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Objectives: To determine the impact of lymph node yield (LNY) in patients undergoing neck dissection at the time of total laryngectomy (TL). To determine the impact of radiation therapy (RT) on LNY.Methods: Retrospective review of LNY and clinical outcomes in 232 patients undergoing primary or salvage total laryngectomy (TL) with ND.Results: Preoperative RT significantly decreased mean LNY from 31.7 to 23.9 nodes (P< .001). In primary TL patients, age (P< .001) and positive margins (P= .044) were associated with decreased OS. In salvage TL patients, only positive margins was associated with poorer OS (P= .009). No LNY cutoff provided significant OS or DFS benefit.Conclusions: Radiotherapy significantly reduces LNY in patients undergoing TL and ND. Within a single institution cohort, positive margins, but not LNY, is associated with survival in both primary and salvage TL patients. Level of Evidence:4

ABSTRACT-->Low voltage-activated Cav1.3 L-type Ca2+-channels are key regulators of neuronal excitability controlling neuronal development and different types of learning and memory. Their physiological functions are enabled by their negative activation voltage-range, which allows Cav1.3 to be active at subthreshold voltages. Alternative splicing in the C-terminus of their pore-forming α1-subunits gives rise to C-terminal long (Cav1.3L) and short (Cav1.3S) splice variants allowing Cav1.3Sto activate at even more negative voltages than Cav1.3L. We discovered that inclusion of exons 8b, 11, and 32 in Cav1.3Sfurther shifts activation (-3 to -4 mV) and inactivation (-4 to -6 mV) to more negative voltages as revealed by functional characterization in tsA-201 cells. We found transcripts of these exons in mouse chromaffin cells, the cochlea, and the brain. Our data further suggest that Cav1.3-containing exons 11 and 32 constitute a significant part of native channels in the brain. We therefore investigated the effect of these splice variants on human disease variants. Splicing did not prevent the gating defects of the previously reported human pathogenic variant S652L, which further shifted the voltage-dependence of activation of exon 11-containing channels by more than -12 mV. In contrast, we found no evidence for gating changes of the CACNA1Dmissense variant R498L, located in exon 11, which has recently been identified in a patient with an epileptic syndrome.Our data demonstrate that alternative splicing outside the C-terminus involving exons 11 and 32 contributes to channel fine-tuning by stabilizing negative activation and inactivation gating properties of wild-type and mutant Cav1.3 channels.

Background: Melanotic schwannoma is a rare tumor with indeterminate biologic behavior and varying treatment recommendations.Methods: We report 2 cases of pigmented melanotic schwannoma of the head and neck and perform literature review. The pathologic and immunohistochemical characteristics of melanotic schwannoma are reviewed.Results: Two cases of melanotic schwannoma are presented. Both cases underwent surgical resection with one patient receiving adjuvant radiation therapy.Conclusions: Melanotic schwannoma is a rare nerve sheath tumor that is frequently mistaken for malignant melanoma. We describe 2 cases of pigmented melanotic schwannoma of the head and neck with different presentations and review the histopathological and immunohistochemical features.

Primary cutaneous angiosarcoma is a rare vasoformative malignant neoplasm that can present a diagnostic and therapeutic challenge. We describe a 76-year-old Caucasian man with right upper eyelid swelling and nodularity, initially suspected clinically to represent either ocular adnexal lymphoma or basal cell carcinoma. Incisional biopsy and wide resection of the mass with frozen section control of margins were interpreted as compatible with hobnail (Dabska-retiform) hemangioendothelioma. Foci of atypia were noted in the tumor, raising speculation of evolution into a more aggressive neoplasm, such as conventional angiosarcoma. The patient subsequently underwent two additional wide resections with frozen section control of margins in an attempt to obtain complete excision of residual tumor, which demonstrated histopathologic features favoring angiosarcoma. The histologic material from the original and subsequent resections was sent in consultation to several soft tissue pathology experts and the final diagnosis of low-grade cutaneous angiosarcoma was established. Despite repeated surgical interventions, there was continued persistence of the tumor in the deep orbital tissues. Various management options, including adjuvant radiotherapy/chemotherapy with and without orbital exenteration, were discussed. The patient decided against further surgical intervention and is currently undergoing adjuvant radiotherapy/chemotherapy. This case illustrates the diagnostic and management difficulties of ocular adnexal angiosarcoma.

