Poor Result Reporting Rate in Cell Therapy Trials Registered at ClinicalTrials.gov.

As research associates in clinical experiments, we have an obligation to disclose clinical methodologies and findings in full transparency in ethics. However, inadequate disclosure in results reporting clinical trials registered on ClinicalTrials.gov has been revealed, with approximately half the trial results not being reported in an applicable manner. Our recent study in clinical trials of regenerative medicine for four kinds of neurological diseases revealed that the rate of result reporting to ClinicalTrials.gov is inadequate for gene and cell therapy (CT) trials. In this path, further curiosity emerged to see what the findings would be if the analysis was conducted for trials in all disease areas, and outcomes if gene therapy (GT) and CT were distinguished in terms. In this study, the scope of analysis was further expanded to include all disease areas, and the drug classification from the AdisInsight database was used for modality classification, with biologic drug trials classified as controls, CT, ex vivo GT, and in vivo GT. To begin, among all interventional clinical trials with registration in the ClinicalTrials.gov registry and with a primary completion between 2010 and 2019, we created a total of 5539 datasets corresponding to trials classified as GT and CT, while biologics (BLG) as controls in the AdisInsight drug classification. The status of reported results of these trials was identified by surveying posting status of ClinicalTrials.gov and publication in journals (PubMed), respectively. Based on the obtained dataset, multivariate analysis was performed on the data on the reporting rate of clinical trial results, aggregated by sponsor, phase, status, and modality (CT, ex vivo GT, in vivo GT, and BLG), respectively. The result shows that CT was identified as an independent factor restraining result reporting ratio in both ClinicalTrials.gov and total disclosures, whereas ex vivo GT as boosting result reporting ratio. Since the result reporting rate of CT results was notably poor, we discussed the causes and solutions in this regard.

A Pilot Study on Result Reporting Rates from Clinical Trials of Regenerative Medicine.

Sharing the methods and results of clinical trials with full transparency is an ethical obligation for those involved in clinical research. In this regard, ClinicalTrials.gov requires reporting of results to the registry within 1 year of completion of the trial. However, a poor result reporting rate has been pointed out, with approximately half the trial results not been reported. It has been suggested that one of the reasons behind this could be the influence of sponsors who conduct the clinical trials. In the course of our previous trend analysis on regenerative medicine for stroke (STR) using ClinicalTrials.gov and the International Clinical Trials Registry Platform (ICTRP) portal site as data sources, we suspected whether the results of gene and/or cell therapy trials are poorly reported. For this reason, a multivariate analysis using data from ClinicalTrials.gov was performed to identify the factors suppressing the result reporting rate, expanding our study to four different kinds of neurological diseases and regenerative medicine as a treatment modality when small-molecule compounds and biologics were set up as controls, in addition to the sponsor type factor. As a result, we found gene and/or cell therapy (therapeutic modality) in addition to STR (disease area), trials completed in 2005-2007, and clinical phases II and IV as independent factors that suppressed the rate of reporting results to ClinicalTrials.gov. On the other hand, big pharmaceutical companies were identified as a factor that increased the reporting result rate to ClinicalTrials.gov. When we applied result reporting publications through PubMed as an index, our study data revealed that the following factors were not identified as the cause for a decrease in the reporting result rate: STR (as disease area), trials completed between 2005 and 2007, and gene/cell therapy (as treatment modality). In this context, our findings indicate that gene/cell therapy has led to the suppression of the result reporting rate to ClinicalTrials.gov. This confirmed our initial suspicion of the low result reporting rate of gene/cell therapy trials. We believe that further studies are required to elucidate the factors affecting the result reporting rate from the perspective of disease area and treatment modality. Impact Statement Several studies have addressed the poor result reporting rate of clinical trials, which still remains an issue. Regenerative medicine holds great promise for the future and the process of its practical application is expected to be challenging. Although having a limited disease area and small sample size, to the best of our knowledge, this is the first study to point out insufficient result reporting of clinical trials of regenerative medicine from the perspective of treatment modality. This report highlights an issue for discussing the path toward its translation through an overview of various factors in comparison with conventional treatment modalities.

Surface Phenotype Changes and Increased Response to Oxidative Stress in CD4+CD25high T Cells.

