS-CoV-2 RNA polymerase. Docking and molecular dynamics simulations suggest that VR17-04 prevents GTP and UTP incorporation. To confirm enisamium's antiviral properties, we conducted a double-blind, randomized, placebo-controlled trial in adult, hospitalized COVID-19 patients, which needed medical care either with or without supplementary oxygen. Patients received either enisamium (500 mg per dose) or placebo for 7 days. A pre-planned interim analysis showed in the subgroup of patients needing supplementary oxygen (n = 77) in the enisamium group a mean recovery time of 11.1 days, compared to 13.9 days for the placebo group (log-rank test; p=0.0259). No significant difference was found for all patients (n = 373) or those only needing medical care (n = 296). These results thus suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis and that enisamium treatment shortens the time to recovery for COVID-19 patients needing oxygen.

Risdiplam treatment has not led to retinal toxicity in patients with spinal muscular atrophy.

OBJECTIVE: Evaluation of ophthalmologic safety with focus on retinal safety in patients with spinal muscular atrophy (SMA) treated with risdiplam (EVRYSDI®), a survival of motor neuron 2 splicing modifier associated with retinal toxicity in monkeys. Risdiplam was approved recently for the treatment of patients with SMA, aged ≥ 2 months in the United States, and is currently under Health Authority review in the EU. METHODS: Subjects included patients with SMA aged 2 months-60 years enrolled in the FIREFISH, SUNFISH, and JEWELFISH clinical trials for risdiplam. Ophthalmologic assessments, including functional assessments (age-appropriate visual acuity and visual field) and imaging (spectral domain optical coherence tomography [SD-OCT], fundus photography, and fundus autofluorescence [FAF]), were conducted at baseline and every 2-6 months depending on study and assessment. SD-OCT, FAF, fundus photography, and threshold perimetry were evaluated by an independent, masked reading center. Adverse events (AEs) were reported throughout the study. RESULTS: A total of 245 patients receiving risdiplam were assessed. Comprehensive, high-quality, ophthalmologic monitoring assessing retinal structure and visual function showed no retinal structural or functional changes. In the youngest patients, SD-OCT findings of normal retinal maturation were observed. AEs involving eye disorders were not suggestive of risdiplam-induced toxicity and resolved with ongoing treatment. INTERPRETATION: Extensive ophthalmologic monitoring conducted in studies in patients with SMA confirmed that risdiplam does not induce ophthalmologic toxicity in pediatric or adult patients with SMA at the therapeutic dose. These results suggest that safety ophthalmologic monitoring is not needed in patients receiving risdiplam, as also reflected in the United States Prescribing Information for risdiplam.

Rewriting the (tran)script: Application to spinal muscular atrophy.

Targeting RNA drastically expands our target space to therapeutically modulate numerous cellular processes implicated in human diseases. Of particular interest, drugging pre-mRNA splicing appears a very viable strategy; to control levels of splicing product by promoting the inclusion or exclusion of exons. After describing the concept of "splicing modulation", this chapter will cover the outstanding progress achieved in this field, by highlighting the breakthrough accomplished recently for the treatment of spinal muscular atrophy using two therapeutic modalities: splice switching oligonucleotides and small molecules. This review discusses the vital but feasible requirement for such drugs to deliver selectivity, and critical safety aspects are highlighted. Transformational medicines such as those developed to treat SMA are likely just the beginning of this story.

Risdiplam distributes and increases SMN protein in both the central nervous system and peripheral organs.

Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small-molecule SMN2 pre-mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.

Discovery of Risdiplam, a Selective Survival of Motor Neuron-2 ( SMN2) Gene Splicing Modifier for the Treatment of Spinal Muscular Atrophy (SMA).

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound 2 was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of 1 (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA.

In Vitro Bioavailability Study of an Antiviral Compound Enisamium Iodide.

An investigation into the biopharmaceutics classification and a study of the in vitro bioavailability (permeability and solubility) of the antiviral compound enisamium iodide (4-(benzylcarbamoyl)-1-methylpyridinium iodide) were carried out. The solubility of enisamium iodide was determined in four different buffers. Apparent intestinal permeability (Papp) of enisamium iodide was assessed using human colon carcinoma (Caco-2) cells at three concentrations. The solubility of enisamium iodide in four buffer solutions from pH 1.2 to 7.5 is about 60 mg/mL at 25 °C, and ranges from 130 to 150 mg/mL at 37 °C, depending on the pH. Based on these results, enisamium iodide can be classified as highly soluble. Enisamium iodide demonstrated low permeability in Caco-2 experiments in all tested concentrations of 10-100 µM with permeability coefficients between 0.2 × 10-6 cm s-1 and 0.3 × 10-6 cm s-1. These results indicate that enisamium iodide belongs to class III of the Biopharmaceutics Classification System (BCS) due to its high solubility and low permeability. The bioavailability of enisamium iodide needs to be confirmed in animal and human studies.