ABSTRACTRecently, we and others identified somatic and germline de novogain-of-function mutations in CACNA1D, the gene encoding the α1-subunit of voltage-gated Cav1.3 Ca2+-channels. While somatic mutations identified in aldosterone producing adenomas (APAs) underlie treatment-resistant hypertension, germline CACNA1Dmutations are associated with a neurodevelopmental disorder characterized by a wide symptomatic spectrum, including autism spectrum disorder. The number of newly identified CACNA1Dmissense mutations is constantly growing, but their pathogenic potential is difficult to predict in silico, making functional studies indispensable to assess their contribution to disease risk.Here we report the functional characterization of previously identified CACNA1DAPA mutations F747L and M1354I using whole-cell patch-clamp electrophysiology upon recombinant expression in tsA-201 cells. We also investigated if alternative splicing of Cav1.3 affects the aberrant gating of the previously characterized APA mutation R990H and two mutations associated with autism spectrum disorder (A479G and G407R). Splice-variant dependent gating changes are of particular interest for germline mutations, since the relative expression of Cav1.3 splice variants differs across different tissues and within brain regions and might therefore result in tissue-specific phenotypes. Our data revealed a complex gain-of-function phenotype for APA mutation F747L confirming its pathogenic role. Furthermore, we found splice-variant dependent gating changes in R990H, A749G and G407R. M1354I did not change channel function of Cav1.3 splice variants and should therefore be considered a rare non-pathogenic variant until further proof for its pathogenicity is obtained. Our new findings together with previously published data allow classification of pathogenic CACNA1Dmutations into four categories based on prototypical functional changes.

ABSTRACTVoltage-dependent calcium channels (CaV) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of CaV1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of CaV1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of CaV1.3 and in VSDs II and IV of CaV1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of CaV1.3 and in two splice variants of CaV1.2. In both channels the D4N (VSD IV) mutation resulted in a  ̴5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in CaV1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined CaV1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle CaV1.1 channel.

Mutations of positively charged amino acids in the S4 transmembrane segment of a voltage-gated ion channel form ion-conducting pathways through the voltage-sensing domain, named ω-current. Here, we used structure modeling and MD simulations to predict pathogenic ω-currents in CaV1.1 and CaV1.3 Ca2+channels bearing several S4 charge mutations. Our modeling predicts that mutations of CaV1.1-R1 (R528H/G, R897S) or CaV1.1-R2 (R900S, R1239H) linked to hypokalemic periodic paralysis type 1 and of CaV1.3-R3 (R990H) identified in aldosterone-producing adenomas conducts ω-currents in resting state, but not during voltage-sensing domain activation. The mechanism responsible for the ω-current and its amplitude depend on the number of charges in S4, the position of the mutated S4 charge and countercharges, and the nature of the replacing amino acid. Functional characterization validates the modeling prediction showing that CaV1.3-R990H channels conduct ω-currents at hyperpolarizing potentials, but not upon membrane depolarization compared with wild-type channels.

High oxidative stress as defined by hydroxyl and peroxyl activity is often found in the stroma of human breast cancers. Oxidative stress induces stromal catabolism, which promotes cancer aggressiveness. Stromal cells exposed to oxidative stress release catabolites such as lactate, which are up-taken by cancer cells to support mitochondrial oxidative phosphorylation. The transfer of catabolites between stromal and cancer cells leads to metabolic heterogeneity between these cells and increased cancer cell proliferation and reduced apoptosis in preclinical models. N-Acetylcysteine (NAC) is an antioxidant that reduces oxidative stress and reverses stromal catabolism and stromal-carcinoma cell metabolic heterogeneity, resulting in reduced proliferation and increased apoptosis of cancer cells in experimental models of breast cancer. The purpose of this clinical trial was to determine if NAC could reduce markers of stromal-cancer metabolic heterogeneity and markers of cancer cell aggressiveness in human breast cancer.

