Jacob Riehm

Glutamine administration reduces Gram-negative bacteremia in severely burned patients: a prospective, randomized, double-blind trial versus isonitrogenous control.

Objective To determine the effect of intravenous glutamine supplementation vs. an isonitrogenous control on infectious morbidity in severely burned patients. Previous clinical studies in seriously ill patients suggest a beneficial effect of glutamine on infectious morbidity, but no trials have examined possible clinical benefits in severely burned patients. Design Prospective, double-blind, randomized trial. Setting Burn intensive care unit of a university hospital. Patients Twenty-six severe burn patients with total burn surface area of 25% to 90% and presence of full-thickness burns. Patients were evaluated for occurrence of bacteremia and antibiotic use during the first 30 days of their burn unit admission. Nutritional status and overall inflammation were also measured. Intervention Either intravenous glutamine or an isonitrogenous control amino acid solution was administered as a continuous infusion during burn intensive care unit stay. Measurements and Main Results The incidence of Gram-negative bacteremia was significantly reduced in the glutamine-supplemented group (8%) vs. control (43%;p < .04). No difference was seen in the incidence of Gram-positive bacteremia or fungemia. Average number of positive blood cultures, antibiotic usage, and mortality rates also were reduced but did not reach statistical significance. Significant improvements in serum transferrin and prealbumin were observed in glutamine-supplemented patients at 14 days after burn injury (p < .01 and .04, respectively). C-reactive protein was also significantly reduced at 14 days after burn injury in the glutamine group (p < .01). Conclusions Significantly fewer bacteremic episodes with Gram-negative organisms occurred in the glutamine-supplemented patients. Glutamine supplementation improved measures of nutrition and decreased measures of overall inflammation. In addition, a trend toward lower mortality rate, decreased overall bacteremia incidence, and antibiotic usage in the glutamine group was observed. Glutamine’s beneficial effects may be a result of improved gut integrity or immune function, but the precise mechanism of glutamine’s protection is unknown.

Glutamine attenuates tumor necrosis factor-α release and enhances heat shock protein 72 in human peripheral blood mononuclear cells

OBJECTIVE Overexpression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) can contribute to multiple organ dysfunction syndrome and septic shock in critically ill patients. We previously found that glutamine (GLN) can attenuate cytokine expression, induce heat shock protein 72 (HSP 72), and protect against endotoxin-induced mortality and organ injury in an in vivo rat model. However, data on the effect of GLN on direct attenuation of cytokine release and HSP 72 expression in human peripheral blood polymorphonuclear cells (PBMCs) is lacking. METHODS In this study, we assessed the effect of GLN on TNF-alpha and HSP 72 expression in human PBMCs. After treating with various doses of GLN, human PBMCs were stimulated with lipopolysaccharide (LPS). TNF-alpha release was analyzed via enzyme-linked immunosorbent assay and HSP 72 via western blot. RESULTS GLN at doses greater than 4 mM decreased TNF-alpha release at 4 and 24 h after LPS stimulation. Sublethal heating of PBMCs before LPS also markedly decreased TNF-alpha after LPS. Doses of GLN greater than 2 to 4 mM led to an increase in HSP 72 expression after LPS. CONCLUSION These results indicate that GLN, which may improve outcomes in critically ill patients, can directly attenuate pro-inflammatory cytokine release in PBMCs. This effect may be related to enhanced HSP 72 expression.

Single dose of glutamine enhances myocardial tissue metabolism, glutathione content, and improves myocardial function after ischemia-reperfusion injury

