The human application of gene therapy to re-program T-cell specificity using chimeric antigen receptors

The adoptive transfer of T cells is a promising approach to treat cancers. Primary human T cells can be modified using viral and non-viral vectors to promote the specific targeting of cancer cells via the introduction of exogenous T-cell receptors(TCRs) or chimeric antigen receptors(CARs). This gene transfer displays the potential to increase the specificity and potency of the anticancer response while decreasing the systemic adverse effects that arise from conventional treatments that target both cancerous and healthy cells. This review highlights the generation of clinical-grade T cells expressing CARs for immunotherapy, the use of these cells to target B-cell malignancies and, particularly, the first clinical trials deploying the Sleeping Beauty gene transfer system, which engineers T cells to target CD19+ leukemia and non-Hodgkin's lymphoma.

Nitric oxide and geriatrics:Implications in diagnostics and treatment of the elderly

The nation’s aging population is growing rapidly.By 2030,the number of adults age 65 and older will nearly double to 70 million.Americans are living longer and older adults can now live for many years with multiple chronic illnesses but with a substantial cost to health care.Twenty percent of the Medicare population has at least five chronic conditions i.e.,hypertension,diabetes,arthritis,etc.Studies in experimental models and even humans reveal that constitutive production of nitric oxide(NO)is reduced with aging and this circumstance may be relevant to a number of diseases that plague the aging population.NO is a multifunctional signaling molecule,intricately involved with maintaining a host of physiological processes including,but not limited to,host defense,neuronal communication and the regulation of vascular tone.NO is one of the most important signaling molecules in our body,and loss of NO function is one of the earliest indicators or markers of disease.Clinical studies provide evidence that insufficient NO production is associated with all major cardiovascular risk factors,such as hyperlipidemia,diabetes,hypertension,smoking and severity of atherosclerosis,and also has a profound predictive value for disease progression including cardiovascular and Alzheimers disease.Thirty plus years after its discovery and over 13 years since a Nobel Prize was awarded for its discovery,there have been no hallmark therapeutic breakthroughs or even NO based diagnostics.We will review the current state of the science surrounding NO in the etiology of a number of different diseases in the geriatric patient.From these observations,it can be concluded that enzymatic production of NO declines steadily with increasing age in healthy human subjects.Implementing strategies to diagnose and treat NO insufficiency may provide enormous benefit to the geriatric patient.

An Investigation of the Required MR Bone Attenuation Correction for Quantitative Whole-Body PET/MR Imaging Using Clinical NaF PET/CT Studies

Tissue-classification-based attenuation correction strategies have been previously proposed to correct for bone attenuation in PET/MR imaging and simulated using computed tomography. However, the complication of voxel averaging uniquely associated with bone has not been considered explicitly in the past. This study investigated the effect of voxel averaging between bone and soft tissue in attenuation images and determined how accurately bone must be detected in MR images in order to perform acceptable attenuation correction of PET data by using CT-simulated attenuation correction. We found out that treating bone as soft tissue caused a mean quantification difference of -9.9% ± 5.5% in all 119 bone lesions. There were no significant differences between lesions in the pelvis and the vertebrae. The nominal difference in lesions in the ribs was significantly lower, likely due to the spatial misregistration between the emission and attenuation images. Interestingly, a non-monotonic relationship between the bone imaging ability and the absolute PET quantification accuracy was observed, with the minimal quantification difference achieved at a BVF around 40% for skull lesions (2.6% ± 2.1%), and 30% for non-skull lesions (1.4% ± 1.1%) and all lesions (1.5% ± 1.3%). This study established that a bone classification sensitivity of approximately 30% BVF is required in order for MR-based attenuation correction methods to achieve optimal quantification in whole-body PET/MR studies. For this purpose, higher bone imaging ability of MR may not be necessary.

