Cytostatic hypothermia and its impact on glioblastoma and survival.

Patients with glioblastoma (GBM) have limited options and require novel approaches to treatment. Here, we studied and deployed nonfreezing "cytostatic" hypothermia to stunt GBM growth. This growth-halting method contrasts with ablative, cryogenic hypothermia that kills both neoplastic and infiltrated healthy tissue. We investigated degrees of hypothermia in vitro and identified a cytostatic window of 20° to 25°C. For some lines, 18 hours/day of cytostatic hypothermia was sufficient to halt division in vitro. Next, we fabricated an experimental tool to test local cytostatic hypothermia in two rodent GBM models. Hypothermia more than doubled median survival, and all rats that successfully received cytostatic hypothermia survived their study period. Unlike targeted therapeutics that are successful in preclinical models but fail in clinical trials, cytostatic hypothermia leverages fundamental physics that influences biology broadly. It is a previously unexplored approach that could provide an additional option to patients with GBM by halting tumor growth.

Physical therapy assessment and whole-body magnetic resonance imaging findings in children with glycogen storage disease type IIIa: A clinical study and review of the literature.

INTRODUCTION: Early recognized manifestations of GSD III include hypoglycemia, hepatomegaly, and elevated liver enzymes. Motor symptoms such as fatigue, muscle weakness, functional impairments, and muscle wasting are typically reported in the 3rd to 4th decade of life. OBJECTIVE: In this study, we investigated the early musculoskeletal findings in children with GSD IIIa, compared to a cohort of adults with GSD IIIa. METHODS: We utilized a comprehensive number of physical therapy outcome measures to cross-sectionally assess strength and gross motor function including the modified Medical Research Council (mMRC) scale, grip and lateral/key pinch, Gross Motor Function Measure (GMFM), Gait, Stairs, Gowers, Chair (GSGC) test, 6 Minute Walk Test (6MWT), and Bruininks-Oseretsky Test of Motor Proficiency Ed. 2 (BOT-2). We also assessed laboratory biomarkers (AST, ALT, CK and urine Glc4) and conducted whole-body magnetic resonance imaging (WBMRI) to evaluate for proton density fat fraction (PDFF) in children with GSD IIIa. Nerve Conduction Studies and Electromyography results were analyzed where available and a thorough literature review was conducted. RESULTS: There were a total of 22 individuals with GSD IIIa evaluated in our study, 17 pediatric patients and 5 adult patients. These pediatric patients demonstrated weakness on manual muscle testing, decreased grip and lateral/key pinch strength, and decreased functional ability compared to non-disease peers on the GMFM, 6MWT, BOT-2, and GSGC. Additionally, all laboratory biomarkers analyzed and PDFF obtained from WBMRI were increased in comparison to non-diseased peers. In comparison to the pediatric cohort, adults demonstrated worse overall performance on functional assessments demonstrating the expected progression of disease phenotype with age. CONCLUSION: These results demonstrate the presence of early musculoskeletal involvement in children with GSD IIIa, most evident on physical therapy assessments, in addition to the more commonly reported hepatic symptoms. Muscular weakness in both children and adults was most significant in proximal and trunk musculature, and intrinsic musculature of the hands. These findings indicate the importance of early assessment of patients with GSD IIIa for detection of muscular weakness and development of treatment approaches that target both the liver and muscle.

Immuno-suppressive hydrogels enhance allogeneic MSC survival after transplantation in the injured brain.

Traumatic brain injury (TBI) triggers multiple biochemical and cellular processes that exacerbate brain tissue damage through a secondary injury. Therapies that prevent or limit the evolution of secondary injury could significantly reduce the neurological deficits associated with TBI. Mesenchymal stem cell (MSC) transplantation after TBI can ameliorate neurological deficits by modulating inflammation and enhancing the expression of neurotrophic factors. However, transplanted MSCs can be actively rejected by host immune responses, such as those mediated by cytotoxic CD8+ T cells, thereby limiting their therapeutic efficacy. Here, we designed an agarose hydrogel that releases Fas ligand (FasL), a protein that can induce apoptosis of cytotoxic CD8+ T cells. We studied the immunosuppressive effect of this hydrogel near the allogeneic MSC transplantation site and its impact on the survival of transplanted MSCs in the injured brain. Agarose-FasL hydrogels locally reduced the host cytotoxic CD8+ T cell population and enhanced the survival of allogeneic MSCs transplanted near the injury site. Furthermore, the expression of crucial neurotrophic factors was elevated in the injury penumbra, suggesting an enhanced therapeutic effect of MSCs. These results suggest that the development of immunosuppressive hydrogels for stem cell delivery can enhance the benefits of stem cell therapy for TBI.