Conversion of CD4+CD25+FOXP3+ T regulatory cells (Tregs) from the immature (CD45RA+) to mature (CD45RO+) phenotype has been shown during development and allergic reactions. The relative frequencies of these Treg phenotypes and their responses to oxidative stress during development and allergic inflammation were analysed in samples from paediatric and adult subjects. The FOXP3lowCD45RA+ population was dominant in early childhood, while the percentage of FOXP3highCD45RO+ cells began increasing in the first year of life. These phenotypic changes were observed in subjects with and without asthma. Further, there was a significant increase in phosphorylated ERK1/2 (pERK1/2) protein in hydrogen peroxide (H2O2)-treated CD4+CD25high cells in adults with asthma compared with those without asthma. Increased pERK1/2 levels corresponded with increased Ca2+ response to T cell receptor stimulation. mRNA expression of peroxiredoxins declined in Tregs from adults with asthma. Finally, CD4+CD25high cells from paediatric subjects were more sensitive to oxidative stress than those from adults in vitro. The differential Treg sensitivity to oxidative stress observed in children and adults was likely dependent on phenotypic CD45 isoform switching. Increased sensitivity of Treg cells from adults with asthma to H2O2 resulted from a reduction of peroxiredoxin-2, -3, -4 and increased pERK1/2 via impaired Ca2+ response in these cells.

Trends in clinical trials for stroke by cell therapy: data mining ClinicalTrials.gov and the ICTRP portal site.

Definitive treatment of stroke constitutes an important thesis of regenerative medicine in the cerebrovascular field. However, to date, no cell therapy products for stroke are yet on the market. In this study, we examined the clinical research trends related to cell therapy products in the stroke field based on data obtained from the ClinicalTrials.gov website and International Clinical Trials Research Platform (ICTRP) portal site. These data do not offer results of clinical trials comprehensively but provide information regarding various attributes of planned clinical trials including work in progress. We selected 78 cell therapy studies related to the field of stroke treatment from ClinicalTrial.gov and ICTRP. These were analyzed according to, e.g., the reporting countries, origin (autologous or allogeneic), of cell used, cell types and source organs, the progress of translational phases, target phase of the disease (acute or chronic stroke), and route of administration. This analysis revealed a trend whereby in the acute phase, mesenchymal stem cells were administered intravenously at a relatively higher dose, whereas in the chronic phase a small number of cells were administered intracranially. Only two randomized controlled Phase III studies with over 100 patients are registered, but none of them has been completed. Thus, cell therapy against stroke appears to constitute a premature area compared with cartilage repair as assessed in our previous report. In addition, tracking by means of the ID number of each trial via PubMed revealed that 44% of clinical studies in this field have corresponding published results, which was also discussed.

Trends in clinical trials for articular cartilage repair by cell therapy.

Focal and degenerative lesions of articular cartilage greatly reduce the patient's quality of life. Various therapies including surgical treatment have been developed, but a definitive therapy is not yet known. Several cell therapy products have already been developed and are available in the market. In this study, we examined the clinical research trends related to cell therapy products in the cartilage repair field based on data obtained from the ClinicalTrial.gov website. Although this website does not provide comprehensive results of clinical trials, it offers information on prospective clinical trials, including work in progress, and thus allows for chronological analysis of the data. We selected 203 studies related to the field of cartilage regeneration from ClinicalTrial.gov. The results showed a shift in the clinical translational trend in utilized cells from cartilage- and bone marrow- to adipose tissue-based cells. Whereas the studies that used cartilage as the cell source included many phase III trials, fewer studies using bone marrow and adipose tissue cells progressed to phase III, suggesting that most clinical developments using the latter sources have not been successful so far. One product covered the entire period from the start of phase I to the completion of phase III, with a time to completion of more than 100 months. Translational trends in autologous chondrocyte implantation were also discussed. The use of ClinicalTrials.gov as the sole data source can yield a perspective view of the global clinical translational trends, which has been difficult to observe up to this point.

Induced Pluripotent Stem Cells: Global Research Trends.

