The role of clonal haematopoiesis in cardiovascular diseases: epidemiology and experimental studies.

Clonal haematopoiesis results from acquired mutations in haematopoietic stem and progenitor cells (HSPCs). These mutations can confer the HSPC with a competitive advantage, leading to their clonal expansion within the limiting bone marrow niche. This process is often insufficient to produce a haematologic malignancy; however, the expanding HSPC clones increasingly give rise to progeny leucocytes whose phenotypes can be altered by the somatic mutations that they harbour. Key findings from multiple human studies have shown that clonal haematopoiesis in the absence of overt haematologic alterations is common amongst the ageing population and associated with mortality and cardiovascular disease. Key findings from experimental studies have provided evidence for a causative role for clonal haematopoiesis in cardiovascular diseases, and aspects of these mechanisms have been elucidated. Whilst our understanding of the impact and biology of clonal haematopoiesis is in its infancy, analyses of some of the most commonly mutated driver genes suggest promising clinical scenarios involving the development of personalized therapies with immunomodulatory drugs that exploit the perturbation caused by the particular mutation. Herein, we review the accumulating epidemiological and experimental evidence, and summarize our current understanding of the importance of clonal haematopoiesis as a new causal risk factor for atherosclerotic cardiovascular disease and heart failure.

Talbot-Lau interferometry-based x-ray imaging system with retractable and rotatable gratings for nondestructive testing.

We develop an x-ray imaging system based on Talbot-Lau interferometry equipped with a mechanical structure for retracting and rotating gratings from the optical axis, which enables not only x-ray phase contrast imaging but also conventional x-ray imaging with high-magnification such as microcomputed tomography (µCT). We investigate the characterization of carbon fiber reinforced plastic (CFRP) laminates using this apparatus. Microcracks and fiber orientations are visualized in the dark-field images. Compared with the obtained µCT images, the relationship between the CFRP microstructures and the contrasts in the dark-field images are recognizable.

Novel reuterin-related compounds suppress odour by periodontopathic bacteria.

OBJECTIVE: Halitosis is caused by volatile sulphur compounds including methyl mercaptan (CH3 SH) in the oral cavity and is a serious problem that limits interpersonal social communication. The aim of study was to evaluate the effects of reuterin-related compounds (RRCs) on halitosis-related periodontopathic bacteria in vitro. MATERIALS AND METHODS: RRC-01, RRC-02 and RRC-03 (32 and 64 µg ml-1 ) in culture media containing Fusobacterium nucleatum JCM8523 and Porphyromonas gingivalis ATCC33277 were used. The effects of RRCs on CH3 SH production and detectable odour by F. nucleatum and P. gingivalis were examined by CH3 SH production assay and organoleptic test, respectively. The number of bacterial cells was also measured using an ATP assay. In P. gingivalis treated with RRCs, the expression of mgl gene, which is responsible for CH3 SH production, was examined by qRT-PCR. RESULTS: CH3 SH production and the score of detectable odour from F. nucleatum and P. gingivalis culture media containing RRCs were significantly lower than that without RRCs (P < 0.05). The expression of mgl gene in P. gingivalis was significantly downregulated by RRC-01 (P < 0.01), but not by RRC-02 or RRC-03. CONCLUSIONS: RRCs are potent oral care products for preventing halitosis via reducing CH3 SH production.

Pim-2 kinase is an important target of treatment for tumor progression and bone loss in myeloma.

Pim-2 kinase is overexpressed in multiple myeloma (MM) cells to enhance their growth and survival, and regarded as a novel therapeutic target in MM. However, the impact of Pim-2 inhibition on bone disease in MM remains unknown. We demonstrated here that Pim-2 expression was also upregulated in bone marrow stromal cells and MC3T3-E1 preosteoblastic cells in the presence of cytokines known as the inhibitors of osteoblastogenesis in MM, including interleukin-3 (IL-3), IL-7, tumor necrosis factor-α, transforming growth factor-ß (TGF-ß) and activin A, as well as MM cell conditioned media. The enforced expression of Pim-2 abrogated in vitro osteoblastogenesis by BMP-2, which suggested Pim-2 as a negative regulator for osteoblastogenesis. Treatment with Pim-2 short-interference RNA as well as the Pim inhibitor SMI-16a successfully restored osteoblastogenesis suppressed by all the above inhibitory factors and MM cells. The SMI-16a treatment potentiated BMP-2-mediated anabolic signaling while suppressing TGF-ß signaling. Furthermore, treatment with the newly synthesized thiazolidine-2,4-dione congener, 12a-OH, as well as its prototypic SMI-16a effectively prevented bone destruction while suppressing MM tumor growth in MM animal models. Thus, Pim-2 may have a pivotal role in tumor progression and bone loss in MM, and Pim-2 inhibition may become an important therapeutic strategy to target the MM cell-bone marrow interaction.

Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) has an essential role in multiple signaling pathways, including cell proliferation and migration, through extracellular ligand binding and subsequent activation of its intracellular tyrosine kinase (TK) domain. The non-small cell lung cancer (NSCLC)-associated EGFR mutants, L858R and G719S, are constitutively active and oncogenic. They display sensitivity to TK inhibitors, including gefitinib and erlotinib. In contrast, the secondary mutation of the gatekeeper residue, T790M, reportedly confers inhibitor resistance on the oncogenic EGFR mutants. In this study, our biochemical analyses revealed that the introduction of the T790M mutation confers gefitinib resistance on the G719S mutant. The G719S/T790M double mutant has enhanced activity and retains high gefitinib-binding affinity. The T790M mutation increases the ATP affinity of the G719S mutant, explaining the acquired drug resistance of the double mutant. Structural analyses of the G719S/T790M double mutant, as well as the wild type and the G719S and L858R mutants, revealed that the T790M mutation stabilizes the hydrophobic spine of the active EGFR-TK conformation. The Met790 side chain of the G719S/T790M double mutant, in the apo form and gefitinib- and AMPPNP-bound forms, adopts different conformations that explain the accommodation of these ligands. In the L858R mutant structure, the active-site cleft is expanded by the repositioning of Phe723 within the P-loop. Notably, the introduction of the F723A mutation greatly enhanced the gefitinib sensitivity of the wild-type EGFR in vivo, supporting our hypothesis that the expansion of the active-site cleft results in enhanced gefitinib sensitivity. Taken together, our results provide a structural basis for the altered drug sensitivities caused by distinct NSCLC-associated EGFR mutations.

Clear association between serum levels of adipokines and T-helper 17-related cytokines in patients with psoriasis.

BACKGROUND: Psoriasis represents one of the T-helper (Th)17-mediated autoimmune diseases, and has been shown to be associated with metabolic syndrome (MetS). It has been reported that some adipokines and Th17-related cytokines are altered in patients with psoriasis. AIM: To examine the relationship between levels of adipokines and Th17-related cytokines in the serum of patients with psoriasis compared with healthy controls. METHODS: We enrolled 30 patients with psoriasis and 30 normal controls in the study, and used ELISA to measure serum adipokines and Th17-related cytokines. The association between each adipokine and each cytokine was determined using Pearson correlation analysis. Multiple regression analysis using all adipokines and Th17-related cytokines as covariates was also performed. RESULTS: Pearson correlation analysis showed a strong positive association between chemerin and resistin levels and between adiponectin and high molecular weight adiponectin in normal controls. By contrast, in patients with psoriasis, resistin levels were significantly positively associated with tumour necrosis factor-α, while there was a strong negative association between retinol binding protein-4 and interleukin (IL)-6 levels. Interestingly, a marked positive correlation between IL-22 and adiponectin was also found in patients with psoriasis. Leptin levels correlated positively with IL-6 in patients with psoriasis, but this did not reach significance. The correlations identified by the multiple regression analyses were almost identical to those from the Pearson analyses. CONCLUSIONS: These data suggest that distinct interaction between adipokines and Th17 cytokines is involved in the pathogenesis of psoriasis.

Extracorporeal membrane oxygenation following pediatric cardiac surgery: development and outcomes from a single-center experience.