Radiostereometric migration measurement of an uncemented Cerafit® femoral stem: 26 patients followed for 10 years.

Radiostereometric analysis (RSA) is the gold standard for evaluating micromotions of orthopaedic implants. The method is applied for identifying novel design weaknesses in endoprostheses. Current research frequently assesses relatively short time periods. Short-term RSA studies have been widely used for predicting the long-term stability of many hip prosthetic designs, but only a few studies have focused on uncemented hip implants, especially for extended periods. The purpose of this study was to analyse the migration pattern of the Cerafit® femoral stem within 10 years and to verify the predictive value of short-term RSA after 2 years for this uncemented femoral hip stem. Twenty-six patients were followed for 10 years. Ten years after implantation, a mean subsidence of 0.22 mm±0.56 mm, a mean internal rotation of 0.59°±1.67° and a mean maximum total point motion (MTPM) of 1.28 mm±0.54 mm were detected. The main migration took place in the first 6 weeks after surgery (subsidence of 0.36 mm±0.73 mm; internal rotation of 0.62°±1.49°, MTPM of 1.05 mm±0.68 mm). All the migration values measured were small. No late-onset migration was observed. This study suggests that the Cerafit® implants are stable after 10 years. Thus, RSA could be the best tool to assess long-term implant behaviour.

Specific Correction of Alternative Survival Motor Neuron 2 Splicing by Small Molecules: Discovery of a Potential Novel Medicine To Treat Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and exclusion of exon 7, producing an unstable, truncated SMNΔ7 protein. Herein, we report the identification of a pyridopyrimidinone series of small molecules that modify the alternative splicing of SMN2, increasing the production of full-length SMN2 mRNA. Upon oral administration of our small molecules, the levels of full-length SMN protein were restored in two mouse models of SMA. In-depth lead optimization in the pyridopyrimidinone series culminated in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to enter human clinical trials.

Analysis of direct clinical consequences of MLC positional errors in volumetric-modulated arc therapy using 3D dosimetry system.

In advanced, intensity-modulated external radiotherapy facility, the multileaf collimator has a decisive role in the beam modulation by creating multiple segments or dynamically varying field shapes to deliver a uniform dose distribution to the target with maximum sparing of normal tissues. The position of each MLC leaf has become more critical for intensity-modulated delivery (step-and-shoot IMRT, dynamic IMRT, and VMAT) compared to 3D CRT, where it defines only field boundaries. We analyzed the impact of the MLC positional errors on the dose distribution for volumetric-modulated arc therapy, using a 3D dosimetry system. A total of 15 VMAT cases, five each for brain, head and neck, and prostate cases, were retrospectively selected for the study. All the plans were generated in Monaco 3.0.0v TPS (Elekta Corporation, Atlanta, GA) and delivered using Elekta Synergy linear accelerator. Systematic errors of +1, +0.5, +0.3, 0, -1, -0.5, -0.3 mm were introduced in the MLC bank of the linear accelerator and the impact on the dose distribution of VMAT delivery was measured using the COMPASS 3D dosim-etry system. All the plans were created using single modulated arcs and the dose calculation was performed using a Monte Carlo algorithm in a grid size of 3 mm. The clinical endpoints D95%, D50%, D2%, and Dmax,D20%, D50% were taken for the evaluation of the target and critical organs doses, respectively. A significant dosimetric effect was found for many cases even with 0.5 mm of MLC positional errors. The average change of dose D 95% to PTV for ± 1 mm, ± 0.5 mm, and ±0.3mm was 5.15%, 2.58%, and 0.96% for brain cases; 7.19%, 3.67%, and 1.56% for head and neck cases; and 8.39%, 4.5%, and 1.86% for prostate cases, respectively. The average deviation of dose Dmax was 5.4%, 2.8%, and 0.83% for brainstem in brain cases; 8.2%, 4.4%, and 1.9% for spinal cord in H&N; and 10.8%, 6.2%, and 2.1% for rectum in prostate cases, respectively. The average changes in dose followed a linear relationship with the amount of MLC positional error, as can be expected. MLC positional errors beyond ± 0.3 mm showed a significant influence on the intensity-modulated dose distributions. It is, therefore, recommended to have a cautious MLC calibration procedure to sufficiently meet the accuracy in dose delivery.