The induced pluripotent stem cell (iPSC) was first described more than 10 years ago and is currently used in various basic science and clinical research fields. The aim of this report is to examine the trends in research using iPSCs over the last 10 years. The 2006-2016 PubMed database was searched using the MeSH term "induced pluripotent stem cells." Only original research articles were selected, with a total of 3323 articles. These were classified according to research theme into reprogramming, differentiation protocols for specific cells and/or tissues, pathophysiological research on diseases, and discovery of new drugs, and then the trends over the years were analyzed. We also focused on 232 research publications on the pathophysiological causes of diseases and drug discovery with impact factor (IF; Thomson Reuters) of six or more. The IF of each article was summed up by year, by main target disease, and by country, and the total IF score was expressed as trends of research. The trends of research activities of reprogramming and differentiation on specific cells and/or tissues reached maxima in 2013/2014. On the other hand, research on pathophysiology and drug discovery increased continuously. The 232 articles with IF ≥6 dealt with neurological, immunological/hematological, cardiovascular, and digestive tract diseases, in that order. The majority of articles were published from the United States, followed by Japan, Germany, and United Kingdom. In conclusion, iPSCs have become a general tool for pathophysiological research on disease and drug discovery.

Tyrphostin AG-related compounds attenuate H2O2-induced TRPM2-dependent and -independent cellular responses.

PURPOSE: TRPM2 is a Ca2+-permeable channel that is activated by H2O2. TRPM2-mediated Ca2+ signaling has been implicated in the aggravation of inflammatory diseases. Therefore, the development of TRPM2 inhibitors to prevent the aggravation of these diseases is expected. We recently reported that some Tyrphostin AG-related compounds inhibited the H2O2-induced activation of TRPM2 by scavenging the intracellular hydroxyl radical. In the present study, we examined the effects of AG-related compounds on H2O2-induced cellular responses in human monocytic U937 cells, which functionally express TRPM2. METHODS: The effects of AG-related compounds on H2O2-induced changes in intracellular Ca2+ concentrations, extracellular signal-regulated kinase (ERK) activation, and CXCL8 secretion were assessed using U937 cells. RESULTS: Ca2+ influxes via TRPM2 in response to H2O2 were blocked by AG-related compounds. AG-related compounds also inhibited the H2O2-induced activation of ERK, and subsequent secretion of CXCL8 mediated by TRPM2-dependent and -independent mechanisms. CONCLUSION: Our results show that AG-related compounds inhibit H2O2-induced CXCL8 secretion following ERK activation, which is mediated by TRPM2-dependent and -independent mechanisms in U937 cells. We previously reported that AG-related compounds blocked H2O2-induced TRPM2 activation by scavenging the hydroxyl radical. The inhibitory effects of AG-related compounds on TRPM2-independent responses may be due to scavenging of the hydroxyl radical.

The TAR-RNA binding protein is required for immunoresponses triggered by Cardiovirus infection.

LGP2 and MDA5 cooperate to detect viral RNA in the cytoplasm of Picornavirus-infected cells and activate innate immune responses. To further define regulatory components of RNA recognition by LGP2/MDA5, a yeast two-hybrid screen was used to identify LGP2-interacting proteins. The screening has identified the TAR-RNA binding protein (TRBP), which is known to be an essential factor for RNA interference (RNAi). Immuno-precipitation experiments demonstrated that TRBP interacted specifically with LGP2 but not with related RIG-I-like receptors, RIG-I or MDA5. siRNA knockdown experiments indicate that TRBP is important for Cardiovirus-triggered interferon responses, but TRBP is not involved in Sendai virus-triggered interferon response that is mediated mainly by RIG-I. To support functional interaction with LGP2, overexpressed TRBP increased Cardiovirus-triggered interferon promoter activity only when LGP2 and MDA5 are co-expressed but not MDA5 alone. Together, our findings illustrate a possible connection between an RNAi-regulatory factor and antiviral RNA recognition that is specifically required for a branch of the virus induced innate immune response.

Suppressive Effects of Glucose-Dependent Insulinotropic Polypeptide on Cardiac Hypertrophy and Fibrosis in Angiotensin II-Infused Mouse Models.