Exposure of human PBMC to substance P results in production of chemokines, including MCP-1, which increases migration of human monocytes. The neuropeptide SP has physiologic and pathophysiologic roles in CNS and peripheral tissues and is involved in crosstalk between nervous and immune systems in various conditions, including HIV and SIV infection. Increased SP levels were demonstrated in plasma of HIV+individuals as well as in the CNS of SIV-infected, nonhuman primates. SP increases HIV infection in macrophages through interaction with its receptor, NK1R. The SP effect on immune system is both pro- and anti-inflammatory and includes up-regulation of a number of cytokines and cell receptors. The main goal of this study was to determine whether there is interplay between monocyte exposure to SP and recruitment into sites of inflammation. We now demonstrate that exposure of either human macrophages or PBMCs to SP leads to increased production of chemokines, including MCP-1, for which expression is limited to cells of the myeloid lineage. This effect is inhibited by the NK1R antagonist, aprepitant. Exposure to conditioned medium derived from SP-treated PBMCs resulted in increased monocyte migration through semipermeable membranes and an in vitro human BBB model. Monocyte migration was blocked by anti–MCP-1 antibodies. Our results suggest that increased SP levels associated with HIV and other inflammatory conditions may contribute to increased monocyte migration into the CNS and other tissues through a MCP-1–dependent mechanism.

Integrase interactor 1 (INI1) is a tumor suppressor gene that is ubiquitously expressed in all nucleated cells. The loss of INI1 protein activity was first demonstrated in aggressive pediatric tumors, including atypical teratoid/rhabdoid (AT/RT) tumor of the central nervous system and malignant rhabdoid tumor of the kidney. Subsequently, INI1 deficiency was discovered in other pediatric and some adult neoplasms. The spectrum of INI1-negative tumors includes a wide variety of neoplasms that occur over a wide age range, are variably aggressive, and have a variable rhabdoid component on histopathologic evaluation. In this report, the authors describe a 27-year-old gravid woman with INI1-deficient carcinoma of the lacrimal gland, previously not described in this location.Integrase interactor 1–deficient tumors are a rare and aggressive group of neoplasms that exhibit typical rhabdoid morphology on histopathological evaluation. Herein, the authors describe the first case of integrase interactor 1–deficient carcinoma of the lacrimal gland.

A patient diagnosed with metastatic melanoma developed the paraneoplastic syndrome of humoral hypercalcemia of malignancy and cachexia after receiving ipilumumab. The cause of the hypercalcemia was thought to be secondary to parathyroid hormone-related peptide (PTHrP) as plasma levels were found to be elevated. The patient underwent two tumor biopsies: at diagnosis (when calcium levels were normal) and upon development of hypercalcemia and cachexia. PTHrP expression was higher in melanoma cells when hypercalcemia had occurred than prior to its onset. Metabolic characterization of melanoma cells revealed that, with development of hypercalcemia, there was high expression of monocarboxylate transporter 1 (MCT1), which is the main importer of lactate and ketone bodies into cells. MCT1 is associated with high mitochondrial metabolism. Beta-galactosidase (β-GAL), a marker of senescence, had reduced expression in melanoma cells upon development of hypercalcemia compared to pre-hypercalcemia.

Anaplastic thyroid cancer (ATC) is one of the most aggressive human cancers. Key signal transduction pathways that regulate mitochondrial metabolism are frequently altered in ATC. Our goal was to determine the mitochondrial metabolic phenotype of ATC by studying markers of mitochondrial metabolism, specifically monocarboxylate transporter 1 (MCT1) and translocase of the outer mitochondrial membrane member 20 (TOMM20). Staining patterns of MCT1 and TOMM20 in 35 human thyroid samples (15 ATC, 12 papillary thyroid cancer [PTC], and eight non-cancerous thyroid) and nine ATC mouse orthotopic xenografts were assessed by visual and Aperio digital scoring. Staining patterns of areas involved with cancer versus areas with no evidence of cancer were evaluated independently where available. MCT1 is highly expressed in human anaplastic thyroid cancer when compared to both non-cancerous thyroid tissues and papillary thyroid cancers (P<.001 for both). TOMM20 is also highly expressed in both ATC and PTC compared to non-cancerous thyroid tissue (P<.01 for both). High MCT1 and TOMM20 expression is also found in ATC mouse xenograft tumors compared to non-cancerous thyroid tissue (P<.001). These xenograft tumors have high 13C- pyruvate uptake. ATC has metabolic features that distinguish it from PTC and non-cancerous thyroid tissue, including high expression of MCT1 and TOMM20. PTC has low expression of MCT1 and non-cancerous thyroid tissue has low expression of both MCT1 and TOMM20. This work suggests that MCT1 blockade may specifically target ATC cells presenting an opportunity for a new drug target.

