Efficacy and correlative analyses of avelumab plus axitinib versus sunitinib in sarcomatoid renal cell carcinoma: post hoc analysis of a randomized clinical trial.

BACKGROUND: Among patients with advanced renal cell carcinoma (RCC), those with sarcomatoid histology (sRCC) have the poorest prognosis. This analysis assessed the efficacy of avelumab plus axitinib versus sunitinib in patients with treatment-naive advanced sRCC. METHODS: The randomized, open-label, multicenter, phase III JAVELIN Renal 101 trial (NCT02684006) enrolled patients with treatment-naive advanced RCC. Patients were randomized 1 : 1 to receive either avelumab plus axitinib or sunitinib following standard doses and schedules. Assessments in this post hoc analysis of patients with sRCC included efficacy (including progression-free survival) and biomarker analyses. RESULTS: A total of 108 patients had sarcomatoid histology and were included in this post hoc analysis; 47 patients in the avelumab plus axitinib arm and 61 in the sunitinib arm. Patients in the avelumab plus axitinib arm had improved progression-free survival [stratified hazard ratio, 0.57 (95% confidence interval, 0.325-1.003)] and a higher objective response rate (46.8% versus 21.3%; complete response in 4.3% versus 0%) versus those in the sunitinib arm. Correlative gene expression analyses of patients with sRCC showed enrichment of gene pathway scores for cancer-associated fibroblasts and regulatory T cells, CD274 and CD8A expression, and tumors with The Cancer Genome Atlas m3 classification. CONCLUSIONS: In this subgroup analysis of JAVELIN Renal 101, patients with sRCC in the avelumab plus axitinib arm had improved efficacy outcomes versus those in the sunitinib arm. Correlative analyses provide insight into this subtype of RCC and suggest that avelumab plus axitinib may increase the chance of overcoming the aggressive features of sRCC.

Age-dependent changes in intervertebral disc cell mitochondria and bioenergetics.

Robust cellular bioenergetics is vital in the energy-demanding process of maintaining matrix homeostasis in the intervertebral disc. Age-related decline in disc cellular bioenergetics is hypothesised to contribute to the matrix homeostatic perturbation observed in intervertebral disc degeneration. The present study aimed to measure how ageing impacted disc cell mitochondria and bioenergetics. Age-related changes measured included matrix content and cellularity in disc tissue, as well as matrix synthesis, cell proliferation and senescence markers in cell cultures derived from annulus fibrosus (AF) and nucleus pulposus (NP) isolated from the discs of young (6-9 months) and older (36-50 months) New Zealand White rabbits. Cellular bioenergetic parameters were measured using a Seahorse XFe96 Analyzer, in addition to quantitating mitochondrial morphological changes and membrane potential. Ageing reduced mitochondrial number and membrane potential in both cell types. Also, it significantly reduced glycolytic capacity, mitochondrial reserve capacity, maximum aerobic capacity and non-glucose-dependent respiration in NP. Moreover, NP cells exhibited age-related decline in matrix synthesis and reduced cellularity in older tissues. Despite a lack of changes in mitochondrial respiration with age, AF cells showed an increase in glycolysis and altered matrix production. While previous studies report age-related matrix degenerative changes in disc cells, the present study revealed, for the first time, that ageing affected mitochondrial number and function, particularly in NP cells. Consequently, age-related bioenergetic changes may contribute to the functional alterations in aged NP cells that underlie disc degeneration.

Effects of pH, conductivity, host cell protein, and DNA size distribution on DNA clearance in anion exchange chromatography media.

