Mitochondrial dysfunction in peripheral blood mononuclear cells in pediatric septic shock

OBJECTIVES: 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. DESIGN: Prospective observational study. SETTING: Academic PICU. PATIENTS: Thirteen pediatric patients with septic shock and greater than or equal to two organ failures and 11 PICU controls without sepsis or organ failure. INTERVENTIONS: Ex vivo measurements of mitochondrial oxygen consumption and membrane potential (DeltaPsim) were performed in intact peripheral blood mononuclear cells on day 1-2 and day 5-7 of septic illness and in controls. The Pediatric Logistic Organ Dysfunction score, inotrope score, and organ failure-free days were determined from medical records. MEASUREMENTS AND MAIN RESULTS: Spare respiratory capacity, an index of bioenergetic reserve, was lower in septic peripheral blood mononuclear cells on day 1-2 (median, 1.81; interquartile range, 0.52-2.09 pmol O2/s/10 cells) compared with controls (5.55; 2.80-7.21; p = 0.03). Spare respiratory capacity normalized by day 5-7. Patients with sepsis on day 1-2 exhibited a higher ratio of LEAK to maximal respiration than controls (17% vs \u3c 1%; p = 0.047) with normalization by day 5-7 (1%; p = 0.008), suggesting mitochondrial uncoupling early in sepsis. However, septic peripheral blood mononuclear cells exhibited no differences in basal or adenosine triphosphate-linked oxygen consumption or DeltaPsim. Oxygen consumption did not correlate with Pediatric Logistic Organ Dysfunction score, inotrope score, or organ failure-free days (all p \u3e 0.05). Although there was a weak overall association between DeltaPsim on day 1-2 and organ failure-free days (Spearman rho = 0.56, p = 0.06), patients with sepsis with normal organ function by day 7 exhibited higher DeltaPsim on day 1-2 compared with patients with organ failure for more than 7 days (p = 0.04). CONCLUSIONS: Mitochondrial dysfunction was present in peripheral blood mononuclear cells in pediatric sepsis, evidenced by decreased bioenergetic reserve and increased uncoupling. Mitochondrial membrane potential, but not respiration, was associated with duration of organ injury

The human channel gating–modifying A749G CACNA1D (Cav1.3) variant induces a neurodevelopmental syndrome–like phenotype in mice

Germline de novo missense variants of the CACNA1D gene, encoding the pore-forming α1 subunit of Cav1.3 L-type Ca2+ channels (LTCCs), have been found in patients with neurodevelopmental and endocrine dysfunction, but their disease-causing potential is unproven. These variants alter channel gating, enabling enhanced Cav1.3 activity, suggesting Cav1.3 inhibition as a potential therapeutic option. Here we provide proof of the disease-causing nature of such gating-modifying CACNA1D variants using mice (Cav1.3AG) containing the A749G variant reported de novo in a patient with autism spectrum disorder (ASD) and intellectual impairment. In heterozygous mutants, native LTCC currents in adrenal chromaffin cells exhibited gating changes as predicted from heterologous expression. The A749G mutation induced aberrant excitability of dorsomedial striatum–projecting substantia nigra dopamine neurons and medium spiny neurons in the dorsal striatum. The phenotype observed in heterozygous mutants reproduced many of the abnormalities described within the human disease spectrum, including developmental delay, social deficit, and pronounced hyperactivity without major changes in gross neuroanatomy. Despite an approximately 7-fold higher sensitivity of A749G-containing channels to the LTCC inhibitor isradipine, oral pretreatment over 2 days did not rescue the hyperlocomotion. Cav1.3AG mice confirm the pathogenicity of the A749G variant and point toward a pathogenetic role of altered signaling in the dopamine midbrain system

Monocarboxylate Transporter 4 (MCT4) Knockout Mice Have Attenuated 4NQO Induced Carcinogenesis; A Role for MCT4 in Driving Oral Squamous Cell Cancer

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common human cancer and affects approximately 50,000 new patients every year in the US. The major risk factors for HNSCC are tobacco and alcohol consumption as well as oncogenic HPV infections. Despite advances in therapy, the overall survival rate for all-comers is only 50%. Understanding the biology of HNSCC is crucial to identifying new biomarkers, implementing early diagnostic approaches and developing novel therapies. As in several other cancers, HNSCC expresses elevated levels of MCT4, a member of the SLC16 family of monocarboxylate transporters. MCT4 is a H+-linked lactate transporter which functions to facilitate lactate efflux from highly glycolytic cells. High MCT4 levels in HNSCC have been associated with poor prognosis, but the role of MCT4 in the development and progression of this cancer is still poorly understood. In this study, we used 4-nitroquinoline-1-oxide (4NQO) to induce oral cancer in MCT4−/− and wild type littermates, recapitulating the disease progression in humans. Histological analysis of mouse tongues after 23 weeks of 4NQO treatment showed that MCT4−/− mice developed significantly fewer and less extended invasive lesions than wild type. In mice, as in human samples, MCT4 was not expressed in normal oral mucosa but was detected in the transformed epithelium. In the 4NQO treated mice we detected MCT4 in foci of the basal layer undergoing transformation, and progressively in areas of carcinoma in situ and invasive carcinomas. Moreover, we found MCT4 positive macrophages within the tumor and in the stroma surrounding the lesions in both human samples of HNSCC and in the 4NQO treated animals. The results of our studies showed that MCT4 could be used as an early diagnostic biomarker of HNSCC. Our finding with the MCT4−/− mice suggest MCT4 is a driver of progression to oral squamous cell cancer and MCT4 inhibitors could have clinical benefits for preventing invasive HNSCC