We evaluated safety, antiviral, immunomodulatory and anti-inflammatory properties of aprepitant – a neurokinin 1 receptor antagonist.

Salivary duct carcinoma (SDC) is a prototypic aggressive salivary gland carcinoma. Our aim is to determine the prevalence of histologic variants (micropapillary, basal-like) and androgen receptor (AR) expression in a large multi-institutional series of SDC. AR status was determined by immunohistochemistry (IHC). Most SDCs were characterized by an apocrine phenotype and AR expression. Cases with a nonapocrine phenotype and AR-negative status were studied by additional IHC and fluorescence in situ hybridization for ETV6or MYBNFIB. The diagnosis of SDC was confirmed in 187 of 199 (94) cases. Variant morphologies were identified in 12 cases: micropapillary (n=6), sarcomatoid (n=3), mucinous (n=2), and basal-like (n=1). AR IHC was performed in 183 cases, of which 179 (97.8) showed AR expression. On the basis of morphologic appearance and results of additional studies, 12 cases were reclassified as squamous cell carcinoma (SCC) (n=4), epithelial-myoepithelial carcinoma with high-grade transformation (HGT) (n=2), myoepithelial carcinoma (n=2), mammary analogue secretory carcinoma, high grade (ETV6translocated; n=1), adenoid cystic carcinoma with HGT (n=1), acinic cell carcinoma with HGT (n=1), and adenosquamous carcinoma (n=1). AR-negative SDC is extremely rare, and the majority of such cases are more accurately classified as other entities. HGTs of other salivary carcinomas and squamous cell carcinoma are the most common mimics of SDC. SDCs with variant morphologies still show at least a minor component of conventional apocrine appearance. Thus, apocrine morphology defines SDC.

CaV1.1e is the voltage-gated calcium channel splice variant of embryonic skeletal muscle. It differs from the adult CaV1.1a splice variant by the exclusion of exon 29 coding for 19 amino acids in the extracellular loop connecting transmembrane domains IVS3 and IVS4. Like the adult splice variant CaV1.1a, the embryonic CaV1.1e variant functions as voltage sensor in excitation-contraction coupling, but unlike CaV1.1a it also conducts sizable calcium currents. Consequently, physiological or pharmacological modulation of calcium currents may have a greater impact in CaV1.1e expressing muscle cells. Here, we analyzed the effects of L-type current modulators on whole-cell current properties in dysgenic (CaV1.1-null) myotubes reconstituted with either CaV1.1a or CaV1.1e. Furthermore, we examined the physiological current modulation by interactions with the ryanodine receptor using a chimeric CaV1.1e construct in which the cytoplasmic II-III loop, essential for skeletal muscle excitation-contraction coupling, has been replaced with the corresponding but nonfunctional loop from the Musca channel. Whereas the equivalent substitution in CaV1.1a had abolished the calcium currents, substitution of the II-III loop in CaV1.1e did not significantly reduce current amplitudes. This indicates that CaV1.1e is not subject to retrograde coupling with the ryanodine receptor and that the retrograde coupling mechanism in CaV1.1a operates by counteracting the limiting effects of exon 29 inclusion on the current amplitude. Pharmacologically, CaV1.1e behaves like other L-type calcium channels. Its currents are substantially increased by the calcium channel agonist Bay K 8644 and inhibited by the calcium channel blocker nifedipine in a dose-dependent manner. With an IC50 of 0.37 μM for current inhibition by nifedipine, CaV1.1e is a potential drug target for the treatment of myotonic dystrophy. It might block the excessive calcium influx resulting from the aberrant expression of the embryonic splice variant CaV1.1e in the skeletal muscles of myotonic dystrophy patients.

Suboptimal vitamin D status is prevalent in HIV-infected patients and associated with increased risk of disease severity and morbidity. We aimed to determine 12-month safety and efficacy of daily 7000 IU vitamin D3(vitD3) versus placebo to sustain increased serum 25-hydroxyvitamin D (25(OH)D) and improve immune status in HIV-infected subjects.

Mitochondrial dysfunction in peripheral blood mononuclear cells has been linked to immune dysregulation and organ failure in adult sepsis, but pediatric data are limited. We hypothesized that pediatric septic shock patients exhibit mitochondrial dysfunction within peripheral blood mononuclear cells which in turn correlates with global organ injury.