Detection of RASSF1A promoter hypermethylation in serum from gastric and colorectal adenocarcinoma patients

AIM:To evaluate the diagnostic role of serum RASSF1A promoter hypermethylation in gastric and colorectal ad- enocarcinoma. METHODS:Methylation-specific polymerase chain reac- tion (MSPCR) was used to examine the promoter meth- ylation status of the serum RASSF1A gene in 47 gastric adenocarcinoma patients, 45 colorectal adenocarcinoma patients, 60 patients with benign gastrointestinal dis- ease (30 with benign gastric disease and 30 with benign colorectal disease), and 30 healthy donor controls. Apaired study of RASSF1A promoter methylation status in primary tumor, adjacent normal tissue, and postopera- tive serum were conducted in 25 gastric and colorectal adenocarcinoma patients who later were underwent sur- gical therapy. RESULTS:The frequencies of detection of serum RASSF1A promoter hypermethylation in gastric (34.0%) and colorectal (28.9%) adenocarcinoma patients were significantly higher than those in patients with benign gastric (3.3%) or colorectal (6.7%) disease or in healthy donors (0%) (P < 0.01). The methylation status of RASSF1A promoter in serum samples was consistent with that in paired primary tumors, and the MSPCR results for RASSF1A promoter methylation status in paired preoperative samples were consistent with those in postoperative serum samples. The serum RASSF1A promoter hypermethylation did not correlate with patient sex, age, tumor differentiation grade, surgical therapy, or serum carcinoembryonic antigen level. Although the serum RASSF1A promoter hypermethylation frequency tended to be higher in patients with distant metastases, there was no correlation between methylation status and metastasis. CONCLUSION:Aberrant CpG island methylation within the promoter region of RASSF1A is a promising biomark- er for gastric and colorectal cancer.

Nonmetabolic functions of pyruvate kinase isoform M2 in controlling cell cycle progression and tumorigenesis

Pyruvate kinase catalyzes the rate-limiting final step of glycolysis, generating adenosine triphosphate (ATP) and pyruvate. The M2 tumor-specific isoform of pyruvate kinase (PKM2) promotes glucose uptake and lactate production in the presence of oxygen, known as aerobic glycolysis or the Warburg effect. As recently reported in Nature, PKM2, besides its metabolic function, has a nonmetabolic function in the direct control of cell cycle progression by activating β-catenin and inducing expression of the β-catenin downstream gene CCND1 (encoding for cyclin D1). This nonmetabolic function of PKM2 is essential for epidermal growth factor receptor (EGFR) activation-induced tumorigenesis.

Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-,and threonine-rich sequence(PEST)

Protein tyrosine phosphatase (PTP)-proline-,glutamate-,serine-,and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration.PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications,including phosphorylation,oxidation,and caspase-dependent cleavage.PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins.Dephos-phorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process.

Translational genomics in cancer research: converting profiles into personalized cancer medicine

Cancer genomics is a rapidly growing discipline in which the genetic molecular basis of malignancy is studied at the scale of whole genomes.While the discipline has been successful with respect to identifying specific oncogenes and tumor suppressors involved in oncogenesis,it is also challenging our approach to managing patients suffering from this deadly disease.Specifically cancer genomics is driving clinical oncology to take a more molecular approach to diagnosis,prognostication,and treatment selection.We review here recent work undertaken in cancer genomics with an emphasis on translation of genomic findings.Finally,we discuss scientific challenges and research opportunities emerging from findings derived through analysis of tumors with high-depth sequencing.

Metabolic alterations in cancer cells and therapeutic implications

Cancer metabolism has emerged as an important area of research in recent years.Elucidation of the metabolic differences between cancer and normal cells and the underlying mechanisms will not only advance our understanding of fundamental cancer cell biology but also provide an important basis for the development of new therapeutic strategies and novel compounds to selectively eliminate cancer cells by targeting their unique metabolism.This article reviews several important metabolic alterations in cancer cells,with an emphasis on increased aerobic glycolysis (the Warburg effect) and glutamine addiction,and discusses the mechanisms that may contribute to such metabolic changes.In addition,metabolic alterations in cancer stem cells,mitochondrial metabolism and its influence on drug sensitivity,and potential therapeutic strategies and agents that target cancer metabolism are also discussed.