Extracorporeal membrane oxygenation (ECMO) has emerged as an effective mechanical support following cardiac surgery with respiratory and cardiac failure. However, there are no clear indications for ECMO use after pediatric cardiac surgery. We retrospectively reviewed medical records of 76 pediatric patients [mean age, 10.8 months (0-86); mean weight, 5.16 kg (1.16-16.5)] with congenital heart disease who received ECMO following cardiac surgery between January 1997 and October 2010. Forty-five patients were treated with an aggressive ECMO approach (aggressive ECMO group, April 2005-October 2010) and 31 with a delayed ECMO approach (delayed ECMO group, January 1997-March 2005). Demographics, diagnosis, operative variables, ECMO indication, and duration of survivors and non-survivors were compared. Thirty-four patients (75.5%) were successfully weaned from ECMO in the aggressive ECMO group and 26 (57.7%) were discharged. Conversely, eight patients (25.8%) were successfully weaned from ECMO in the delayed ECMO group and two (6.5%) were discharged. Forty-five patients with shunted single ventricle physiology (aggressive: 29 patients, delayed: 16 patients) received ECMO, but only 15 (33.3%) survived and were discharged. The survival rate of the aggressive ECMO group was significantly better when compared with the delayed ECMO group (p<0.01). Also, ECMO duration was significantly shorter among the aggressive ECMO group survivors (96.5 ± 62.9 h, p<0.01). Thus, the aggressive ECMO approach is a superior strategy compared to the delayed ECMO approach in pediatric cardiac patients. The aggressive ECMO approach improved our outcomes of neonatal and pediatric ECMO.

Pediatric perfusion in Japan: 2010 practice survey.

We report here Japan's first pediatric perfusion survey. It covers practices from January 2007 through December 2009. Of the 70 congenital heart centers contacted, 53 (76%) completed the survey. They reported performing 3,379 pediatric cardiopulmonary bypass (CPB) procedures in 2009, 3,408 in 2008, and 3,358 in 2007.Twenty-eight percent of all centers used CPB circuits with a priming volume between 151-200 ml. All centers used pre-bypass ultrafiltration and only 6% used retrograde autologous priming. A biomaterial-coated circuit was used by 78% of the centers, a roller pump as the arterial pump by 91%, vacuum-assisted venous drainage by 39%, dilutional ultrafiltration by 48%, and modified ultrafiltration at the end of the procedure by 30%. A regional oxygen saturation monitor was used by 69% of the centers and high flow (150-200 ml/kg/min) management with alpha-stat blood gas control was standard during moderate to normothermic CPBs. Crystalloid cardioplegia solution was used as myocardial protection by 56% of the centers, electronic recording of monitoring data by 51%. The centers performed 98 pediatric extracorporeal membrane oxygenation procedures in 2007, 109 in 2008, and 119 in 2009; 58% of the centers used a centrifugal pump. This survey provides a description of the current practice in Japan. Future surveys will identify trends and rate of change in practice.

Carbenoxolone alters the morphology of adipose tissues and downregulates genes involved in adipogenesis, glucose transport and lipid metabolism in high-fat diet-fed mice.

Glucocorticoid (GC) excess promotes adipose tissue accumulation, and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) plays an important role in the local amplification of GC. Therefore, in this study, we investigated the effects of carbenoxolone (CBX), an 11ß-HSD1 inhibitor, on morphological changes in visceral fat, and the expression of genes involved in adipogenesis and lipid metabolism in high-fat (HF) diet-fed mice. Mice were fed a HF diet from 5 weeks of age. At 10 weeks of age, the mice received an intraperitoneal injection of CBX or vehicle every day for 2 weeks. CBX decreased body weight and visceral fat mass, and improved insulin sensitivity in HF-fed mice. This was accompanied by reduced adipocyte size and a decrease in large-sized adipocytes in visceral fat. The expression of adipogenesis (PPARγ and C/EBPα), glucose transport (GLUT4) and lipid metabolism (LPL, ATGL, and HSL)-related genes were suppressed in CBX mice. CBX treatment induced beneficial morphological changes in visceral fat and decreased the expression of adipogenesis, glucose transport and lipid metabolism-related genes. These findings reveal a potential mechanism underling the effects of CBX on reduced fat accumulation and improved insulin sensitivity.

Diminished regulatory T cells in cutaneous lesions of thymoma-associated multi-organ autoimmunity: a newly described paraneoplastic autoimmune disorder with fatal clinical course.