This review will discuss the evolution of the role of chemotherapy in the treatment of locally advanced head and neck cancer (HNC), over the last few decades. Studies were identified by searching PubMed electronic databases. Surgery followed by radiotherapy (RT) or definitive RT are potentially curative approaches for locally advanced HNC. While chemotherapy itself is not curative, it can improve cure rates when given as an adjunct to RT. The benefit of combining chemotherapy with RT is related to the timing of the chemotherapy. Several prospective randomized trials have demonstrated that concurrent delivery of chemotherapy and RT (CRT) is the most promising approach, given that locoregional recurrence is the leading pattern of failure for patients with locally advanced HNC. Induction chemotherapy before CRT has not been shown to be superior to CRT alone and the added toxicity may negatively impact the compliance with CRT. Sequential chemotherapy administration, in the form of induction chemotherapy followed by RT or CRT, has been successful as a strategy for organ preservation in patients with potentially resectable laryngeal and hypopharyngeal cancer. Systemic chemotherapy delivered concurrently with RT is used as a standard treatment for locally advanced HNC.

Thrombotic events occurring in either arteries or veins are the primary causes of fatal perioperative cardiovascular events. Risk factors for deep vein thrombosis, several of which are evidently associated with specific surgical procedures, are quite different from those for arterial thrombosis (e.g., aging or atherosclerotic diseases). Thrombus formed in arteries consists mainly of platelets coated with fibrin (i.e., white thrombus), while venous thrombus formed at relatively lower shear stress consists of all blood components including erythrocytes as well as leukocytes infiltrated with fibrin (red thrombus). Clinical evidence indicates beneficial roles of neuraxial anesthesia/ analgesia in the prevention of VTE for patients undergoing high risk surgical procedures. To date, mechanisms of action of drugs used for neuraxial anesthesia/analgesia to prevent venous thrombosis are uncertain. However, accumulation of clinical as well as experimental findings points to the involvement of immune cells (especially monocytes) in red thrombus generation and to the interaction of anesthetics with these cells. We also suggest that adhesion molecules associated with the formation of monocyte platelet aggregates as well as substance P: neurokinin-1 receptor (SP/NK1R) pathway that involves neurogenic inflammation are crucial. Local anesthetics and NK1R antagonists are candidate drugs that may possess the capability to prevent venous thrombotic disorders in perioperative settings.

This article covers, in a comprehensive way, thyroid cytopathology. The Bethesda System for Reporting Thyroid Cytology is reviewed, with emphasis on the atypical and indeterminate diagnostic categories. Immunohistochemistry stains and molecular tests panels applicable to cytology specimens are described.

Objective. To describe the dietary intake of HIV-infected urban children and young adults and to evaluate their diet quality. Methods. Participants were children and youth with both perinatally and behaviorally acquired HIV infection participating in a study of vitamin D supplementation. Data collected included dietary intake, anthropometrics, and HIV status, with medical history abstracted from participants’ medical records. Results. Of 55 participants, 38 were male, 46 were African American, with a mean age of 20.7 ± 3.8 years. Growth and nutritional status were comparable to reference norms. Only 22% either met or exceeded their estimated energy requirement at low-active and 40% at sedentary activity levels. Fiber, potassium, and intakes of vitamins D and E were <50% of recommended dietary allowance/adequate intake (RDA/AI), whereas vitamins A and K, choline, potassium, calcium, and magnesium were ≤75% of RDA/AI. Sodium intake exceeded the dietary reference intake upper limit in 92%. Vitamin D intake was significantly and positively associated with CD4% HIV status indicator. Conclusion. Dietary intake was inadequate for multiple micronutrients, particularly vitamin D, calcium, and potassium. Overall poor dietary quality was observed (in terms of intake of sodium, sugar, saturated fat, cholesterol, and fiber). Participants had high rates of overweight and obesity. These dietary and body weight concerns were similar to those seen in healthy American peers. The vitamin D intake and immunological status correlation deserves further investigation. HIV-infected individuals may benefit from routine dietary assessment and counseling.