Flowthrough anion exchange chromatography is commonly used as a polishing step in downstream processing of monoclonal antibodies and other therapeutic proteins to remove process-related impurities and contaminants such as host cell DNA, host cell proteins, endotoxin, and viruses. DNA with a wide range of molecular weight distributions derived from Chinese Hamster Ovary cells was used to advance the understanding of DNA binding behavior in selected anion exchange media using the resin (Toyopearl SuperQ-650M) and membranes (Mustang® Q and Sartobind® Q) through DNA spiking studies. The impacts of the process parameters pH (6-8), conductivity (2-15 mS/cm), and the potential binding competition between host cell proteins and host cell DNA were studied. Studies were conducted at the least and most favorable experimental conditions for DNA binding based on the anticipated electrostatic interactions between the host cell DNA and the resin ligand. The resin showed 50% higher DNA binding capacity compared to the membrane media. Spiking host cell proteins in the load material showed no impact on the DNA clearance capability of the anion exchange media. DNA size distributions were characterized based on a "size exclusion qPCR assay." Results showed preferential binding of larger DNA fragments (>409 base pairs). © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:141-149, 2018.

Exosomes as biomarkers and therapeutic tools for type 1 diabetes mellitus.

Early diagnosis of diabetes mellitus can significantly improve therapeutic strategies and overall health span. Identifying biomarkers as a tool for determining the risk of developing diabetes as well as a monitoring strategy for progression of the disease state would be useful in predicting potential complications while simultaneously improving our ability to prevent and treat diabetes. Extracellular vesicles (EV) have recently emerged as prominent mediators of intercellular communication and as a potential source for the discovery of novel biomarkers. A deeper understanding of the cargo molecules present in EVs obtained from type 1 diabetes mellitus (T1D) patients may aid in the identification of novel diagnostic and prognostic biomarkers, and can potentially lead to the discovery of new therapeutic targets.

Intracranial monodermal teratoma presenting with growing teratoma syndrome.

Intracranial teratomas are rare. We report a case of a purely monodermal teratoma manifesting as intracranial growing teratoma syndrome. To the best of our knowledge, this is the first report of such nature in the literature.

Adhesion of Pseudomonas fluorescens biofilms to glass, stainless steel and cellulose.

OBJECTIVES: The adhesion of colloidal probes of stainless steel, glass and cellulose to Pseudomonas fluorescens biofilms was examined using atomic force microscopy (AFM) to allow comparisons between surfaces to which biofilms might adhere. RESULTS: Biofilm was grown on a stainless steel substrate and covered most of the surface after 96 h. AFM approach and retraction curves were obtained when the biofilm was immersed in a tryptone/soy medium. On approach, all the colloidal probes experienced a long non-contact phase more than 100 nm in length, possibly due to the steric repulsion by extracellular polymers from the biofilm and hydrophobic effects. Retraction data showed that the adhesion varied from position to position on the biofilm. The mean value of adhesion of glass to the biofilm (48 ± 7 nN) was the greatest, followed by stainless steel (30 ± 7 nN) and cellulose (7.8 ± 0.4 nN). CONCLUSION: The method allows understanding of adhesion between the three materials and biofilm, and development of a better strategy to remove the biofilm from these surfaces relevant to different industrial applications.

NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair.

The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found that deleting one allele of the NF-κB subunit p65 (p65+/-) improved the survival and enhanced the anti-inflammatory capacity of muscle-derived stem cells (MDSCs) following intramuscular transplantation. Factors secreted from p65+/- MDSCs in cell cultures modulated macrophage cytokine expression in an HGF-receptor-dependent manner. Indeed, we found that following genetic or pharmacologic inhibition of basal NF-κB/p65 activity, HGF gene transcription was induced in MDSCs. We investigated the role of HGF in anti-NF-κB therapy in vivo using mdx;p65+/- mice, and found that accelerated regeneration coincided with HGF upregulation in the skeletal muscle. This anti-NF-κB-mediated dystrophic phenotype was reversed by blocking de novo HGF production by myogenic cells following disease onset. HGF silencing resulted in increased inflammation and extensive necrosis of the diaphragm muscle. Proteolytic processing of matrix-associated HGF is known to activate muscle stem cells at the earliest stages of repair, but our results indicate that the production of a second pool of HGF by myogenic cells, negatively regulated by NF-κB/p65, is crucial for inflammation resolution and the completion of repair in dystrophic skeletal muscle. Our findings warrant further investigation into the potential of HGF mimetics for the treatment of DMD.

