[Prognostic value of KIT and other clonal genetic mutations in core-binding factor acute myeloid leukemia].

Objective: To evaluate the prognostic significance of clonal gene mutations using next-generation sequencing in patients with core-binding factor acute myeloid leukemia (CBF-AML) who achieved first complete remission after induction chemotherapy. Methods: The study, which was conducted from July 2011 to August 2017 in First Affiliated Hospital of Soochow University, comprised 195 newly diagnosed patients with CBF-AML, including 190 patients who achieved first complete remission after induction chemotherapy. The cohort included 134 patients with RUNX1-RUNXIT1(+) AML and 56 patients with CBFß-MYH11(+) AML. The cohort age ranged from 15 to 64 years, with a median follow-up of 43.6 months. Overall survival (OS) and disease-free survival (DFS) were assessed by the log-rank test, and the Cox proportional hazards regression model was used to determine the effects of clinical factors and genetic mutations on prognosis. Results: The most common genetic mutations were in KIT (47.6% ) , followed by NRAS (20.0% ) , FLT3 (18.4% ) , ASXL2 (14.3% ) , KRAS (10.7% ) , and ASXL1 (9.7% ) . The most common mutations involved genes affecting tyrosine kinase signaling (76.4% ) , followed by chromatin modifiers (29.7% ) . Among the patients receiving intensive consolidation therapy, the OS tended to be better in patients with CBFß-MYH11(+) AML than in those with RUNX1-RUNXIT1 (+) AML (P=0.062) . Gene mutations related to chromatin modification, which were detected only in patients with RUNX1-RUNXIT1(+) AML, did not affect DFS (P=0.557) . The patients with mutations in genes regulating chromatin conformation who received allo-hematopoietic stem cell transplantation (allo-HSCT) achieved the best prognosis. Multivariate analysis identified KIT exon 17 mutations as an independent predictor of inferior DFS in patients with RUNX1-RUNXIT1(+) AML (P<0.001) , and allo-HSCT significantly prolonged DFS in these patients (P=0.010) . Conclusions: KIT exon 17 mutations might indicate poor prognosis in patients with RUNX1-RUNXIT1(+) AML. Allo-HSCT may improve prognosis in these patients, whereas allo-HSCT might also improve prognosis in patients with mutations in genes related to chromatin modifications.

Sequential therapy for non-thalamus supratentorial hypertensive intracerebral hemorrhages.

OBJECTIVE: We sought to assess the effectiveness of sequential therapy for non-thalamus supratentorial hypertensive intracerebral hemorrhage (NTS-HICH). PATIENTS AND METHODS: We retrospectively analyzed clinical data of 110 patients with HICH. The patients were admitted 72 hours after disease onset, and 43 patients received sequential therapy. The length of hospital stay, treatment costs, incidence of pulmonary infections, mortality rates and Modified Rankin Score (mRS) 1 and 3 months after NTS-HICH were compared between patients who received sequential or non-sequential therapies. RESULTS: The length of hospital stay, treatment costs, and 1-month mortality rates were not significantly different between both groups. However, mortality rates at 3 months, incidence of pulmonary infection, and mRS at both 1 and 3 months were significantly better in patients who received sequential therapy. CONCLUSIONS: Sequential therapy significantly improves the prognosis for patients with NTS-HICH.

Identification of risk factors of Coxiella burnetii (Q fever) infection in veterinary-associated populations in southern Taiwan.

The first case of Q fever in Taiwan was reported in 1993. The disease is considered to be emerging in Taiwan, but the route of transmission has remained unclear. The annual number of confirmed Q fever cases has been increasing up to more than 100 cases since 2005, comparing with less than 30 before 2003. The purpose of this study was to determine the seroprevalence and risk factors of Coxiella burnetii infection in veterinary-associated populations in southern Taiwan. A total of 228 serum samples of high risk individuals engaging in veterinary-related work or animal-farm work, were collected between March and June in 2007. The study individuals were interviewed by a structured questionnaire designed for Q fever investigation. Serum samples from different animal species were also obtained for Q fever analysis in the same study areas. Serological test was conducted by indirect immunofluorescence antibody assay (IFA). The result demonstrated the overall seroprevalence of Q fever was 26.3% in individuals engaging in veterinary and animal-related work in southern Taiwan. After multiple logistic regression analysis, goat exposure was significantly associated with seropositivity of Q fever in the study population in southern Taiwan (adjusted odds ratio: 2.62; 95% CI: 1.06-6.46). In addition, the highest seroprevalence (43.8%) of Q fever was identified in goats (P < 0.05). Finally, this study documented that people with prior knowledge of Q fever were less likely to be seropositive for C. burnetii. It was concluded that goat exposure was the most important risk factor associated with C. burnetii infection and appropriate health education could be useful to prevent high risk individuals from the infection in southern Taiwan.

