Supportive care in the acute phase of Stevens-Johnson syndrome and toxic epidermal necrolysis: an international, multidisciplinary Delphi-based consensus.

BACKGROUND: Supportive care is the cornerstone of management of adult and paediatric Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). However, consensus on the modalities of supportive care is lacking. OBJECTIVES: Our aim in this international multicentric Delphi exercise was to establish a multidisciplinary expert consensus to standardize recommendations regarding supportive care in the acute phase of SJS/TEN. METHODS: Participants were sent a survey via the online tool SurveyMonkey, consisting of 103 statements organized into 11 topics: multidisciplinary team composition, suspect drug management, infection prevention, fluid resuscitation and prevention of hypothermia, nutritional support, pain and psychological distress management, management of acute respiratory failure, local skincare, ophthalmological management, management of other mucosa, and additional measures. Participants evaluated the level of appropriateness of each statement on a scale of 1 (extremely inappropriate) to 9 (extremely appropriate). The results were analysed according to the RAND/UCLA Appropriateness Method. RESULTS: Forty-five participants from 13 countries (on three continents) participated. After the first round, a consensus was obtained for 82.5% of the 103 initially proposed statements. After the second round, a final consensus was obtained for 102 statements. CONCLUSIONS: We have reached an international Delphi-based consensus on best supportive care practice for SJS/TEN. Our expert consensus should help guide physicians in treating patients with SJS/TEN and thereby improve short-term prognosis and the risk of sequelae.

Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer.

BACKGROUND: Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance. METHODS: We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining. RESULTS: The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients. CONCLUSIONS: miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

miR-320c regulates gemcitabine-resistance in pancreatic cancer via SMARCC1.

BACKGROUND: Gemcitabine-based chemotherapy is the standard treatment for pancreatic cancer. However, the issue of resistance remains unresolved. The aim of this study was to identify microRNAs (miRNAs) that govern the resistance to gemcitabine in pancreatic cancer. METHODS: miRNA microarray analysis using gemcitabine-resistant clones of MiaPaCa2 (MiaPaCa2-RGs), PSN1 (PSN1-RGs), and their parental cells (MiaPaCa2-P, PSN1-P) was conducted. Changes in the anti-cancer effects of gemcitabine were studied after gain/loss-of-function analysis of the candidate miRNA. Further assessment of the putative target gene was performed in vitro and in 66 pancreatic cancer clinical samples. RESULTS: miR-320c expression was significantly higher in MiaPaCa2-RGs and PSN1-RGs than in their parental cells. miR-320c induced resistance to gemcitabine in MiaPaCa2. Further experiments showed that miR-320c-related resistance to gemcitabine was mediated through SMARCC1, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. In addition, clinical examination revealed that only SMARCC1-positive patients benefited from gemcitabine therapy with regard to survival after recurrence (P=0.0463). CONCLUSION: The results indicate that miR-320c regulates the resistance of pancreatic cancer cells to gemcitabine through SMARCC1, suggesting that miR-320c/SMARCC1 could be suitable for prediction of the clinical response and potential therapeutic target in pancreatic cancer patients on gemcitabine-based therapy.

Post-acute phase and sequelae management of epidermal necrolysis: an international, multidisciplinary DELPHI-based consensus.

BACKGROUND: Long-term sequelae are frequent and often disabling after epidermal necrolysis (Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN)). However, consensus on the modalities of management of these sequelae is lacking. OBJECTIVES: We conducted an international multicentric DELPHI exercise to establish a multidisciplinary expert consensus to standardize recommendations regarding management of SJS/TEN sequelae. METHODS: Participants were sent a survey via the online tool "Survey Monkey" consisting of 54 statements organized into 8 topics: general recommendations, professionals involved, skin, oral mucosa and teeth, eyes, genital area, mental health, and allergy workup. Participants evaluated the level of appropriateness of each statement on a scale of 1 (extremely inappropriate) to 9 (extremely appropriate). Results were analyzed according to the RAND/UCLA Appropriateness Method. RESULTS: Fifty-two healthcare professionals participated. After the first round, a consensus was obtained for 100% of 54 initially proposed statements (disagreement index < 1). Among them, 50 statements were agreed upon as 'appropriate'; four statements were considered 'uncertain', and ultimately finally discarded. CONCLUSIONS: Our DELPHI-based expert consensus should help guide physicians in conducting a prolonged multidisciplinary follow-up of sequelae in SJS-TEN.

Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells.

Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma B*CaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma B*CaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma B*CaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma B*CaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC-dependent signaling systems.

Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-beta. Possible existence of "vascular natriuretic peptide system".

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is thus far known to be distributed mainly in the central nervous system and is considered to act as a neuropeptide, in contrast to atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which act as cardiac hormones. Recently, we and others have demonstrated that the ANP-B receptor, which is selectively activated by CNP, is localized not only in the central nervous system but in peripheral tissues, including blood vessels. This finding has made us speculate regarding the peripheral production of CNP. In the present study, cultured endothelial cells were examined for CNP production by RIA and Northern blot analysis. CNP-like immunoreactivity was detected in the conditioned media of endothelial cells. Northern blot analysis detected CNPmRNA with a size of 1.2 kb. In addition, transforming growth factor (TGF)-beta, one of the key growth factors for vascular remodeling, markedly stimulated the expression of CNPmRNA and induced a tremendous increase in CNP secretion. We could also detect CNP transcript in the bovine thoracic aorta using the reverse transcription-polymerase chain reaction method. The present study demonstrates the endothelial production of CNP and suggests that a member of the natriuretic peptide family may act as a local regulator in vascular walls. Since evidence for the pathophysiological importance of the vascular renin-angiotensin system has been accumulating and the natriuretic peptide system is known to be antagonistic to the renin-angiotensin system, the possible existence of "vascular natriuretic peptide system" may prove to be of physiological and clinical relevance.

Risk factors for graft dysfunction after adult-to-adult living donor liver transplantation.

The aim of this study was to investigate the risk factors for graft dysfunction after adult-to-adult living donor liver transplantation (LDLT). Thirty-nine adults with chronic cirrhosis underwent LDLT between 1999 and 2004. Their postoperative courses were uneventful with no vascular or bile duct complications early after LDLT, except one mild hepatic artery stenosis. The preoperative MELD scores were significantly higher in the failed graft group (n=5) than the functioning graft group (n=34; P=.004), while the graft liver weight/standard liver volume ratio was similar between these groups. We concluded that a high preoperative MELD score was associated with postoperative graft failure and that graft size had little impact on graft outcome. Although large grafts would seem intuitively more suitable for sick recipients, we did not show a benefit among this cohort; the MELD score was the best predictor, a finding that is also most consistent with donor safety.

Alpha-fetoprotein mRNA detection in peripheral blood for prediction of hepatocellular carcinoma recurrence after liver transplantation.

The aim of this study was to assess the value of alphafeto protein (AFP) mRNA-expressing cells detected in peripheral blood for predicting tumor recurrence after living donor liver transplantation (LDLT) in patients with hepatocellular carcinoma (HCC). The test group consisted of 25 patients who underwent LDLT for end-stage liver disease with HCC while the control group consisted of 37 living donors. Quantitative real-time reverse-transcriptase polymerase chain reaction was used for detection of AFP mRNA-expressing cells in peripheral blood. Nine (36%) of 25 patients developed tumor recurrences (four lung; one liver; one peritoneum; two bone; one adrenal gland) during the follow-up period. Perioperatively, AFP mRNA was positive in peripheral blood of eight patients (32.0%) but only in 1 (2.7%) of the control. Preoperative AFP mRNA was positive in three cases. Univariate analyses revealed that preoperative and perioperative AFP mRNA and microscopical vascular invasion were the significant predictors for HCC recurrence (P = .007, .037, and .005, respectively). In the patients with HCC exceeding Milan criteria (n = 15), the presence of AFP mRNA-positive cells in the peripheral blood correlated significantly with HCC recurrence (P = .033). We concluded that the presence of AFP mRNA-expressing cells could be a useful predictor of HCC recurrence in liver transplant patients.

Optical imaging of the propagation patterns of neural responses in the rat sensory cortex: comparison under two different anesthetic conditions.

Although many studies have reported the influence of anesthetics on the shape of somatic evoked potential, none has evaluated the influence on the spatio-temporal pattern of neural activity in detail. It is practically impossible to analyze neural activities spatially, using conventional electrophysiological methods. Applying our multiple-site optical recording technique for measuring membrane potential from multiple-sites with a high time resolution, we compared the spatio-temporal pattern of the evoked activity under two different anesthetic conditions induced by urethane or α-chloralose. The somatic cortical response was evoked by electrical stimulation of the hindlimb, and the optical signals were recorded from the rat sensorimotor cortex stained with a voltage-sensitive dye (RH414). The evoked activity emerged in a restricted area and propagated in a concentric manner. The spatio-temporal pattern of the evoked activity was analyzed using isochrone maps. There were significant differences in the latency and propagation velocity of the evoked activity, as well as the full width at half maximum of optical signal between the two anesthetic conditions. Differences in the amplitude and the slope of the rising phase were not significant.

Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells--evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells.

We previously demonstrated that C-type natriuretic peptide (CNP), originally isolated from the porcine brain, is produced by endothelial cells and proposed that CNP can exert local control over vascular tone and growth as a local regulator from endothelial cells. Since cytokines play pivotal roles in the control of vascular tone and structure, we have examined effects of various cytokines on CNP secretion from endothelial cells using the specific radioimmunoassay for CNP. While interleukin (IL)-2 had no significant effect on CNP secretion, IL-1 alpha, IL-1 beta and tumor necrosis factor (TNF)-alpha stimulated CNP secretion in a time- and dose-dependent manner. Among them, TNF-alpha, one of the key mediators for inflammation and vascular remodeling, induced more than two orders of magnitude increase in CNP secretion. In addition, lipopolysaccharide (LPS) potently stimulated CNP secretion. These results indicate that IL-1, TNF-alpha and LPS, the endotoxin itself, can regulate local vascular tone and growth through the activation of CNP secretion from endothelial cells. Therefore, CNP could be of clinical relevance as an autocrine/paracrine regulator from endothelial cells for systemic and local cytokine-associated disorders, such as endotoxin shock and atherosclerosis.

C-type natriuretic peptide (CNP) in rats and humans.

We have established a specific radioimmunoassay (RIA) for C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, and have elucidated its tissue distribution and molecular form. In rats, high concentrations of CNP-like immunoreactivity (-LI) were detected in the anterior lobe (19.8 +/- 8.6 pmol/g) and neurointermediate lobe (4.64 +/- 0.74 pmol/g) of the pituitary gland. CNP-LI was present throughout the brain with its high concentrations in the hypothalamus and cerebellum. Small amounts of CNP-LI were also detected in the lower part of gastrointestinal tract and the kidney. However, no significant amount of CNP-LI was present in other organs including the heart. Considerable amounts of CNP-LI were detected throughout the human brain. High performance-gel permeation chromatography coupled with the RIA detected two peaks of CNP-LI in the rat brain; CNP and presumably its N-terminally elongated form with 53 amino-acid residues, CNP-53. These findings indicate that the tissue distribution and processing pattern of CNP are clearly different from those of atrial natriuretic peptide and brain natriuretic peptide and suggest possible roles of CNP as a neurotransmitter or neuromodulator rather than as a cardiac hormone.

Glucocorticoids modulate CD28 mediated pathways for interleukin 2 production in human T cells: evidence for posttranscriptional regulation.

In T cells stimulated through the T cell receptor (TCR), both cyclosporine (CsA) and glucocorticoids (GC) inhibit the transcription of the IL-2 gene. In these cells costimulation via the CD28 cell surface molecule further increases the transcription of IL-2 and stabilizes its mRNA, resulting in a 20-30 fold induction in IL-2 production. This pathway is relatively resistant to the inhibitory effect of CsA. In this study, we asked whether GC interfere with CD28-mediated costimulatory signals for T cell activation. Primary human T cells or Jurkat T cells were stimulated with anti-CD28 and phorbol myristate acetate (PMA) in the presence of dexamethasone (Dex, 10(-10)-10(-5) M). Dex inhibited both the mRNA for IL-2 and IL-2 production in a dose-dependent fashion (minimum effective dose 10(-9) M). In similar experiments employing anti-CD3 mAb and PMA, a 7-20 fold higher concentration of Dex was required to obtain comparable inhibition. To determine if transcriptional modulation is occurring, Jurkat T cells were transfected with a plasmid containing the IL-2 promoter linked to the chloramphenicol acetyl transferase reporter gene. Following stimulation with ionomycin and PMA, high doses (10(-6) M) of Dex inhibited the activity of the IL-2 promoter (approximately 50% inhibition). However, in the presence of anti-CD28 mAb, this promoter became resistant to Dex (< or = 10% inhibition). These results suggest that GC inhibit accessory pathways for IL-2 production via CD28 by predominantly posttranscriptional mechanisms. Inhibition of the CD28 pathway may be an important mechanism for the T cell directed immunosuppressive effects of low-to-moderate doses of GC.

A monoclonal antibody to C-type natriuretic peptide--preparation and application to radioimmunoassay and neutralization experiment.