Renal cell carcinoma to haemangioblastoma metastasis: a rare manifestation of Von Hippel-Lindau syndrome.

Brain metastases are the most common intracranial malignancy in adults and may occasionally deposit within a pre-existing primary brain neoplasm. We describe, in two directly related family members, the rare occurrence of renal cell carcinoma (RCC) metastasis to haemangioblastoma (HB) in the context of Von Hippel-Lindau syndrome. Detection of this phenomenon can be marred by histological overlap between RCC and HB and therefore careful histological examination, and consideration of supportive immunohistochemistry, is required when examining all HB resections. Metastatic RCC to HB upstages a primary RCC and is clinically diagnostic of Von Hippel-Lindau syndrome.

Expression of IL-2 in ß cells by AAV8 gene transfer in pre-diabetic NOD mice prevents diabetes through activation of FoxP3-positive regulatory T cells.

We previously demonstrated that intraperitoneal delivery of adeno-associated virus serotype 8 (AAV8) stably transduces the pancreas, including the ß cells in the pancreatic islets. We further demonstrated the ability to deliver and express target genes specifically in ß cells for at least 6 months using a murine insulin promoter in a double-stranded, self-complementary AAV vector. Recombinant interleukin (IL)-2 has been shown to induce CD4(+)CD25(+) regulatory T cells (Tregs) in several mouse models of autoimmune disease. Here we evaluated the effects of double-stranded adeno-associated virus serotype 8-mouse insulin promoter (dsAAV8-mIP)-mediated delivery of  2 to pancreatic ß cells in non-obese diabetic (NOD) mice. AAV8-mIP-mediated gene expression of IL-2 to pancreatic ß cells of 10-week-old NOD mice prevented the onset of hyperglycemia in NOD mice more in a dose-dependent manner with the lower dose of virus being more effective than a higher dose of AAV-mIP-IL-2 and IL-4. Moreover, the local ß-cell expression of IL-2 increased the number of CD4(+)CD25(+)FoxP3(+) cells in the pancreatic lymph node (PLN) and SPL in both NOD and C57BL/6 mice. Taken together, these results demonstrate that local, low expression of mIL-2 in islets prevents progress of diabetes through the regulation of Tregs.

Immune escape from NY-ESO-1-specific T-cell therapy via loss of heterozygosity in the MHC.

Adoptive immunotherapy of tumors with T cells specific for the cancer-testis antigen NY-ESO-1 has shown great promise in preclinical models and in early stage clinical trials. Tumor persistence or recurrence after NY-ESO-1-specific therapy occurs, however, and the mechanisms of recurrence remain poorly defined. In a murine xenograft model of NY-ESO-1(+) multiple myeloma, we observed tumor recurrence after adoptive transfer of CD8(+) T cells genetically redirected to the prototypic NY-ESO-1157-165 peptide presented by HLA-A*02:01. Analysis of the myeloma cells that had escaped from T-cell control revealed intact expression of NY-ESO-1 and B2M, but selective, complete loss of HLA-A*02:01 expression from the cell surface. Loss of heterozygosity (LOH) in the major histocompatibility complex (MHC) involving the HLA-A locus was identified in the tumor cells, and further analysis revealed selective loss of the allele encoding HLA-A*02:01. Although LOH involving the MHC has not been described in myeloma patients with persistent or recurrent disease after immune therapies such as allogeneic hematopoietic cell transplantation (HCT), it has been described in patients with acute myelogenous leukemia who relapsed after allogeneic HCT. These results suggest that MHC loss should be evaluated in patients with myeloma and other cancers who relapse after adoptive NY-ESO-1-specific T-cell therapy.

Exosomes in the Pathogenesis, Diagnosis and Treatment of Pancreatic Diseases.