BACKGROUND: Activation of glucose-dependent insulinotropic polypeptide receptor (GIPR) has been shown to be protective against atherosclerosis. However, effects of GIP on the heart have remained unclear. To address this question, in vitro and in vivo experiments were conducted. METHODS AND RESULTS: In isolated mouse cardiomyocytes, GIPR mRNA was detected by reverse transcription-polymerase chain reaction, and GIP stimulation increased adenosine 3',5'-cyclic monophosphate production. In apolipoprotein E-knockout mice, infusion of angiotensin II (AngII; 2,000 ng·kg(-1)·min(-1)) significantly increased the heart weights, and co-administration of GIP (25 nmol·kg(-1)·day(-1)) reversed this increase (both P<0.01). In the left ventricular walls, GIP suppressed AngII-induced cardiomyocyte hypertrophy by 34%, apoptosis by 77%, and interstitial fibrosis by 79% (all P<0.01). Furthermore, GIP reduced AngII-induced expression of transforming growth factor-ß1 (TGF-ß1) and hypoxia inducible factor-1α. In wild-type mice, cardiac hypertrophy was induced by AngII to a lesser extent, and prevented by GIP. In contrast, GIP did not show any cardioprotective effect against AngII-induced cardiac hypertrophy in GIPR-knockout mice. In an in vitro experiment using mouse cardiomyocytes, GIP suppressed AngII-induced mRNA expression of B-type natriuretic peptide and TGF-ß1. CONCLUSIONS: It was demonstrated that cardiomyocytes represent a direct target of GIP action in vitro, and that GIP ameliorated AngII-induced cardiac hypertrophy via suppression of cardiomyocyte enlargement, apoptosis, and fibrosis in vivo. (Circ J 2016; 80: 1988-1997).

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation.

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation.

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells.

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30°C to 37°C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca(2+) ([Ca(2+)]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe(2+)-accumulation following pretreatment with FeSO4. Thus intracellular Fe(2+)-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe(2+)-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe(2+)-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37°C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe(2+)-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe(2+)-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2 channels by Fe(2+) may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe(2+) is released by heme degradation under intracerebral hemorrhage.

Inhibitory effects of AG490 on H2O2-induced TRPM2-mediated Ca(2+) entry.

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel that controls Ca(2+) signalling. The activation of Janus kinase 2 (Jak2) by oxidative stress is implicated in the production of inflammatory mediators. We found that AG490, a Jak2 inhibitor, had an inhibitory effect on H2O2-induced TRPM2 activation. The purpose of this study was to examine the underlying mechanisms of the inhibitory effects of AG490. Activation of TRPM2 in TRPM2-expressing human embryonic kidney 293 (TRPM2/HEK) cells or the human monocytic cell line U937 was monitored by fluorescence-based Ca(2+) imaging and patch-clamp techniques. Treatment with AG490 almost completely blocked H2O2-induced increase in intracellular Ca(2+) in TRPM2/HEK and U937 cells. In the patch-clamp study, AG490 inhibited the H2O2-evoked inward current but not the ADP-ribose-induced inward current in TRPM2/HEK cells. In contrast, Jak inhibitor 1 (pyridone 6) and staurosporine, both of which inhibit Jak2, had no effect on H2O2-induced increase in intracellular Ca(2+). Moreover, AG490 decreased intracellular reactive oxygen species level, which was measured by using a hydroperoxide-sensitive fluorescent dye, on incubation with H2O2. In the cell-free assay system, AG490 scavenged hydroxyl radicals but not H2O2. These findings indicate that AG490 significantly reduces H2O2-induced TRPM2 activation, presumably by scavenging hydroxyl radicals rather than Jak2-dependent mechanisms. Although transient receptor potential ankyrin 1 (TRPA1) channel is also activated by H2O2, the H2O2-induced Ca(2+) entry through TRPA1 was only slightly delayed by AG490. This validates the potential use of AG490, as one of the materials for characterizing the role of TRPM2 channels in pathological models.

A simple smart amplification assay for the rapid detection of human cytomegalovirus in the urine of neonates.

Human cytomegalovirus (CMV) is the most common cause of infection-related congenital abnormalities in neonates and the leading cause of non-hereditary sensorineural hearing loss in childhood. In addition, the number of low-birth-weight infants has recently increased, especially in Japan, in association with an increasing frequency of postnatal CMV infections transferred through raw breast milk. The increase in the number of congenital CMV and postnatal CMV infections in low-birth-weight infants requires rapid detection at the bedside in order to ensure a correct diagnosis and provide early anti-viral therapy. In this report, a simplified smart amplification (SMAP) method was developed to detect CMV in the urine of neonates. This method does not require DNA extraction, and the DNA amplification procedure is performed under isothermal conditions. Therefore, it takes only 60 min to detect CMV in a urine sample, and CMV DNA was rapidly detectable in symptomatic infants. In brief, this SMAP-based assay provides a simple, rapid and efficient method for detecting human CMV at the bedside.