The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores

P2Y2 receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemoattractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide. UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2 receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126, supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca2+-ATPases in mammalian cells, the effect of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y2 receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y2 receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca2+ mobilization indicating that nucleotides cause Ca2+ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca2+ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca2+ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca2+ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca2+ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca2+ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y2 receptor can cause Ca2+ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins

Structure-activity relationships of anthraquinone derivatives derived from bromaminic acid as inhibitors of ectonucleoside triphosphate diphosphohydrolases (E-NTPDases)

Reactive blue 2 (RB-2) had been characterized as a relatively potent ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) inhibitor with some selectivity for NTPDase3. In search for the pharmacophore and to analyze structure-activity relationships we synthesized a series of truncated derivatives and analogs of RB-2, including 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinones, 1-amino-2-methyl-4-arylaminoanthraquinones, 1-amino-4-bromoanthraquinone 2-sulfonic acid esters and sulfonamides, and bis-(1-amino-4-bromoanthraquinone) sulfonamides, and investigated them in preparations of rat NTPDase1, 2, and 3 using a capillary electrophoresis assay. Several 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinone derivatives inhibited E-NTPDases in a concentration-dependent manner. The 2-sulfonate group was found to be required for inhibitory activity, since 2-methyl-substituted derivatives were inactive. 1-Amino-2-sulfo-4-p-chloroanilinoanthraquinone (18) was identified as a nonselective competitive blocker of NTPDases1, 2, and 3 (Ki 16–18 μM), while 1-amino-2-sulfo-4-(2-naphthylamino)anthraquinone (21) was a potent inhibitor with preference for NTPDase1 (Ki 0.328 μM) and NTPDase3 (Ki 2.22 μM). Its isomer, 1-amino-2-sulfo-4-(1-naphthylamino)anthraquinone (20), was a potent and selective inhibitor of rat NTPDase3 (Ki 1.5 μM)

A Randomized, Placebo Controlled, Double Masked Phase IB Study Evaluating the Safety and Antiviral Activity of Aprepitant, a Neurokinin-1 Receptor Antagonist in HIV-1 Infected Adults

Neurokinin-1 receptor (NK1R) antagonists have anti-HIV activity in monocyte-derived macrophages, decrease CCR5 expression and improve natural killer cell function ex vivo. Aprepitant is a NK1R antagonist approved by FDA as an antiemetic.We conducted a phase IB randomized, placebo controlled, double masked study to evaluate the safety, antiviral activity, pharmacokinetics and immune-modulatory effects of aprepitant in HIV-infected adults not receiving antiretroviral therapy, with CD4+ cell count ≥350 cells/mm(3) and plasma viral load ≥2,000 copies/ml. Subjects were stratified by viral load (< vs. ≥20,000 copies/ml) and randomized within each stratum to receive aprepitant at 125 mg QD(Low), or 250 mg QD(High), or placebo(PL) for 14 days, and followed for 42 days.Thirty subjects were randomized and 27 completed treatment (9, 8, 10 subjects in 125 (Low), 250 (High), and PL groups). 63% were male; 37% white; mean (SD) age 43 (9.3) years. Geometric mean baseline viral load (copies/ml) for Low, High, and PL was 15,709, 33,013, and 19,450, respectively. Mean (95%CI) change in log10 viral load at day 14 for Low, High, and PL was -0.02(-0.24,+0.20), -0.05(-0.21,+0.10), and +0.04(-0.08,+0.16), respectively. The number of subjects with AEs was 4(44.4%), 5(62.5%), and 1(10%) for Low, High, and PL. No Grade 4 AEs occurred.Adverse events of aprepitant were more common in the treated groups. At the dose used in this two-week phase IB study, aprepitant showed biological activity, but no significant antiviral activity.ClinicalTrials.gov NCT00428519

Purinergic signalling and immune cells

This review article provides a historical perspective on the role of purinergic signalling in the regulation of various subsets of immune cells from early discoveries to current understanding. It is now recognised that adenosine 5'-triphosphate (ATP) and other nucleotides are released from cells following stress or injury. They can act on virtually all subsets of immune cells through a spectrum of P2X ligand-gated ion channels and G protein-coupled P2Y receptors. Furthermore, ATP is rapidly degraded into adenosine by ectonucleotidases such as CD39 and CD73, and adenosine exerts additional regulatory effects through its own receptors. The resulting effect ranges from stimulation to tolerance depending on the amount and time courses of nucleotides released, and the balance between ATP and adenosine. This review identifies the various receptors involved in the different subsets of immune cells and their effects on the function of these cells

Pilot study demonstrating metabolic and anti-proliferative effects of in vivo anti-oxidant supplementation with N-Acetylcysteine in Breast Cancer

Background 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. Methods Subjects with newly diagnosed stage 0 and I breast cancer who were not going to receive neoadjuvant therapy prior to surgical resection were treated with NAC before definitive surgery to assess intra-tumoral metabolic markers. NAC was administered once a week intravenously at a dose of 150 mg/kg and 600 mg twice daily orally on the days not receiving intravenous NAC. Histochemistry for the stromal metabolic markers monocarboxylate transporter 4 (MCT4) and caveolin-1 (CAV1) and the Ki67 proliferation assay and TUNEL apoptosis assay in carcinoma cells were performed in pre- and post-NAC specimens. Results The range of days on NAC was 14–27 and the mean was 19 days. Post-treatment biopsies showed significant decrease in stromal MCT4 and reduced Ki67 in carcinoma cells. NAC did not significantly change stromal CAV1 and carcinoma TUNEL staining. NAC was well tolerated. Conclusions NAC as a single agent reduces MCT4 stromal expression, which is a marker of glycolysis in breast cancer with reduced carcinoma cell proliferation. This study suggests that modulating metabolism in the tumor microenvironment has the potential to impact breast cancer proliferation