Exosomes (EXOs) are small vesicles (30-200 nm) of endocytic origin, which are released by many different cell types into the extracellular space. They may play a key role in facilitating cell-cell communication, under both physiological and pathological conditions. EXOs contain a wide range of RNA molecules and proteins. Their specific molecular signatures make them promising candidates in early diagnosis and prognosis of pancreatic diseases. EXOs could also provide a new method to monitor treatment response in patients suffering from pancreatic cancer and other diseases of the pancreas. Additionally they may help to improve current treatments via personalized medicine approaches using them as therapeutic vehicles themselves.

LIM domain protein-3 (LMP3) cooperates with BMP7 to promote tissue regeneration by ligament progenitor cells.

Gene transfer of key regulators of osteogenesis for mesenchymal stem cells represents a promising strategy to regenerate bone. It has been reported that LMP3, a transcription variant of LIM domain mineralization protein (LMP) lacking LIM domains, can induce osteogenesis in vitro and in vivo. As little is known about the effects of LMP3 gene therapy on periodontal ligament (PDL) cell osteogenic differentiation, this study sought to explore whether gene delivery of LMP3 can promote PDL cell mineralization and bone formation. Our results showed that adenoviral mediated gene transfer of LMP3 (AdLMP3) significantly upregulated ALP (Alkaline Phosphatase), BSP (Bone Sialoprotein) and BMP2 gene expression and increased in vitro matrix mineralization in human PDL. Although AdLMP3 gene delivery to PDL cells did not induce ectopic bone formation in vivo, we found that AdLMP3 augments new bone formation, which co-delivered with AdBMP7 gene transfer. Our study provides the evidence that there is a synergistic effect between LMP3 and BMP-7 in vivo, suggesting that LMP3 delivery may be used to augment BMP-mediated osteogenesis. LMP3 and BMP-7 combinatory gene therapy may also have specific applications for oral and periodontal regenerative medicine.

Exploring the effects of high shear blending on lactose and drug using fluidised bed elutriation.

Powder formulations comprising inhalation grade lactose and a mimic drug (cholesterol) were prepared using a high shear blending process for which the total energy input could be quantified. The formulations were fluidised in a classic fluidised bed system, to determine whether blending-induced changes could be determined through either bulk fluidisation behaviour or the characteristics of elutriated fractions from the powder beds. The evolution of the fluidisation regime within the powder beds (Δ pressure vs. superficial gas velocity) and total mass of elutriated material were not sensitive measures to differentiate between blended and unblended samples. However, blended and unblended material could be distinguished by the size distributions of the elutriated fractions. The study also showed that there were no further changes in the size distribution of the elutriated fractions once a chemically homogenous mixture of lactose and drug had been produced. However, further blending beyond this 'point of homogeneity' continued to change the lactose particle size distribution of the bulk powder; this may have implications for blend end point determination for these types of formulation.

dsAAV8-mediated gene transfer and ß-cell expression of IL-4 and ß-cell growth factors are capable of reversing early-onset diabetes in NOD mice.

Type-I diabetes is a chronic disease mediated by autoimmune destruction of insulin-producing ß-cells. Although progress has been made towards improving diabetes-associated pathologies and the quality of life for those living with diabetes, no therapy has been effective at eliminating disease manifestations or reversing disease progression. Here, we examined whether double-stranded adeno-associated virus serotype 8 (dsAAV8)-mediated gene delivery to endogenous ß-cells of interleukin (IL)-4 in combination with ß-cell growth factors can reverse early-onset diabetes in NOD mice. Our results demonstrate that a single treatment with dsAAV8 vectors expressing IL-4 in combination with glucagon-like peptide-1 or hepatocyte growth factor/NK1 under the regulation of the insulin promoter enhanced ß-cell proliferation and survival in vivo, significantly delaying diabetes progression in NOD mice, and reversing disease in ∼10% of treated NOD mice. These results demonstrate the ability to reverse hyperglycemia in NOD mice with established diabetes by in vivo gene transfer to ß-cells of immunomodulatory factors and ß-cell growth factors.

The antitumor effects of adenoviral-mediated, intratumoral delivery of interleukin 23 require endogenous IL-12.