Potentiated macrophage activation by acid sensing under low adiponectin levels.

Adiponectin can protect against inflammation; one of the mechanisms involves direct, inhibition of macrophages (MΦ). We postulated that adiponectin anti-sense transgenic (AsTg) mice raised in our laboratory are prone to inflammation because of systemic low adiponectin levels. The writhing response to acetic acid was utilized as an in vivo inflammatory model, and using Ca(2)(+), response to the acid was exploited in vitro to evaluate the function of resident peritoneal MΦ. The in vivo response to the acid was increased and the Ca(2)(+) response of MΦ was enhanced in AsTg mice, compared with those in wild type (WT) mice. In parallel with these enhanced responses, MΦ from AsTg mice augmented TNF-α and IL-6 mRNA expression. We further analyzed the enhancement in activity of MΦ from AsTg mice by acid sensing using specific inhibitors, amiloride for acid-sensing ion channels (ASICs) and KB-R7943 for Na(+)/Ca(2)(+) exchangers (NCXs). Our results indicated that in AsTg mice, the Ca(2)(+) response to the acid was facilitated in MΦ by a low threshold of ASIC1 and NCX1 molecules and the activity of these channel was possibly regulated by adiponectin.

Adiponectin enhances calcium dependency of mouse bladder contraction mediated by protein kinase Cα expression.

Adiponectin is an adipose tissue-secreted protein and is a multifunctional adipocytokine. However, the association of adiponectin with bladder contraction has not been investigated. In this study, the adiponectin-sense transgenic mouse (Adip-Sen mouse; age, 16-24 weeks; male) and age-matched controls (C57Bl mouse) were studied. The Adip-Sen mouse showed a significant increase in plasma adiponectin levels (56.2%; P < 0.01), compared with those in the C57Bl mouse, without affecting other lipid parameters. Isometric force development in bladder smooth muscle tissues were detected using an organ-bath system. Although carbachol (CCh)-induced (0.1-100 µM) time- and dose-dependent contractions in Adip-Sen mouse bladder were slightly enhanced, compared with those in the C57Bl mouse during a low range (0.3-1.0 µM) of CCh, differences could not be detected with other CCh concentrations. However, the reduction in contraction under Ca(2+)-replaced conditions was significantly different between Adip-Sen and C57Bl mice (94.1 and 66.3% of normal contraction, respectively; n = 5). A parameter of Ca(2+) sensitivity, the relation between intracellular Ca(2+) concentration and contraction, was increased in the Adip-Sen mouse, compared with that in the C57B1 mouse. This Ca(2+) dependency in the Adip-Sen mouse was reduced by a protein kinase C (PKC) inhibitor, but not by a Rho kinase inhibitor. Expression of the calcium-dependent isoform of PKC, PKCα, was increased in the Adip-Sen mouse bladder, and CCh-induced phosphorylation of PKCα was also enhanced, compared with those in the C57Bl mouse. In conclusion, adiponectin is associated with bladder smooth muscle contraction, which involves an increase in Ca(2+) dependency of contraction mediated by PKCα expression.

Neutrophil TRPM2 channels are implicated in the exacerbation of myocardial ischaemia/reperfusion injury.