HIV infection in macrophages is enhanced by substance P through a mechanism dependent on the haptoglobinhemoglobin scavenger receptor CD163. Activation of NK1R by SP contributes to increased HIV‐1 infection in macrophages. The scavenger receptor CD163 is expressed on cells of monocyte‐macrophage origin. Our main goal was to determine if there is interplay among SP, CD163 expression, and HIV infection in macrophages. We showed that SP triggers intracellular calcium elevation and increased CD163 expression in human monocytes in a time‐ and concentration‐dependent manner. The role of CD163 on HIV infection was examined by RT‐PCR in sorted monocytes (CD163lowand CD163high) and in macrophages having CD163 knocked down using siRNA. We found that the productivity of HIV infection was higher in CD163highcells. Additionally, in macrophages with CD163 expression knocked down, we found a significant decrease of HIV infection. Furthermore, Hb‐Hp complexes, which function as an endogenous ligand for CD163, decreased HIV infection in macrophages in a dose‐dependent manner. Thus, we demonstrate that SP induces higher levels of CD163 in monocytes and that high expression of CD163 is associated with increases HIV infection in macrophages. Thus, in addition to being a prognostic marker of HIV infection, the expression of CD163 on macrophages may be critical in HIV immunopathogenesis.

The tumor microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) is comprised of cancer-associated fibroblasts (CAFs), immune cells, and other supporting cells. Genetic changes in the carcinoma cells, such as alterations to TP53, NOTCH1, and specific gene expression profiles, contribute to derangements in cancer and microenvironment cells such as increased ROS, overproduction of cytokines, and epithelial to mesenchymal transition (EMT). CAFs are among the most critical elements of the TME contributing to proliferation, invasion, and metastasis. The adaptive immune response is suppressed in HNSCC through overexpression of cytokines, triggered apoptosis of T cells, and alterations in antigen processing machinery. Overexpression of critical cytokines, such as transforming growth factor-β (TGF-β), contributes to EMT, immune suppression, and evolution of CAFs. Inflammation and hypoxia are driving forces in angiogenesis and altered metabolism. HNSCC utilizes glycolytic and oxidative metabolism to fuel tumorigenesis via coupled mechanisms between cancer cell regions and cells of the TME. Increased understanding of the TME in HNSCC illustrates that the long-held notion of “condemned mucosa” reflects a process that extends beyond the epithelial cells to the entire tissue comprised of each of these elements.

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4 inhibitor”. Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish “metabolic parasites”, like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted “antibiotics” to selectively starve cancer cells. Our results provide new support for the “seed and soil” hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.

Here, we interrogated head and neck cancer (HNSCC) specimens (n = 12) to examine if different metabolic compartments (oxidative vs. glycolytic) co-exist in human tumors. A large panel of well-established biomarkers was employed to determine the metabolic state of proliferative cancer cells. Interestingly, cell proliferation in cancer cells, as marked by Ki-67 immunostaining, was strictly correlated with oxidative mitochondrial metabolism (OXPHOS) and the uptake of mitochondrial fuels, as detected via MCT1 expression (p < 0.001). More specifically, three metabolic tumor compartments were delineated: (1) proliferative and mitochondrial-rich cancer cells (Ki-67+/TOMM20+/COX+/MCT1+); (2) non-proliferative and mitochondrial-poor cancer cells (Ki-67−/TOMM20−/COX−/MCT1−); and (3) non-proliferative and mitochondrial-poor stromal cells (Ki-67−/TOMM20−/COX−/MCT1−). In addition, high oxidative stress (MCT4+) was very specific for cancer tissues. Thus, we next evaluated the prognostic value of MCT4 in a second independent patient cohort (n = 40). Most importantly, oxidative stress (MCT4+) in non-proliferating epithelial cancer cells predicted poor clinical outcome (tumor recurrence; p < 0.0001; log-rank test), and was functionally associated with FDG-PET avidity (p < 0.04). Similarly, oxidative stress (MCT4+) in tumor stromal cells was specifically associated with higher tumor stage (p < 0.03), and was a highly specific marker for cancer-associated fibroblasts (p < 0.001). We propose that oxidative stress is a key hallmark of tumor tissues that drives high-energy metabolism in adjacent proliferating mitochondrial-rich cancer cells, via the paracrine transfer of mitochondrial fuels (such as L-lactate and ketone bodies). New antioxidants and MCT4 inhibitors should be developed to metabolically target “three-compartment tumor metabolism” in head and neck cancers. It is remarkable that two “non-proliferating” populations of cells (Ki-67−/MCT4+) within the tumor can actually determine clinical outcome, likely by providing high-energy mitochondrial “fuels” for proliferative cancer cells to burn. Finally, we also show that in normal mucosal tissue, the basal epithelial “stem cell” layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and is metabolically programmed to use mitochondrial fuels (MCT1+), such as ketone bodies and L-lactate. Thus, oxidative mitochondrial metabolism (OXPHOS) is a common feature of both (1) normal stem cells and (2) proliferating cancer cells. As such, we should consider metabolically treating cancer patients with mitochondrial inhibitors (such as Metformin), and/or with a combination of MCT1 and MCT4 inhibitors, to target “metabolic symbiosis.”