Effect of genistein and quercetin on proliferation, collagen synthesis, and type I procollagen mRNA levels of rat hepatic stellate cells.

AIM: To study the effects of genistein (GE) and quercetin (QU) on proliferation, collagen synthesis, and procollagen messenger RNA (mRNA) expression of rat hepatic stellate cell line HSC-T6 cells. METHODS: Cell proliferation was measured by crystal violet staining assay. Collagen synthesis was determined by [3H]proline incorporation assay. Type I mRNA level was determined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: GE (25 - 70 micromol/L) and QU (6.25 - 50 micromol/L) concentration-dependently reduced serum-driven HSC-T6 cell proliferation and collagen synthesis associated with a suppression of type I procollagen mRNA level. CONCLUSION: GE and QU inhibited hepatic stellate cell proliferation and collagen synthesis that might have a protective role against liver fibrosis.

Quantitative assessments of physiological and biological parameters in psoriatic lesions and its correlations to the clinical severity of psoriasis.

Twenty-nine psoriatics were examined using a model with clinical, physiological and pathological assessment parameters. The three parts in this assessment model include: (1) clinical assessment: psoriasis area and severity index (PASI); (2) assessment of skin physiology and microcirculation: water content of stratum corneum, water-holding capacity of stratum corneum, transepidermal water loss, intravital dynamic videocapillaroscopy-measuring the capillary diameters and blood cell velocity in proximal nailfold of ring finger, and fluorescence angiography-measuring transcapillary Na-fluorescein(NAF) diffusion; and (3) immunohistochemistry examination: markers of proliferation (Ki67Ag), differentiation (involucrin), and inflammation (neutrophil elastase, intercellular adhesion molecule-1(ICAM-1), endothelial leukocyte adhesion molecule-1(ELAM-1)). Our results showed both the transcapillary diffusion of NAF and the expression of cell markers-dermal neutrophil elastase, epidermal ELAM-1 and Ki67Ag--correlated significantly to PASI scores (P < 0.05, linear regression). According to our results, the increased capillary permeability and inflammation markers, and enhanced expression of Ki67Ag correlated very well with PASI score. These markers could serve as alternative methods for assessment of the clinical severity of psoriatic patients.

Beneficial effect of HCL-31D in murine models of endotoxaemia.

We evaluated the effect of HCL-31D, a novel cAMP-specific phosphodiesterase inhibitor, on the induction of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-treated rat aortic smooth muscle cells (RASMC) and on survival in a murine model of severe endotoxaemia. Treatment of cultured RASMC with LPS and IFN-gamma resulted in an increase of nitrite, tumour necrosis factor (TNF-alpha) production and induction of iNOS mRNA. However, incubation with HCL-31D (1 approximately 50 microM) for 24 h caused significant attenuation of nitrite and TNF-alpha formation as well as iNOS mRNA induction in a dose-dependent manner but no effect on iNOS activity in RASMC. In addition, administration of HCL-31D (5 mg/kg, i.p.) resulted in that the increase of both plasma nitrate and TNF-alpha levels induced by LPS in vivo was significantly reduced in LPS-treated rats. Treatment of conscious mice with a high dose of LPS (60 mg/kg, i.p.) to ICR mice resulted in a 24-h survival rate of only 10%. However, administration of HCL-31D (5 mg/kg, i.p. at 0 h and 6 h after LPS) improved the 24-h survival to 50%, indicating that HCL-31D has a beneficial effect in murine model endotoxaemia. These effects may be mainly due to inhibition of TNF-alpha formation and of the induction of iNOS. We proposed that the elevation of cAMP levels by HCL-31D may be involved in the prevention of TNF-alpha formation and iNOS induction.

[Antifibrotic effects of genistein and quercetin in vitro].

AIM: To study the antifibrotic effects of genistein (GE) and quercetin (QU) on rat hepatic stellate HSC-T6 cell proliferation stimulated with platelet-derived growth factor (PDGF), collagen synthesis and type I procollagen messenger RNA (mRNA) expression stimulated with transforming growth factor beta 1 (TGF beta 1). METHODS: Cell proliferation was measured by crystal violet staining assay. Collagen synthesis was determined by 3H-proline incorporation assay. Type I procollagen mRNA level was determined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: GE (25-70 mumol.L-1) and QU (6.25-50 mumol.L-1) concentration-dependently attenuated PDGF-drive HSC-T6 cell proliferative activity. TGF beta 1-stimulated collagen synthesis was also reduced. This was associated with a decrease of type I procollagen mRNA, indicating an effect at a pretranslational level. CONCLUSION: GE and QU may have therapeutic potential against liver fibrosis by regulating PDGF and TGF beta 1 actions.

Gene expression profiling in the human hypothalamus-pituitary-adrenal axis and full-length cDNA cloning.