Patient-Specific and Generic Immobilization Devices for Prostate Radiotherapy

The purpose of our study was to compare interfractional bony setup variations in pelvic anatomy with two immobilization devices, the patient-specific Vac-Lok and the generic Dual Leg Positioner system (both Civco Medical Solutions, Kalona, IA), for bilateral proton radiotherapy of the prostate. Two groups of 10 patients were studied. Computed tomography (CT) was performed three times a week, yielding 233 CT image sets for the vacuum system group and 252 for the other group. The translational shifts of the pelvic bone and prostate and rotation of the upper femurs of the femoral heads with respect to the simulation CT images were analyzed. Along the anterior-posterior and lateral axes, mean and systematic translational variations of the pelvic bone and prostate, relative to skin fiducials, were significantly lower in the Vac-Lok group (all p 0.01) than in the Dual Leg Positioner group. Abduction of the upper femur, the dominant rotation, had random rotational variations of 1.9° and 2.0° and systematic rotations of 3.1&#176 and 2.9&#176 for the vacuum and generic system groups, respectively. Femoral abduction was highly correlated with anterior prostate displacement for both femurs in both groups (p tion introduced during simulation CT, particularly with the generic immobilization system. High degrees of femoral rotation may introduce prostate translation and distal misalignment of lateral proton beams with the prostate.

Amino acid transporter SLC7A11/ xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer

Cancer cells often upregulate nutrient transporters to fulfill their increased biosynthetic and bioenergetic needs,and to maintain redox homeostasis.One nutrient transporter frequently overexpressed in human cancers is the cystine/glutamate antiporter solute carrier family 7 member 11(SLC7A11;also known as xCT).SLC7A11 promotes cystine uptake and glutathione biosynthesis,resulting in protection from oxidative stress and ferroptotic cell death.Recent studies have unexpectedly revealed that SLC7A11 also plays critical roles in glutamine metabolism and regulates the glucose and glutamine dependency of cancer cells.This review discusses the roles of SLC7A11 in regulating the anti-oxidant response and nutrient dependency of cancer cells,explores our current understanding of SLC7A11 regulation in cancer metabolism,and highlights key open questions for future studies in this emerging research area.A deeper understanding of SLC7A11 in cancer metabolism may identify new therapeutic opportunities to target this important amino acid transporter for cancer treatment.

Crosstalk of the Wnt/b-catenin pathway with other pathways in cancer cells

Many cancers have similar aberrations in various signaling cascades with crucial roles in cellular proliferation,differentiation,and morphogenesis.Dysregulation of signal cascades that play integral roles during early cellular development is well known to be a central feature of many malignancies.One such signaling cascade is the Wnt/b-catenin pathway,which has a profound effect on stem cell proliferation,migration,and differentiation.This pathway is dysregulated in numerous cell types,underscoring its global oncogenetic potential.This review highlights regulators and downstream effectors of this receptor cascade and addresses the increasingly apparent crosstalk of Wnt with other tumorigenic signaling pathways.As understanding of the genetic and epigenetic changes unique to these malignancies increases,identifying the regulatory mechanisms unique to the Wnt/b-catenin pathway and similarly aberrant receptor pathways will be imperative.

Survival and maintenance of regulatory T cells require the kinase TAK1

Regulatory T （Treg） cells play a central role in regulating peripheral immune tolerance and preventing autoimmunity. Despite the extensive studies on the development of Treg cells, the molecular mechanisms that maintain the population of committed Treg cells remain poorly understood. We show here that Treg-conditional ablation of the kinase TAK1 reduced the number of Treg cells in the peripheral lymphoid organs, causing abnormal activation of conventional T cells and autoimmune symptoms. Using an inducible gene knockout approach, we further demonstrate that TAK1 is crucial for the survival of Treg cells. Expression of a constitutively active IKB kinase partially restored the level of Treg cells in the TAKITreg-KO mice. These results suggest a crucial role for TAK1 for maintaining the survival of committed Treg cells under physiololzical conditions.

Dynamic Behavior and Function of Foxp3^+ Regulatory T Cells in Tumor Bearing Host

Regulatory T cells （Tregs） expressing forkhead/winged-helix transcription factor Foxp3 represent a distinct lineage of lymphocytes which play a central role in protecting the host from autoimmune diseases. However, Tregs also pose a major problem to anti-tumor immunity. Growing body of evidence from both laboratory and clinical investigations has demonstrated that expansion and accumulation of these immunosuppressive cells correlates with advanced tumor growth and predicts poor disease prognosis. How tumor development subverts normal self-tolerance function of Tregs thereby thwarts host anti-tumor immunity remains elusive. This review will discuss our current knowledge in understanding the dynamics and plasticity of Foxp3~ Treg activation and induction in tumor bearing hosts and their interaction with various antigen presenting cells （APCs） in tumor microenvironment leading to the establishment of active local and systemic immune suppression. Cellular ＆ Molecular Immunology.