Thymoma-associated multi-organ autoimmunity is a rare, autoimmune disease that causes colitis, liver dysfunction and cutaneous graft-versus-host (GVH)-like skin damage. This paraneoplastic autoimmune disorder may be due to inadequate T cell selection in the tumour environment of the thymus. Although sporadic case reports have revealed its clinical features, little is known about its pathological mechanism. By comparing the skin-infiltrating T cell subsets with those of GVH disease (GVHD) and other inflammatory skin diseases, we sought to elucidate the pathological mechanism of thymoma-associated multi-organ autoimmunity. Histopathological and immunohistochemical analysis of skin biopsies was performed for three patients with thymoma-associated multi-organ autoimmunity. Histopathological findings of thymoma-associated multi-organ autoimmunity were indistinguishable from those of patients with acute GVHD, although the aetiologies of these diseases are completely different. The frequency of regulatory T cells (T(regs)) is reduced in cutaneous lesions and CD8+ cytotoxic T lymphocytes that massively infiltrate into the epidermis of patients with thymoma-associated multi-organ autoimmunity. Additionally, the ratio of T helper type 17 (Th17) cells to CD4+ cells in patients with thymoma-associated multi-organ autoimmunity and acute GVHD was higher than that in healthy controls, but similar to that in psoriasis vulgaris patients. Similarity of the skin-infiltrating T cell subsets with those of acute GVHD suggested that skin damage in patients with thymoma-associated multi-organ autoimmunity might be induced by self-reactive cytotoxic T lymphocytes under the diminished suppressive capacity of T(regs).

Altered gene expressions of ghrelin, PYY, and CCK in the gastrointestinal tract of the hyperphagic intrauterine growth restriction rat offspring.

Intrauterine growth restriction (IUGR) is associated with a substantially greater incidence of metabolic syndrome in adulthood. Animal studies have shown that IUGR offspring are hyperphagic during the early postnatal period and therefore exhibit obesity. The molecular mechanisms underlying food intake regulation in the gastrointestinal tract have not been clarified in IUGR. In the present study, we utilized a rat model of IUGR by restricting the food intake of the mother (50% of the normal intake, ad libitum; FR group) from day 7 of gestation until delivery. Pups from undernourished mothers were fostered by control mothers. We examined the food intake and assessed the gene expressions of ghrelin, peptide YY (PYY), and cholecystokinin (CCK) in the alimentary tract of male newborns (postnatal day1) and adult offspring (age, 7 months). Compared to the offspring whose mothers received the standard diet ad libitum (CON offspring), FR offspring were hyperphagic from the weaning time until the end of the experiment, and resulted in a heavier final weight. Both newborn and adult FR offspring had higher ghrelin gene expression in the stomach and higher ghrelin plasma levels than did the controls. Although the gastrointestinal gene expressions and plasma levels of the anorexic peptides, PYY and CCK, were elevated in the FR newborns, they decreased in the FR adults. Our findings suggest that the altered gene expressions of orexigenic and anorexigenic gut peptides in the gastrointestinal tract in the maternal undernutrition-induced IUGR offspring provide a potential mechanism to explain hyperphagia and obesity seen in these offspring.

Induction of antibacterial activity in larvae of the blowfly Lucilia sericata by an infected environment.

Maggot debridement therapy (MDT) is a method for the treatment of intractable, infected and necrotic wounds. In MDT, sterile larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) are applied to infected wounds, where they exert antibacterial effects. Once the larvae are placed in the wound, they are no longer germ-free. This study analysed the influence of infected environments on larval antibacterial activities. Sterile larvae were mixed in a test tube containing a bacterial suspension of Staphylococcus aureus or Pseudomonas aeruginosa, transferred to liver puree agar, and incubated at 25 °C for set periods. To collect the larval extracts, the incubated larvae were transferred to a test tube containing phosphate buffered saline (PBS), cut into multiple pieces with scissors, and centrifuged. The supernatant was used to test antibacterial activities. The results showed that infected larvae had better antibacterial capacities than sterile larvae. Antibacterial activities were induced by pretreatment with a single bacterial species, S. aureus or P. aeruginosa, within 24 h and 12 h, respectively, and disappeared after 36 h. The activities were effective against S. aureus, but not against P. aeruginosa. This natural infection model is very similar to the clinical wound context in MDT and will be a powerful tool with which to study the antibacterial activities of L. sericata larvae in MDT.

Effects of maternal high-fat diet on serum lipid concentration and expression of peroxisomal proliferator-activated receptors in the early life of rat offspring.