The primary neuroendocrine interface, hypothalamus and pituitary, together with adrenals, constitute the major axis responsible for the maintenance of homeostasis and the response to the perturbations in the environment. The gene expression profiling in the human hypothalamus-pituitary-adrenal axis was catalogued by generating a large amount of expressed sequence tags (ESTs), followed by bioinformatics analysis (http://www.chgc.sh.cn/ database). Totally, 25,973 sequences of good quality were obtained from 31,130 clones (83.4%) from cDNA libraries of the hypothalamus, pituitary, and adrenal glands. After eliminating 5,347 sequences corresponding to repetitive elements and mtDNA, 20,626 ESTs could be assembled into 9, 175 clusters (3,979, 3,074, and 4,116 clusters in hypothalamus, pituitary, and adrenal glands, respectively) when overlapping ESTs were integrated. Of these clusters, 2,777 (30.3%) corresponded to known genes, 4,165 (44.8%) to dbESTs, and 2,233 (24.3%) to novel ESTs. The gene expression profiles reflected well the functional characteristics of the three levels in the hypothalamus-pituitary-adrenal axis, because most of the 20 genes with highest expression showed statistical difference in terms of tissue distribution, including a group of tissue-specific functional markers. Meanwhile, some findings were made with regard to the physiology of the axis, and 200 full-length cDNAs of novel genes were cloned and sequenced. All of these data may contribute to the understanding of the neuroendocrine regulation of human life.

Antimicrobial activity of synthetic all-D mastoparan M.

Mastoparan M, a tetradecapeptide toxin (INKAIAALAKKLL-NH2) from hornet venom and its D-form mastoparan M were synthesized chemically. All D- and L-mastoparan M forms were found to adopt 28% alpha-helical structures in a 30% trifluroethanol solution as shown by the circular dichroism spectrum. All-D mastoparan M caused 3H-thymidine release from labeled bacterial cells after incubation for 1 h and complete cell lysis by 4 h. Both L- and D-mastoparan M showed strong activity against gram-positive and gram-negative bacteria. All-D mastoparan M showed 2-fold higher antibacterial activity than L-mastoparan M. The effects of all-D mastoparan M on the surface morphology of Staphylococcus aureus (ATCC29213) and Escherichia coli (ATCC25922) were studied by scanning-beam electron microscopy. Blast-like bleb extrusions on the surface of some S. aureus and swellings on the end of E. coli were seen after culture with all-D mastoparan M. These findings indicated the all-D mastoparan M could kill bacteria by disrupting cells.

Inhibitory effect of hexapeptide (RGRHGD) on platelet aggregation.

The B chain of beta-bungarotoxin 1-6 sequence, RGRHGD, presents the highest local average hydrophilicity measured by Kyte and Doolittle modeling analysis. The RGRHGD holds parts of both RGD and KGD peptides, which have been reported as having high binding affinity to GPIIb-IIIa. The present study evaluates whether the synthesized hexapeptide, RGRHGD, has an antiplatelet effect and further elucidates the possible mechanisms of action. RGRHGD dose-dependently inhibited rabbit platelet aggregation and adenosine triphosphate release induced by arachidonic acid, collagen, platelet-activating factor, thrombin, or U46619 with the IC50 range of 82.7 to 510 microg/mL. The platelet thromboxane B2 formation induced by collagen or thrombin was also significantly decreased by RGRHGD, but there was no effect on arachidonic acid-induced thromboxane B2 formation. In addition, RGRHGD also inhibited the rise of intracellular calcium level stimulated by arachidonic acid, collagen, or thrombin in Fura 2-AM-loaded platelets. The adenosine 3',5'-cyclic monophosphate level of washed platelets was not affected by RGRHGD. In conclusion, these data indicate that the inhibitory effect of RGRHGD on platelet aggregation may be due to the attenuation of thromboxane A2 formation and intracellular calcium mobilization. In addition, this study may provide a useful method of finding potential therapeutic agents by using molecular modeling analysis.

Mechanism of inhibition of platelet aggregation by HCL-31D.

The antiplatelet effect of the pyridazinone analogue, 4, 5-dihydro-6-[4-[2-hydroxy-3-(3,4 dimethoxybenzylamino)propoxy]naphth-1-yl]-3(2H)-pyridazinone (HCL-31D), was investigated in vitro with rabbit platelets. HCL-31D dose-dependently inhibited the platelet aggregation and ATP release induced by collagen (10 microg/ml), arachidonic acid (100 microM) or thrombin (0.1 U/ml) with an IC(50) of about 0.95-5.41 microM. HCL-31D (0.5-5 microM) increased the platelet cyclic AMP level in a dose-dependent manner. Furthermore, HCL-31D potentiated cyclic AMP formation caused by prostaglandin E(1) but not that caused by 3-isobutyl-1-methylxanthine (IBMX). HCL-31D also attenuated phosphoinositide breakdown and intracellular Ca(2+) elevation induced by collagen, arachidonic acid or thrombin. HCL-31D inhibited the formation of thromboxane B(2) induced by collagen or thrombin but not by arachidonic acid. In addition, HCL-31D did not affect platelet cylooxygenase and thromboxane synthase activity. These data indicate that HCL-31D is an inhibitor of phosphodiesterase and that its antiplatelet effect is mainly mediated by elevation of cyclic AMP levels.