Fusion genes in solid tumors: an emerging target for cancer diagnosis and treatment

Studies over the past decades have uncovered fusion genes, a class of oncogenes that provide immense diagnostic and therapeutic advantages because of their tumor-specific expression. Originally associated with hemotologic cancers, fusion genes have recently been discovered in a wide array of solid tumors, including sarcomas, carcinomas, and tumors of the central nervous system. Fusion genes are attractive as both therapeutic targets and diagnostic tools due to their inherent expression in tumor tissue alone. Therefore, the discovery and elucidation of fusion genes in various cancer types may provide more effective therapies in the future for cancer patients.

Dextran sulfate sodium-induced acute colitis impairs dermal lymphatic function in mice

AIM: To investigate whether dermal lymphatic function and architecture are systemically altered in dextran sulfate sodium(DSS)-induced acute colitis.METHODS: Balb/c mice were administered 4% DSS in lieu of drinking water ad libitum for 7 d and monitored to assess disease activity including body weight, diarrhea severity, and fecal bleeding. Control mice received standard drinking water with no DSS. Changes in mesenteric lymphatics were assessed following oral administration of a fluorescently-labelled fatty acid analogue, while dermal lymphatic function and architecture was longitudinally characterized using dynamic near-infrared fluorescence(NIRF) imaging following intradermal injection of indocyanine green(ICG) at the base of the tail or to the dorsal aspect of the left paw prior to, 4, and 7 d after DSSadministration. We also measured dye clearance rate after injection of Alexa680-bovine serum albumin(BSA). NIRF imaging data was analyzed to reveal lymphatic contractile activity after selecting fixed regions of interest(ROIs) of the same size in fluorescent lymphatic vessels on fluorescence images. The averaged fluorescence intensity within the ROI of each fluorescence image was plotted as a function of imaging time and the lymphatic contraction frequency was computed by assessing the number of fluorescent pulses arriving at a ROI. RESULTS: Mice treated with DSS developed acute inflammation with clinical symptoms of loss of body weight, loose feces/watery diarrhea, and fecal blood, all of which were aggravated as disease progressed to 7 d. Histological examination of colons of DSS-treated mice confirmed acute inflammation, characterized by segmental to complete loss of colonic mucosa with an associated chronic inflammatory cell infiltrate that extended into the deeper layers of the wall of the colon, compared to control mice. In situ intravital imaging revealed that mice with acute colitis showed significantly fewer fluorescent mesenteric lymphatic vessels, indicating impaired uptake of a lipid tracer within mesenteric lymphatics. Our in vivo NIRF imaging data demonstrated dilated dermal lymphatic vessels, which were confirmed by immunohistochemical staining of lymphatic vessels, and significantly reduced lymphatic contractile function in the skin of mice with DSS-induced acute colitis. Quantification of the fluorescent intensity remaining in the depot as a function of time showed that there was significantly higher Alexa680-BSA fluorescence in mice with DSSinduced acute colitis compared to pre-treatment with DSS, indicative of impaired lymphatic drainage.CONCLUSION: The lymphatics are locally and systemically altered in acute colitis, and functional NIRF imaging is useful for noninvasively monitoring systemic lymphatic changes during inflammation.

Deubiquitinases as pivotal regulators of T cell functions

T cells efficiently respond to foreign antigens to mediate immune responses against infections but are tolerant to self-tissues. Defect in T cell activation is associated with severe immune deficiencies, whereas aberrant T cell activation contributes to the pathogenesis of diverse autoimmune and inflammatory diseases. An emerging mechanism that regulates T cell activation and tolerance is ubiquitination, a reversible process of protein modification that is counter-regulated by ubiquitinating enzymes and deubiquitinases （DUBs）. DUBs are isopeptidases that cleave polyubiquitin chains and remove ubiquitin from target proteins, thereby controlling the magnitude and duration of ubiquitin signaling. It is now well recognized that DUBs are crucial regulators of T cell responses and serve as potential therapeutic targets for manipulating immune responses in the treatment of immunological disorders and cancer. This review will discuss the recent progresses regarding the functions of DUBs in T cells.