BACKGROUND Myocardial ischemia and reperfusion (I/R) injury causes significant morbidity and mortality. Protection against I/R injury may occur via preservation of tissue metabolism and ATP content, preservation of reduced glutathione, and stimulation of heat shock protein (HSP) synthesis. Supplementation with glutamine (GLN) has been reported to have beneficial effects on all of these protective pathways. Thus, we hypothesized that GLN pretreatment given to the rat in vivo would protect the myocardium against I/R-induced dysfunction. METHODS GLN (0.52 g/kg, intraperitoneally, given as alanine-glutamine dipeptide), alanine alone (0.23 g/kg), or a Ringers lactate solution (control) was administered to Sprague-Dawley rats 18 hours before heart excision, perfusion, exposure to global ischemia (15 minutes) and reperfusion (1 hour). Tissue metabolites were analyzed via magnetic resonance spectroscopy. RESULTS In control and alanine-treated animals, I/R injury resulted in cardiac dysfunction, indicated by a decrease in cardiac output. Administration of GLN 18 hours before I/R injury preserved cardiac output after reperfusion. Metabolic analysis of the myocardial tissue revealed that [/R injury led to significant diminution of myocardial tissue glutamate, ATP content, accumulation of myocardial lactate, and a reduction in reduced glutathione content in control animals. GLN significantly reduced the deleterious changes in myocardial metabolism and improved reduced glutathione content. No changes in pre- or post-I/R injury HSP expression were observed after GLN administration. CONCLUSIONS These observations demonstrate that remote in vivo administration of GLN before cardiac I/R injury can improve post-I/R cardiac function. This effect may be mediated via improved myocardial metabolism and enhanced reduced glutathione content.

Met gene amplification and protein hyperactivation is a mechanism of resistance to both first and third generation EGFR inhibitors in lung cancer treatment

The 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs; e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants, represent very promising therapeutic options for patients with non-small cell lung cancer (NSCLC) that has become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. The current study has demonstrated that Met amplification and hyperactivation is a resistance mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed amplified Met gene and hyperactivated Met, and were cross-resistant to AZD9291 or erlotinib. Met inhibition overcame the resistance of these cell lines to AZD9291 both in vitro and in vivo, including enhancement of apoptosis or G1 cell cycle arrest. Hence, we suggest that Met inhibition is also an effective strategy to overcome resistance of certain EGFR-mutated NSCLCs with Met amplification to AZD9291, warranting the further clinical validation of our findings.

Unique fractal evaluation and therapeutic implications of mitochondrial morphology in malignant mesothelioma.

Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.

PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent.

Whole-animal mounts of Caenorhabditis elegans for 3D imaging using atomic force microscopy

UNLABELLED The 3D surface of Caenorhabditis elegans was imaged at nanometer resolution using atomic force microscopy (AFM). Oscillation of a medium stiffness silicon AFM cantilever at the upper second amplitude peak, typically 6 times above the fundamental frequency, vastly improved image quality on the moist, sticky, and soft worms. Whole-animal mounts of normal and double-headed mutants of the nematode worm were prepared and scanned. Well-preserved anatomical features including annuli, furrows, alae, and rows of never before seen nanometer-sized pores dotting the molted worms outermost surface coat were resolved. Well-preserved anatomical features including annuli, furrows, alae, and rows of nanometer-sized pores or struts dotting the molted worms outermost surface were resolved. This AFM method represents a simple and rapid new approach for nanometer-resolved 3D imaging and analysis of whole-animal specimens of C. elegans. FROM THE CLINICAL EDITOR In this interesting article the authors describe a new AFM sampling method to allow better images on whole-animal mounts such as C. elegans. This method would generate more information and in the future may be useful for differentiating even individual animals with different genetic backgrounds.

Extracellular Vesicles from Caveolin-Enriched Microdomains Regulate Hyaluronan-Mediated Sustained Vascular Integrity.