One of the earliest steps in the development of the neuromuscular junction (NMJ) is the pre-patterning of acetylcholine receptors (AChR) in the center of muscle fibers. This process has recently been proposed to depend on L-type calcium currents. But its feeble current properties make the skeletal muscle calcium channel (Cav1.1) a poor candidate for this function. Independently a new Cav1.1e splice variant with greatly distinct current properties has been described. But so far this channel lacked a function. Could this orphan channel be responsible for regulating AChR pre-patterning? Here we compare the properties of the two splice variants and argue that the newly discovered variant Cav1.1e, is dominantly expressed at the proper time in development and has the essential current properties to accomplish the proposed role in the formation of the NMJ.

Ligation of neurokinin 1 receptor by substance P in NK cells can reduce calcium flux and cytotoxicity through interference with activation‐induced ERK phosphorylation. SP is a potent neuroimmunomodulator that functions through ligating members of the neurokinin receptor family, one of which, NK1R, is widely expressed in immune cells. As in humans, circulating SP levels are increased in pathologic states associated with impairment of NK cell functions, such as depression and HIV infection, we hypothesized that SP has a direct, inhibitory effect upon NK cells. We have studied a clonal human NK cell line (YTS) as well as ex vivo human NK cells and have determined that truncated and full‐length NK1R isoforms are expressed in and SP bound by ex vivo NK cells and the YTS NK cell line. Incubation of YTS cells with 10−6M SP and ex vivo NK cells with 10−5M SP inhibited cytotoxic ability by ∼20% and reduced degranulation. This inhibitory effect upon cytotoxicity was partially prevented by the NK1R antagonist CP96,345. The treatment of YTS or ex vivo NK cells with SP neither down‐modulated NCR expression nor affected triggering receptor‐induced NF‐κB activation. Preincubation of YTS cells with SP, however, did abbreviate the typically prolonged intracellular calcium increase induced by target cell engagement and reduced triggering receptor‐induced pERK. Thus, SP has the potential to regulate NK cell functions and acts downstream from neurokinin receptors to modulate NK cell activation signaling. This mechanism may contribute to impairment of NK cell function in certain disease states associated with increased circulating SP. Antagonism of this system may present an opportunity to augment NK cell function therapeutically in selected human diseases.

Recent studies suggest a link between neuropsychiatric disorders and HIV/SIV infection. Most evidence indicates that monocytes/macrophages are the primary cell type infected within the CNS and that they contribute to CNS inflammation and neurological disease. Substance P (SP), a pleotropic neuropeptide implicated in inflammation, depression, and immune modulation via interaction with its cognate receptor, the neurokinin 1 receptor (NK1-R), is produced by monocyte/macrophages. While the presence of NK1-R on neurons is well known, its role on cells of the immune system such as monocyte/macrophages is just beginning to emerge. Therefore, we have examined the expression of SP and NK1-R and their relationship to SIV/HIV encephalitis (SIVE/HIVE) lesions and SIV-infected cells. These studies demonstrated intense expression of SP and NK1-R in SIVE lesions, with macrophages being the principal cell expressing NK1-R. Interestingly, all of the SIV-infected macrophages expressed NK1-R. Additionally, we examined the functional role of SP as a proinflammatory mediator of monocyte activation and chemotaxis. These studies demonstrated that treatment of monocytes with SP elicited changes in cell-surface expression for CCR5 and NK1-R in a dose-dependent manner. Moreover, pretreatment with SP enhanced both SP- and CCL5-mediated chemotaxis. All of these findings suggest that SP and NK1-R are important in SIV infection of macrophages and the development of SIVE lesions.