Cryopreservation does not alter main characteristics of Good Manufacturing Process-grade human multipotent mesenchymal stromal cells including immunomodulating potential and lack of malignant transformation.

BACKGROUND AIMS: The immunomodulating capacity of multipotent mesenchymal stromal cells (MSCs) qualifies them as a therapeutic tool in several diseases. However, repeated transplantation with products of reproducible characteristics may be required. This could be achieved with cryopreserved aliquots of Good Manufacturing Practice (GMP)-grade MSCs. However, the impact of cryopreservation on the characteristics of GMP-MSCs is ill defined. METHODS: We produced fresh and cryopreserved MSCs from human donors with a xenogen-free GMP protocol. Immunogenicity and immunomodulating capacity were tested in co-culture with putative recipient-specific peripheral blood mononuclear cells (PBMCs). Risk of malignant transformation was assessed in vitro and in vivo. RESULTS: Cryopreservation had no impact on viability and consensus criteria of MSCs. In co-culture with PBMCs, MSCs showed low immunogenicity and suppressed mitogen-stimulated proliferation of PBMC irrespective of cryopreservation. Cytogenetic aberrations were not observed consistently in fresh and cryopreserved products, and no signs of malignant transformation occurred in functional assays. MSC products from an elderly pretreated donor showed reduced functional quality, but imminent failure of functional criteria could be detected by an increased population doubling time in early passages. DISCUSSION: This study is the first systematic analysis on cryopreservation of xenogen-free human bone marrow-derived GMP-MSCs. The data support that cryopreservation does not alter the characteristics of the cells and thus may allow the generation of products for serial transplantation. In addition, the protocol allowed early detection of MSC products with low functional capacity.

Radiostereometric migration analysis of the Lubinus SP II hip stem: 59 hips followed for 2 years.

The aim of this prospective study was to analyze the migration pattern of the Lubinus SP II hip stem and to evaluate the clinical results. Fifty-nine patients were followed for 2 years. Translational and rotational micromotion of the implant was measured by radiostereometric analysis (RSA) and the Harris hip score (HHS), and the Charnley classification was used to assess the clinical outcome. Although there was a very small, but statistically significant, distal migration of 0.04±0.83 mm, the prosthesis was found stable at 2 years of follow-up. The main migration in this direction took place between 6 months and 1 year. Maximum total point motion (MTPM) showed a mean of 0.99±0.69 mm. Good clinical outcome with HHS results of 42±11 before and 79±16 at 2 years after surgery was observed. The Charnley classification showed increasing additional impairments in the 2-year interval, which is likely to influence the HHS results of future follow-ups. The migration values measured in the present study are far below the thresholds considered clinically relevant in literature. Thus, the conclusion can be drawn that the implant is not at risk for early aseptic loosening. Long-term RSA is required to assess possible late migration.

Monitoring of xenograft tumor growth and response to chemotherapy by non-invasive in vivo multispectral fluorescence imaging.

A continuous monitoring of the whole tumor burden of individuals in orthotopic tumor models is a desirable aim and requires non-invasive imaging methods. Here we investigated whether quantification of a xenograft tumor intrinsic fluorescence signal can be used to evaluate tumor growth and response to chemotherapy. Stably fluorescence protein (FP) expressing cell clones of colorectal carcinoma and germ cell tumor lines were generated by lentiviral transduction using the FPs eGFP, dsRed2, TurboFP635, and mPlum. Applying subcutaneous tumor models in different experimental designs, specific correlations between measured total fluorescence intensity (FI) and the tumor volume (V) could be established. The accuracy of correlation of FI and V varied depending on the cell model used. The application of deep-red FP expressing xenografts (TurboFP635, mPlum) was observed to result in improved correlations. This was also reflected by the results of a performed error analysis. In a model of visceral growing mPlum tumors, measurements of FI could be used to follow growth and response to chemotherapy. However, in some cases final necropsy revealed the existence of additional, deeper located tumors that had not been detected in vivo by their mPlum signal. Consistently, only the weights of the tumors that were detected in vivo based on their mPlum signal correlated with FI. In conclusion, as long as tumors are visualized by their fluorescence signal the FI can be used to evaluate tumor burden. Deep-red FPs are more suitable for in vivo applications as compared to eGFP and dsRed2.