C-type natriuretic peptide (CNP), the third member of natriuretic peptides, has recently been discovered from the porcine brain. Using a polyclonal antiserum to CNP, we demonstrated that CNP-like immunoreactivity (CNP-LI) is present mainly in the central nervous system. Recently, however, we have discovered the production and secretion of CNP in vascular endothelial cells. These observations suggested that CNP may act not only as a neuropeptide but also as a local regulator of vascular tone or growth. In order to further clarify the pathophysiological significance of CNP, we aimed at the preparation of a monoclonal antibody to CNP. A monoclonal antibody to CNP, KY-CNP-I, has been produced. This monoclonal antibody belongs to the immunoglobulin G1 subclass and has high affinity for CNP. Using this monoclonal antibody, we established a specific radioimmunoassay (RIA) for CNP. The RIA detected CNP-LI in rat brain extracts and culture media conditioned with bovine endothelial cells. In addition, the pretreatment of cultured aortic smooth muscle cells with KY-CNP-I attenuated cyclic GMP production induced by CNP in vitro. The preadministration of KY-CNP-I to rats also attenuated plasma cyclic GMP increase after intravenous injection of CNP in vivo. These results indicate that this monoclonal antibody is a useful tool to clarify the pathophysiological role of CNP as a neuropeptide and as a local vascular regulator.

Changes in atrial natriuretic peptide and brain natriuretic peptide associated with hypobaric hypoxia-induced pulmonary hypertension in rats.

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) both have diuretic, natriuretic, and hypotensive effects, and both are involved in cardiovascular homeostasis as cardiac hormones. To study the effects of HHE on the natriuretic peptide synthesis system, 170 male Wistar rats were housed in a chamber at the equivalent of the 5500-m altitude level for 1-12 weeks. After 1 week of HHE, pulmonary arterial pressure was significantly raised, and the ratio of left ventricle plus septum over right ventricle of the heart showed a significant decrease (compared with those of ground-level control rats). In both ventricular tissues, the expression of ANP messenger (m)RNA and BNP mRNA increased after exposure to HHE. The amounts of ANP and BNP had decreased significantly in right atrial tissue at 12 weeks of HHE (compared with those of the controls), whereas in ventricular tissues at the same time point, both levels had increased significantly. In in situ hybridization and immunohistochemical studies, the staining of the mRNAs for ANP and BNP and of ANP and BNP themselves was more intense in both ventricular tissues after exposure to HHE than before (i.e., in the controls). The results suggest that, in response to HHE, the changes in ventricular synthesis are similar for ANP and BNP. These changes may play a role in modulating pulmonary hypertension in HHE. However, under our conditions, pulmonary hypertension increased progressively throughout the HHE period.

C-type natriuretic peptide in chronic renal failure and its action in humans.

We have previously reported that C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is produced in vascular endothelial cells and acts as an endothelium-derived relaxing peptide. To clarify the clinical significance of CNP in renal disorders, we examined the plasma level of CNP in patients with various cardiovascular diseases, including chronic renal failure (CRF) patients who were under hemodialysis therapy. We also investigated biological effects of intravenously-administered CNP (0.43 nmol/kg) by bolus injection from the peripheral vein in healthy volunteers and measured systemic hemodynamic variables, plasma levels of CNP, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cGMP, aldosterone and also urine volume, urinary excretions of sodium, potassium, chloride and cGMP. The plasma CNP levels in healthy humans (N = 13) was 1.4 +/- 0.6 fmol/ml. In CRF patients, the plasma CNP significantly increased up to 3.0 +/- 1.1 fmol/ml. The administration of CNP elicited significant increase of plasma cGMP level (from 4.77 +/- 1.25 to 8.33 +/- 1.59 pmol/ml 15 min after the administration) and of urinary cGMP excretion (from 30.7 +/- 4.3 to 74.9 +/- 13.4 nmol/30 min). Intravenously-administered CNP exerted significant diuretic (% increase: +117 +/- 85.0), natriuretic, kalliuretic and chloriuretic actions with the increase of endogenous creatinine clearance. CNP also elicited significant hypotensive actions (delta BPs/delta BPd: -4.3 +/- 1.3/-4.1 +/- 1.0 mm Hg) with the concomitant increase of heart rate (+7.6 +/- 2.6 bpm). Plasma aldosterone concentration significantly decreased from 45.4 +/- 2.3 to 35.4 +/- 4.9 pg/ml 30 minutes after the administration. Taken together, these results suggest a role for CNP in human renal function.