位于氧化还原稳态调控和癌症营养依赖性十字路口的氨基酸转运蛋白SLC7A11/xCT

癌细胞经常会上调营养转运蛋白的表达,以满足它们日益增加的生物合成和生物能量需求,并维持氧化还原的动态平衡。在人类癌症中有一种经常过表达的营养转运蛋白,是胱氨酸/谷氨酸逆向转运蛋白溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11,也称为xCT)。SLC7A11促进胱氨酸摄取和谷胱甘肽生物合成,从而防止氧化应激和细胞铁死亡。最近的研究意外地揭示了SLC7A11在谷氨酰胺代谢中也起着关键作用,并调节癌细胞的葡萄糖和谷氨酰胺依赖性。本文综述了SLC7A11在调节癌细胞抗氧化反应和营养依赖中的作用,探讨了SLC7A11在肿瘤代谢中的调控作用,并提出了这一新兴研究领域中有待进一步研究的关键问题。对癌症代谢中SLC7A11的更深入了解可能会为这种重要的氨基酸转运蛋白靶向癌症治疗提供新的治疗机会。

Amiloride and guggulsterone suppression of esophageal cancer cell growth in vitro and in nude mouse xenografts

Esophageal adenocarcinoma is increasing in the US and Western countries and frequent gastresophageal reflux or gastresophageal reflux disease carrying gastric acid and bile acid could contribute to esophageal adenocarcinogenesis. This study was designed to detect the expression of gastric acid-inducing gene Na＋/H＋ exchanger-1 （NHE-1） ex vivo and then to explore targeting of NHE-1 expression or activity to control esophageal cancer cell viability in vitro and in nude mouse xenografts. The data showed that NHE-1 was highly expressed in esophageal adenocarcinoma tissues （66 of 101 cases [65.3%｜, but not in normal esophageal squamous cell epithelium （1 of 26 cases [3.8~0]）. Knockdown of NHE-1 expression using NHE-1 shRNA or inhibition of NHE-1 activity using the NHE-1 inhibitor amiloride suppressed viability and induced apoptosis in esophageal cancer cells. Molecularly, amiloride inhibited expression of cyclooxygenase-2 and matrix metallopeptidase-9 but not NHE-1 mRNA in esophageal cancer cells. A combination of amiloride and guggulsterone （a natural bile acid receptor inhibitor） showed more than additive effects in suppressing esophageal cancer cell growth in vitro and in nude mouse xenografts. This study suggests that inhibition of NHE-1 expression or activity or combination of amiloride and guggulsterone could be useful in control of esophageal adenocarcinoma.

Connexins in neurons and glia: targets for intervention in disease and injury

Both neurons and glia throughout the central nervous system are organized into networks by gap junctions. Among glia, gap junctions facilitate metabolic homeostasis and intercellular communication. Among neurons, gap junctions form electrical synapses that function primarily for communication. However, in neurodegenerative states due to disease or injury gap junctions may be detrimental to survival. Electrical synapses may facilitate hyperactivity and bystander killing among neurons, while gap junction hemichannels in glia may facilitate inflammatory signaling and scar formation. Advances in understanding mechanisms of plasticity of electrical synapses and development of molecular therapeutics to target glial gap junctions and hemichannels offer new hope to pharmacologically limit neuronal degeneration and enhance recovery.

Lymphatic endothelial cells regulate B-cell homing to lymph nodes via a NIK-dependent mechanism

B cells home to the lymph nodes(LNs)via high endothelial venules(HEVs)under the guidance of chemokines,particularly CXCL13.However,as CXCL13 is not directly made in HEVs,the molecular mechanism mediating B-cell homing to LNs has remained unclear.We show here that nuclear factor(NF)-κB-inducing kinase(NIK),a kinase mediating activation of the noncanonical NF-κB pathway,functions in lymphatic endothelial cells(LECs)to regulate B-cell homing to LNs.LEC-conditional deletion of NIK in mice did not affect the integrity or global function of lymphatic vessels but caused a severe reduction in the frequency of B cells in LNs.The LEC-specific NIK deficiency did not affect the survival of B cells or the frequency of B cells in the spleen.B-cell adoptive transfer studies revealed that the LEC-specific NIK deletion impairs the ability of LNs to recruit B cells.We further show that NIK mediates expression of the chemokines CXCL13 and CCL19 in LECs.Although CCL19 is also expressed in blood endothelial cells(BECs),CXCL13 is not produced in BECs.These results suggest that NIK regulates naive B-cell homing to LNs via mediating production of the B-cell homing chemokine CXCL13 in LECs.