Defects in vascular integrity are an initiating factor in several disease processes. We have previously reported that high molecular weight hyaluronan (HMW-HA), a major glycosaminoglycan in the body, promotes rapid signal transduction in human pulmonary microvascular endothelial cells (HPMVEC) leading to barrier enhancement. In contrast, low molecular weight hyaluronan (LMW-HA), produced in disease states by hyaluronidases and reactive oxygen species (ROS), induces HPMVEC barrier disruption. However, the mechanism(s) of sustained barrier regulation by HA are poorly defined. Our results indicate that long-term (6–24 hours) exposure of HMW-HA induced release of a novel type of extracellular vesicle from HLMVEC called enlargeosomes (characterized by AHNAK expression) while LMW-HA long-term exposure promoted release of exosomes (characterized by CD9, CD63, and CD81 expression). These effects were blocked by inhibiting caveolin-enriched microdomain (CEM) formation. Further, inhibiting enlargeosome release by annexin II siRNA attenuated the sustained barrier enhancing effects of HMW-HA. Finally, exposure of isolated enlargeosomes to HPMVEC monolayers generated barrier enhancement while exosomes led to barrier disruption. Taken together, these results suggest that differential release of extracellular vesicles from CEM modulate the sustained HPMVEC barrier regulation by HMW-HA and LMW-HA. HMW-HA-induced specialized enlargeosomes can be a potential therapeutic strategy for diseases involving impaired vascular integrity.

Focal adhesion kinase a potential therapeutic target for pancreatic cancer and malignant pleural mesothelioma

ABSTRACT The non-receptor cytoplasmic tyrosine kinase, Focal Adhesion Kinase (FAK) is known to play a key role in a variety of normal and cancer cellular functions such as survival, proliferation, migration and invasion. It is highly active and overexpressed in various cancers including Pancreatic Ductal Adenocarcinoma (PDAC) and Malignant Pleural Mesothelioma (MPM). Here, initially, we demonstrate that FAK is overexpressed in both PDAC and MPM cell lines. Then we analyze effects of two small molecule inhibitors PF-573228, and PF-431396, which are dual specificity inhibitors of FAK and proline rich tyrosine kinase 2 (PYK2), as well as VS-6063, another small molecule inhibitor that specifically inhibits FAK but not PYK2 for cell growth, motility and invasion of PDAC and MPM cell lines. Treatment with PF-573228, PF-431396 and VS-6063 cells resulted in a dose-dependent inhibition of growth and anchorage-independent colony formation in both cancer cell lines. Furthermore, these compounds suppressed the phosphorylation of FAK at its active site, Y397, and functionally induced significant apoptosis and cell cycle arrest in both cell lines. Using the ECIS (Electric cell-substrate impedance sensing) system, we found that treatment of both PF compounds suppressed adherence and migration of PDAC cells on fibronectin. Interestingly, 3D-tumor organoids derived from autochthonous KC (Kras;PdxCre) mice treated with PF-573228 revealed a significant decrease in tumor organoid size and increase in organoid cell death. Taken together, our results show that FAK is an important target for mesothelioma and pancreatic cancer therapy that merit further translational studies.

Abstract 2105: c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors

c-Met amplification and acquisition of a second T790M mutation are key mechanisms accounting for majority of resistant cases to first generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs; i.e., erlotinib). The third generation EGFR-TKIs (e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants while sparing wild-type EGFR, represent very promising therapeutic options for NSCLC patients who have become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. We show that c-Met amplification and hyperactivation is an universal mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed elevated levels of c-Met (due to gene amplification) and p-c-Met and were cross-resistant to AZD9291 or erlotinib. Both chemical and genetic inhibition of c-Met overcame the resistance of these cell lines to AZD9291 including enhancement of apoptosis or G1 cell cycle arrest. Consistently the combination of AZD9291 and c-Met inhibition effectively inhibited the growth of both erlotinib- and AZD9291-resistant HCC827 xenografts in nude mice. Hence, we suggest that inhibition of c-Met is also an effective strategy to overcome resistance of EGFR-mutated NSCLCs with c-Met amplification or hyperactivation to AZD9291, providing the rationale for clinical development of this novel combination strategy. (SSR, TKO and SYS are Georgia Research Alliance Distinguished Cancer Scientists) Citation Format: Puyu Shi, You-Take Oh, Guojing Zhang, Weilong Yao, Ping Yue, Rajani Kanteti, Jacob Riehm, Ravi Salgia, Taofeek Owonikoko, Suresh S. Ramalingam, Mingwei Chen, Shi-Yong Sun. c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2105.