Evaluation of M2-like macrophage enrichment after diffuse traumatic brain injury through transient interleukin-4 expression from engineered mesenchymal stromal cells.

BACKGROUND: Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities for the millions of those inflicted annually. In TBI, cellular mediators of inflammation, including macrophages and microglia, possess a range of phenotypes relevant for an immunomodulatory therapeutic approach. It is thought that early phenotypic modulation of these cells will have a cascading healing effect. In fact, an anti-inflammatory, "M2-like" macrophage phenotype after TBI has been associated with neurogenesis, axonal regeneration, and improved white matter integrity (WMI). There already exist clinical trials seeking an M2-like bias through mesenchymal stem/stromal cells (MSCs). However, MSCs do not endogenously synthesize key signals that induce robust M2-like phenotypes such as interleukin-4 (IL-4). METHODS: To enrich M2-like macrophages in a clinically relevant manner, we augmented MSCs with synthetic IL-4 mRNA to transiently express IL-4. These IL-4 expressing MSCs (IL-4 MSCs) were characterized for expression and functionality and then delivered in a modified mouse TBI model of closed head injury. Groups were assessed for functional deficits and MR imaging. Brain tissue was analyzed through flow cytometry, multi-plex ELISA, qPCR, histology, and RNA sequencing. RESULTS: We observed that IL-4 MSCs indeed induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression after TBI. However, here we demonstrate that acute enrichment of M2-like macrophages did not translate to improved functional or histological outcomes, or improvements in WMI on MR imaging. To further understand whether dysfunctional pathways underlie the lack of therapeutic effect, we report transcriptomic analysis of injured and treated brains. Through this, we discovered that inflammation persists despite acute enrichment of M2-like macrophages in the brain. CONCLUSION: The results demonstrate that MSCs can be engineered to induce a stronger M2-like macrophage response in vivo. However, they also suggest that acute enrichment of only M2-like macrophages after diffuse TBI cannot orchestrate neurogenesis, axonal regeneration, or improve WMI. Here, we also discuss our modified TBI model and methods to assess severity, behavioral studies, and propose that IL-4 expressing MSCs may also have relevance in other cavitary diseases or in improving biomaterial integration into tissues.

Use of Preoperative Apparent Diffusion Coefficients to Predict Brain Tumor Grade.

Introduction The apparent diffusion coefficient (ADC) sequence is based on the diffusion properties of water molecules within tissues and correlates with tissue cellularity. ADC may have a role in predicting tumor grade for gliomas, and may in turn assist in identifying tumor biopsy sites. The purpose of this investigation was to assess the competence of preoperative ADC values in predicting tumor grades. Methods This was a retrospective investigation. We calculated the ADC values in the areas of greatest restriction in solid tumor components, and we recorded the pattern of contrast enhancement. Pathology reports masked to the imaging results were reviewed independently. We calculated the differences in the mean values of different tumor grades and high-grade and low-grade gliomas. A receiver operator curve (ROC) analysis assessed the predictive potential of ADC values for low-grade gliomas. Results Forty-eight cases of glioma were included in our study. We noted a statistically significant difference in the lowest mean ADC values for the tumor regions of Grade IV lesions (333.83 ± 295.47) compared with Grade I lesions (653.20 ± 145.07). On ROC analysis, we noted an area under the curve (AUC) of 0.80 for the lowest ADC value in the whole tumor region, which was a predictor of low-grade glioma with 95 % confidence interval (CI) of 0.675-0.926. The sensitivity of the lowest ADC value was 84.5% for high-grade lesions. Conclusion Given our findings that the means of the lowest ADC value are significantly different between low and high-grade gliomas with an AUC of 0.80 for ADC as a predictor of low-grade lesions and a sensitivity of 84.5% for high-grade lesions, ADC values contain some predictive properties of tumor grading. ADC values may be a valuable parameter in the assessment and treatment of tumors.