The Ca2+ channel α1S subunit (CaV1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca2+ release from internal stores and conducts a slowly activating Ca2+ current. However, this Ca2+ current is not essential for skeletal muscle EC coupling. Here, we identified a CaV1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged α1S subunit and expressed it in dysgenic (α1S-null) myotubes. GFP-α1SΔ29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca2+ currents through GFP-α1SΔ29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca2+ influx contributed substantially to the depolarization-induced Ca2+ transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary α2δ-1 subunit. Thus, characterizing the CaV1.1Δ29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca2+ channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.

Substance P (SP) is a potent modulator of monocyte/macrophage function. The SP‐preferring receptor neurokinin‐1 receptor (NK1R) has two forms: a full‐length NK1R (NK1R‐F) isoform and a truncated NK1R (NK1R‐T) isoform, which lacks the terminal cytoplasmic 96‐aa residues. The distribution of these receptor isoforms in human monocytes is not known. We previously identified an interaction among SP, NK1R, and HIV viral strains that use the chemokine receptor CCR5 as a coreceptor, suggesting crosstalk between NK1R and CCR5. The purpose of this study was to determine which form(s) of NK1R are expressed in human peripheral blood monocytes and to determine whether SP affects proinflammatory cellular responses mediated through the CCR5 receptor. Human peripheral blood monocytes were found to express NK1R‐T but not NK1R‐F. SP interactions with NK1R‐T did not mobilize calcium (Ca2+), but SP mobilized Ca2+when the NK1R‐F was transfected into monocytes. However, the NK1R‐T was functional in monocytes, as SP enhanced the CCR5 ligand CCL5‐elicited Ca2+mobilization, a response inhibited by the NK1R antagonist aprepitant. SP interactions with the NK1R‐T also enhanced CCL5‐mediated chemotaxis, which was ERK1/2‐dependent. NK1R‐T selectively activated ERK2 but increased ERK1 and ERK2 activation by CCL5. Activation of NK1R‐T elicited serine phosphorylation of CCR5, indicating that crosstalk between CCL5 and SP may occur at the level of the receptor. Thus, NK1R‐T is functional in human monocytes and activates select signaling pathways, and the NK1R‐T‐mediated enhancement of CCL5 responses does not require the NK1R terminal cytoplasmic domain.

Abstract: The skeletal muscle dihydropyridine receptor is a slowly-activating calcium channel that functions as the voltage sensor in excitation-contraction coupling. In addition to the pore-forming α1S subunit it contains the transmembrane α2δ-1 and γ1 subunits and the cytoplasmic β1a subunit. Although the roles of the auxiliary subunits in calcium channel function have been intensively studied in heterologous expression systems, their functions in excitation-contraction coupling has only recently been elucidated in muscle cells of various null-mutant animal models. In this article we will briefly outline the current state of these investigations.

The activated platelet surface serves as an integral part of the prothrombinase complex upon activation by potent platelet agonists such as thrombin and collagen. We determined the receptor specificity through which thrombin was enhancing collagen‐induced thrombin generation. Whereas SFLLRN or AYPGKF alone produced minimal thrombin generation or phosphatidylserine exposure through protease activated receptor (PAR) stimulation, they caused a leftward shift in the collagen‐induced thrombin generation dose–response curve. Although SFLLRN or AYPGKF potentiated collagen‐induced thrombin generation, neither of them potentiated to the same extent as thrombin. However, SFLLRN and AYPGKF together potentiated collagen‐induced thrombin generation to the same extent as thrombin. We conclude that thrombin mediates its procoagulant activity through activation of both PAR1 and PAR4 receptors. Similarly, neither PAR1 nor PAR4 stimulation alone mimicked the annexin V‐binding response caused by thrombin stimulation. The combination of PAR activating peptides caused minimal increases in annexin V binding, but caused significant thrombin generation, suggesting that events other than phosphatidylserine exposure may play a role in platelet prothrombinase complex formation. We also investigated the ability of ADP to potentiate agonist‐induced thrombin generation. Whereas P2Y1antagonism did not affect collagen or thrombin‐induced thrombin generation, P2Y12antagonism did decrease both collagen‐ and thrombin‐induced thrombin generation, suggesting that ADP potentiates thrombin generation primarily through the P2Y12receptor. Collectively, these results suggest that stimulation of both the PAR1 and PAR4 receptors are necessary for thrombin‐induced procoagulant activity, and that the P2Y12receptor, but not the P2Y1receptor, is responsible for the potentiation of agonist‐induced platelet procoagulant activity.