Peroxisomal proliferator-activated receptors (PPARs) play an important role in the regulation of lipid metabolism. The aim of this study was to investigate the effects of a maternal high-fat (HF) diet on serum lipid concentration and PPAR gene expression in liver and adipose tissue in the early life of the rat offspring. Female Sprague-Dawley rats were fed either an HF or control (CON) diet 6 weeks before mating and throughout gestation and lactation. Blood and tissue samplings of male offspring were carried out at birth or weaning. Birth weights were similar and serum triglyceride (TG) and nonesterified fatty acid (NEFA) levels showed no significant difference between HF and CON newborns, despite greatly increased hepatic PPARα mRNA expression in the HF newborns (p<0.05). Both HF newborns and weanlings revealed significantly decreased hepatic PPARγ expression compared with controls (p<0.0001). Hepatic PPARα expression in the HF weanlings was reduced markedly compared with CON weanlings (p<0.0001) and showed a negative correlation with serum TG levels (r=-0.743, p<0.05). However, epididymal expression of PPARγ in the HF weanlings was upregulated significantly compared with controls (p<0.05) and demonstrated a positive correlation with epididymal fat mass (r=0.733, p<0.05). These were accompanied by obesity as well as a rise in serum TG by 79% (p<0.05) and NEFA concentration by 36% (p<0.05) in these HF weanlings. Our findings suggest that maternal HF diet leads to alterations in PPAR gene expression in the weanling offspring, which is associated with the disturbed lipid homeostasis.

Síndrome de Cowden: caso clínico y revisión de la literatura / Cowden´s syndrome: a clinical case and review of the literature

El síndrome de Cowden es una enfermedad autosómica dominante caracterizada por múltiples tumores hamartomatosos, cuyos hallazgos mucocutáneos benignos comotriquilemomas, queratosis acrales y pápulas o papilomas bucales son frecuentemente la clave para el diagnóstico. Los dos sitios de compromiso tumoral (benigno y maligno)más importantes son tiroides y mama. La enfermedad es debida a mutaciones en el gen PTEN. Se describe un caso clínico correspondiente a esta entidad.

Constitutive activation and targeted disruption of signal transducer and activator of transcription 3 (Stat3) in mouse epidermis reveal its critical role in UVB-induced skin carcinogenesis.

In this study, the potential role of Stat3 in UVB-induced skin carcinogenesis was examined using skin-specific gain and loss of function transgenic mice, that is, K5.Stat3C and K5Cre.Stat3(fl/fl) mice, respectively. The epidermis of Stat3-deficient mice was highly sensitive to UVB-induced apoptosis, whereas the epidermis of K5.Stat3C mice was more resistant to UVB-induced apoptosis. In particular, the status of Stat3 influenced the survival of ultraviolet-photoproduct cells, including those located in the hair follicles. K5.Stat3C mice exhibited significantly increased epidermal proliferation and hyperplasia in response to UVB irradiation, whereas Stat3-deficient mice showed reduced epidermal proliferation and hyperplasia. Expression of target genes regulated by Stat3, such as cyclin D1 and Bcl-x(L), was increased in epidermis of both control and UVB-irradiated K5.Stat3C mice, and downregulated in epidermis of both control and UVB-irradiated K5Cre.Stat3(fl/fl) mice. Following UVB irradiation, the formation of skin tumors in K5.Stat3C mice was accelerated and both the incidence and multiplicity of skin tumors were significantly greater than wild-type controls. In contrast, Stat3-deficient mice were resistant to UVB skin carcinogenesis. These results show that Stat3 plays an important role in the development of UVB-induced skin tumors through its effects on both survival and proliferation of keratinocytes during carcinogenesis.

[Surgical treatment of transposition of great arteries: the coronary buttons transfer using medially based trapdoor flaps].

The arterial switch operation (ASO) has become the primary surgical approach used for correction of transposition of the great arteries. All the prerequisites for a successful ASO were recognized in time and dealt with, which allowed general acceptation of the technique. We report on our technique for the procedure and the result to date. From January 1991 to January 2008, a total of 100 patients underwent ASO at our unit using medially-based trapdoor flap method. The neo-pulmonary artery (PA) was reconstructed using a single rectangular pericardial patch. The initial patient having intramural coronary artery died due to ischemic event after Aubert procedure. Three patients had re-right ventricular out flow tract repair (RVOTR) in a long-term follow-up period. There was no significant aortic insufficiency, no ischemic event and no lethal arrhythmia.