The importance of Evolutionary Medicine in developing countries: A case for Pakistan's medical schools.

Evolutionary Medicine (EM) is a fundamental science exploring why our bodies are plagued with disease and hindered by limitations. EM views the body as an assortment of benefits, mistakes, and compromises molded over millennia. It highlights the role of evolution in numerous diseases encountered in community and family medicine clinics of developing countries. It enables us to ask informed questions and develop novel responses to global health problems. An understanding of the field is thus crucial for budding doctors, but its study is currently limited to a handful of medical schools in high-income countries. For the developing world, Pakistan's medical schools may be excellent starting posts as the country is beset with communicable and non-communicable diseases that are shaped by evolution. Remarkably, Pakistani medical students are open to studying and incorporating EM into their training. Understanding the principles of EM could empower them to tackle growing health problems in the country. Additionally, some difficulties that western medical schools face in integrating EM into their curriculum may not be a hindrance in Pakistan. We propose solutions for the remaining challenges, including obstinate religious sentiments. Herein, we make the case that incorporating EM is particularly important in developing countries such as Pakistan and that it is achievable in its medical student body.

Enrichment of endogenous fractalkine and anti-inflammatory cells via aptamer-functionalized hydrogels.

Early recruitment of non-classical monocytes and their macrophage derivatives is associated with augmented tissue repair and improved integration of biomaterial constructs. A promising therapeutic approach to recruit these subpopulations is by elevating local concentrations of chemoattractants such as fractalkine (FKN, CX3CL1). However, delivering recombinant or purified proteins is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we report an approach to enrich endogenous FKN, obviating the need for delivery of exogenous proteins. In this study, modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels ("aptagels") that localize, dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate significant local increases in Ly6CloCX3CR1hi non-classical monocytes and CD206+ M2-like macrophages. The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins or cells and FKN-aptamer functionalized biomaterials may be a promising approach to recruit anti-inflammatory subpopulations to sites of injury. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic.

Predicting outcomes of decompressive craniectomy: use of Rotterdam Computed Tomography Classification and Marshall Classification.

BACKGROUND: Data on the evaluation of the Rotterdam Computed Tomography Classification (RCTS) as a predictor of outcomes in patients undergoing decompressive craniectomy (DC) for trauma is limited and lacks clarity. OBJECTIVE: To explore the role of RCTS in predicting unfavourable outcomes, including mortality in patients undergoing DC for head trauma. METHODS: This was an observational cohort study conducted from 1 January 2009 to 31 March 2013. CT scans of adults with head trauma prior to emergency DC were scored according to RCTS. A receiver operating characteristic curve analysis was performed to identify the optimal cut-off RCTS for predicting unfavourable outcomes [Glasgow outcome scale (GOS) = 1-3]. Binary logistic regression analysis was performed to evaluate the relationship between RCTS and unfavourable outcomes including mortality. RESULTS: One hundred ninety-seven patients (mean age: 31.4 ± 18.7 years) were included in the study. Mean Glasgow coma score at presentation was 8.1 ± 3.6. RCTS was negatively correlated with GOS (r = -0.370; p < 0.001). The area under the curve was 0.687 (95% CI: 0.595-0.779; p < 0.001) and 0.666 (95% CI: 0.589-0.742; p < 0.001) for mortality and unfavourable outcomes, respectively. RCTS independently predicted both mortality (adjusted odds ratio for RCTS >3 compared with RCTS ≤3: 2.792, 95% CI: 1.235-6.311) and other unfavourable outcomes (adjusted odds ratio for RCTS >3 compared with RCTS ≤3: 2.063, 95% CI: 1.056-4.031). CONCLUSION: RCTS is an independent predictor of unfavourable outcomes and mortality among patients undergoing emergency DC.

Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Niches for Neural Stem Cells.

Neural stem cells (NSCs) possess great potential for neural tissue repair after traumatic injuries to the central nervous system (CNS). However, poor survival and self-renewal of NSCs after injury severely limits its therapeutic potential. Sulfated chondroitin sulfate glycosaminoglycans (CS-GAGs) linked to CS proteoglycans (CSPGs) in the brain extracellular matrix (ECM) have the ability to bind and potentiate trophic factor efficacy, and promote NSC self-renewal in vivo. In this study, we investigated the potential of CS-GAG hydrogels composed of monosulfated CS-4 (CS-A), CS-6 (CS-C), and disulfated CS-4,6 (CS-E) CS-GAGs as NSC carriers, and their ability to create endogenous niches by enriching specific trophic factors to support NSC self-renewal. We demonstrate that CS-GAG hydrogel scaffolds showed minimal swelling and degradation over a period of 15 days in vitro, absorbing only 6.5 ± 0.019% of their initial weight, and showing no significant loss of mass during this period. Trophic factors FGF-2, BDNF, and IL10 bound with high affinity to CS-GAGs, and were significantly (p < 0.05) enriched in CS-GAG hydrogels when compared to unsulfated hyaluronic acid (HA) hydrogels. Dissociated rat subventricular zone (SVZ) NSCs when encapsulated in CS-GAG hydrogels demonstrated ∼88.5 ± 6.1% cell viability in vitro. Finally, rat neurospheres in CS-GAG hydrogels conditioned with the mitogen FGF-2 demonstrated significantly (p < 0.05) higher self-renewal when compared to neurospheres cultured in unconditioned hydrogels. Taken together, these findings demonstrate the ability of CS-GAG based hydrogels to regulate NSC self-renewal, and facilitate growth factor enrichment locally.

Congenital malignant melanoma of the scalp in a 25-day-old neonate.

We present a case of congenital malignant melanoma of the scalp in a neonate. The child was born through caesarean section with a swelling, the size of a tennis ball, on the posterior scalp. At presentation to the clinic at 25 days after birth, the swelling had significantly increased in size and ulcerated. An excision was carried out but, because of extensive haemorrhage and haemodynamic instability, the procedure was limited to subtotal resection. Later on, completion of the excision and flap coverage of the wound were performed. After an initial stable course of a few months, the child came back with local recurrence. A re-excision was planned but the child developed pneumonia resulting in sepsis leading to the demise of the child. The report adds to the literature by describing a rare entity and challenges of managing large vascular scalp lesions with complete excision and defect coverage.

Sampling bacterial biodiversity from a highly contaminated stream flowing through a densely populated urban area in Karachi.

BACKGROUND: Few studies have attempted to understand the complexity of microbial populations in Pakistan where infectious diseases are prevalent. This study was undertaken to assess bacterial biodiversity in Nehr-e-Khayyam a heavily polluted stream connected to the Arabian Gulf, which runs through a densely populated urban area in Karachi, Pakistan. METHODS: Employing a universal pair of oligonucleotides capable of amplifying species-specific segments of 16S rRNA gene from all Eubacteria, we generated a library of PCR products using total DNA purified from the collected sample, cloned the amplifers into pGEM-T-Easy and sequenced each recombinant clone. The obtained DNA sequences were subjected to bio-informatic analyses. RESULTS: A total of 71 recombinant clones were obtained from the amplified 16S rDNA products and sequenced. Bioinformatics analyses revealed that 54 (out of 71) were unique sequences from which 42 shared > 97% and 12 shared < 97% homology to their database counterparts. One sequence originated from the plastid DNA of eukaryote Pyramimonas disomata. From the remaining 53 sequences, 45 were Proteo-bacteria and 8 Fermicute in origin. Among 71 sequences, Alpha-, Beta- and Gamma-proteobacteria species constituted -86% of Proteo-bacteria identified in the sample while only -13% were Fermicutes. CONCLUSIONS: The microbial niche in Nehr-e-Khayyam is occupied predominantly by heterotrophic Proteo-bacterial and Firmicute strains, some of which are known human pathogens.