Interleukin (IL)-23 is a member of the IL-12 family of heterodimeric cytokines, comprised of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. We have demonstrated previously that adenoviral-mediated, intratumoral delivery of IL-23 (Ad.IL-23) was able to induce systemic antitumor immunity. Here we demonstrate that Ad.IL-23 requires endogenous IL-12 for conferring an antitumor effect after adenoviral-mediated, intratumoral delivery. In contrast, Ad.IL-12 does not require IL-23 for its antitumor effects although endogenous IL-23 appears important for induction of systemic antitumor immunity by IL-12. However, despite the requirement for endogenous IL-12, co-delivery of IL-23 and IL-12 does not provide even an additive local or systemic antitumor effect, regardless of the dose. We further demonstrate that although the use of a single-chain IL-23 (scIL-23) results in higher level of expression and a more pronounced IL-23-mediated antitumor effect, there is still no synergy with IL-12. These results demonstrate that although significant antitumor effects are achieved by intratumoral injection of adenovirus expressing either scIL-23 or IL-12 alone and that IL-23 requires endogenous IL-12 for maximum antitumor benefit, the combined use of these cytokines provides no additive or synergistic effect.

Orthopedic gene therapy--lost in translation?

Orthopedic gene therapy has been the topic of considerable research for two decades. The preclinical data are impressive and many orthopedic conditions are well suited to genetic therapies. But there have been few clinical trials and no FDA-approved product exists. This paper examines why this is so. The reasons are multifactorial. Clinical translation is expensive and difficult to fund by traditional academic routes. Because gene therapy is viewed as unsafe and risky, it does not attract major funding from the pharmaceutical industry. Start-up companies are burdened by the complex intellectual property environment and difficulties in dealing with the technology transfer offices of major universities. Successful translation requires close interactions between scientists, clinicians and experts in regulatory and compliance issues. It is difficult to create such a favorable translational environment. Other promising fields of biological therapy have contemplated similar frustrations approximately 20 years after their founding, so there seem to be more general constraints on translation that are difficult to define. Gene therapy has noted some major clinical successes in recent years, and a sense of optimism is returning to the field. We hope that orthopedic applications will benefit collaterally from this upswing and move expeditiously into advanced clinical trials.

Bioplasty for vertebral fractures: preliminary results of a pre-clinical study on goats using autologous modified skin fibroblasts.

The debate is still ongoing about the long term effects of the mininvasive vertebral augmentation techniques and their usefulness in treating more complex cases where a bone inducing effect more than a merely bone substitution would be suitable, such as the vertebral fractures in young patients. We previously developed a clinically relevant gene therapy approach using modified dermal fibroblasts for inducing bone healing and bone formation in different animal models. The aim of this study is to show the feasibility of a minimally invasive percutaneous intrasomatic ex vivo gene therapy approach to treat thoracolumbar vertebral fractures and anterior column bone defects in a goat model.

Reflexive assessment of practical and holistic sanitation development tools using the rural and peri-urban case of Mexico.

Lack of sanitation affects the lives of billions of people worldwide. It is now generally agreed that sustainable solutions to this complex problem require social and cultural factors to be addressed in addition to the habitual economic and technical aspects. Increasingly, sector professionals view the fragmented approaches to sanitation as a limiting factor. This refers to the fragmentation of the knowledge on the subject among often hermetic disciplines and to the distribution of political mandates on sanitation across many institutions, which independently tackle specific aspects of the issue. Holistic approaches have often been suggested as a solution. This paper presents the development of such a holistic approach, designed to assess sanitation development in rural and peri-urban settings. Tested in three Mexican communities, it relies on qualitative research tools to identify critical influences to sanitation development. This article presents generic results about micro and macro-factors affecting sanitation development in Mexican villages, and reflexively examines the research process as well as the strengths and limitations of the approach. The conceptual map developed for each case study successfully highlights the interconnectedness of all factors affecting sanitation development. Despite some weaknesses, these maps constitute a practical assessment tool for interdisciplinary teams deployed in integrated water and sanitation development programs and a valuable didactic tool for training activities.