AIMS: Transient receptor potential melastatin 2 (TRPM2) highly expressed in immunocytes is a Ca(2+)-permeable non-selective cation channel activated by oxidative stress. Myocardial ischaemia/reperfusion (I/R) injury is characterized by acute inflammation associated with the augmentation of oxidative stress. We hypothesized that TRPM2 is implicated in the exacerbation of myocardial I/R injury. METHODS AND RESULTS: Wild-type (Trpm2(+/+)) and Trpm2 knockout (Trpm2(-/-)) mice were subjected to ligation of the left main coronary artery followed by reperfusion. Myocardial infarction following I/R, but not ischaemia alone, was reduced more in Trpm2(-/-)mice than in Trpm2(+/+) mice and cardiac contractile functions were also improved in Trpm2(-/-)mice. TRPM2 was highly expressed in the polymorphonuclear leucocytes (PMNs) rather than in the heart. The number of neutrophils and myeloperoxidase (MPO) activity in the reperfused area following ischaemia was lowered in Trpm2(-/-) mice. When Trpm2(+)(/+) or Trpm2(-/-) PMNs were administered to the Trpm2(-/-) heart ex vivo through the perfusate or in vivo by iv injection, Trpm2(+)(/+) PMNs produced enlargement of the infarct size. Following in vitro regional I/R, a pharmacological inhibitor of TRPM2 reduced the infarct size. The combination of H(2)O(2) and leukotriene B(4) (LTB(4)) increased intracellular Ca(2+) concentration and their adhesion to endothelial cells in Trpm2(+)(/+) but not in Trpm2(-/-)PMNs. CONCLUSION: These findings indicate that neutrophil TRPM2 is implicated in the exacerbation of myocardial reperfusion injury. Accumulation of neutrophils in the reperfused area mediated by TRPM2 activation is likely to play a crucial role in myocardial I/R injury.

Low HCMV DNA copies can establish infection and result in significant symptoms in extremely preterm infants: a prospective study.

Breast milk (BM) is the main source of human cytomegalovirus (HCMV) infection. We examined whether the number of HCMV DNA copies in BM is related to HCMV infection in very low birth weight (VLBW) infants. We identified 11 pairs of VLBW infants and mothers. BM samples were collected every week until 10 weeks postpartum. Urine samples were collected from the infants within 1 week, at 6 to 8 weeks, at discharge, and whenever HCMV infection was suspected. HCMV DNA in BM was positive in 7 of 11 mothers and reached a peak at 4 to 5 weeks postpartum. Of the 11, 5 infants were determined to be infected from positive HCMV DNA in the urine, despite the fact that BM was used after being frozen. Of the five, four infected infants exhibited symptoms between 35 and 60 days of age. Symptomatic infants had longer stays and slower weight gain. The HCMV infection rate is high in very preterm infants. A new strategy to prevent HCMV infection other than freezing should therefore be established.

Impaired Ca²âº regulation of CD4⁺CD25⁺ regulatory T cells from pediatric asthma.

BACKGROUND: CD4(+)CD25(+) regulatory T (T(reg)) cells can control the allergic response to allergen, airway eosinophilia and airway hypersensitivity. We speculated that chronic inflammation persisting in asthma airways is dependent on abnormalities of these T(reg) cells. There are differences in the pathology of asthma in adults and children, and the airways of pediatric asthma are considered to be more naive than those of adults. Therefore, we analyzed the functionality of T(reg) cells in pediatric asthma and the relationship between T(reg) function and asthma symptoms. METHODS: The anergic state, which is one of the defining properties of T(reg), was analyzed by measuring intracellular Ca(2+) influx following T cell receptor (TCR) stimulation. FOXP3-positive cells and FOXP3 mRNA expression were measured by flow analysis and real-time PCR with the SYBR method, respectively. RESULTS: CD45RO(+) cells make up approximately 99% of CD4(+)CD25(high) T cells and 89% of CD4(+)CD25(low) T cells in human adult blood. The proportion of CD45RO(+) cells in CD4(+)CD25(+) (high + low) T cells from pediatric asthma was much smaller (about 56%). Interestingly, our data indicated that CD45RO(+) T(reg) cells from pediatric asthma aberrantly increased intracellular Ca(2+) concentrations following TCR activation compared with pediatric nonasthma controls. CONCLUSION: These impaired CD45RO(+) T(reg) cell functions were correlated with asthma symptoms. The correlation was observed in the group with a highly expressed atopic phenotype and longer duration of asthma. We suggest that chronic inflammation in pediatric asthma airways may be the result of impaired regulatory functions of CD45RO(+) T(reg) cells.

TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration.

Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca(2+)-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H(2)O(2)) evokes Ca(2+) influx through TRPM2 to activate Ca(2+)-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-kappaB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H(2)O(2)-induced Ca(2+) influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca(2+) influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.