Stimulation of TNF-alpha, IL-1beta and nitrite release from mouse cultured spleen cells and lavaged peritoneal cells by mastoparan M.

Chemically synthesized mastoparan M, a tetradecapeptide toxin of venom (INLKAIAALAKKLL), was used in the experiments described. After addition of mastoparan M to cultures of mouse macrophages in vitro, tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) were detected in the culture fluids by 12 h and their highest accumulation was observed by 24 h. Mastoparan M induced increases in both TNF-alpha secretion and mRNA level at the same time. Nitrite levels, which reflect nitric oxide synthesis, were also found to increase in the macrophage cultures at 24 h after mastoparan M addition. In vivo studies showed that mastoparan M induced the formation and accumulation of TNF-alpha, IL-1beta and nitrite in the peritoneal exudates of mice much faster at 90 min, 120 min and 180 min after mastoparan M injection, respectively. Similarly, significant increases in myeloperoxidase activity, a marker for neutrophil and macrophage content, were observed in the peritoneal lavage cells after intraperitoneal injection of mastoparan M. However, induction of nitrite by mastoparan M was completely inhibited by simultaneous addition of antimouse TNF-alpha antibody to the macrophage cultures. These results suggest that modulation of both neutrophil and macrophage influx by mastoparan M may be conveyed through TNF-alpha and IL-1beta secretion accompanied by nitrite formation.

The cytolytic action of all-D mastoparan M on tumor cell lines.

All-D and all-L mastoparan M, a tetradecapeptide toxin (INLKAIAALAKKLL-NH2) from hornet venom, were chemically synthesized in this study. Under circular dichroism investigation, all-D and L-mastoparan M adopted a 30% alpha-helical structure in 30% trifluroethanol solution but only a 10% á-helical structure in phosphate solution. After being added to the cultures of tumor cell lines in vitro, D-mastoparan (12.5 micrograms/ml) directly inhibited the growth of the tumor cell lines Colo 225 (59%), KB (38%), Hep-2 (63%), H226Br (43%) and HeLa (54%) determined by the MTT assay. We also found that D-mastoparan M has a higher potency of antitumor cell activity in vitro than L-mastoparan M. When examined under the scanning-beam electron microscope, hollow, shrunk and collapsed structures of tumor cells could be seen, after being mixed with D-mastoparan M. The appearance of these morphological alterations indicated that mastoparan M inhibits the growth of tumor cells by direct lysis of target cells.

Inhibitory effect of quercetin on tumor necrosis factor and interleukin-1 beta pro-osteoclastic activities.

AIM: To study the effects of quercetin on tumor necrosis factor (TNF) and interleukin-1 beta (IL-1 beta) pro-osteoclastic activities. METHODS: [3H] TdR uptake by osteoblasts was used to measure cell proliferation, microspectrophotometer for cellular AIP activity using p-nitrophenyl phosphate as enzyme substrate, and radioimmunoassay for prostaglandin E2. RESULTS: Quercetin 5-40 mumol.L-1 reduced the inhibition of cell proliferation and AIP activity induced by TNF or IL-1 beta in a concentration-dependent manner. PGE2 production stimulated by either cytokines was also reduced by quercetin at 20 and 40 mumol.L-1. CONCLUSION: quercetin exerted a marked inhibitory effect on TNF and IL-1 activities, related to their pro-osteoclastic function.

[Inhibitory effects of protein kinase C inhibitors on tumor necrosis factor induced bovine pulmonary artery endothelial cell injuries].

The effects of protein kinase C(PKC) inhibitors 1-(5-isoquino-linylsulfonyl)-2-methylpeperazine (H-7) and quercetin on tumor necrosis factor (TNF) were studied in cultured bovine pulmonary artery endothelial cells (BPAEC) in vitro. Incubation of BPAEC with TNF caused a significant increase in percent lactate dehydrogenase (LDH) release, stimulation of EC-dependent neutrophils (PMN) adhesion to BPAEC and inhibition of BPAEC DNA synthesis and proliferation. All these were restored by both H-7 and quercetin. The IC50 of H-7 and quercetin was 9.7 and 10.8 mumol.L-1 for the inhibition of LDH% release; 19.5 and 16.7 mumol.L-1 for the inhibition of TNF-induced PMN-EC adhesion; 7.0 and 6.1 mumol.L-1 for TNF-induced inhibition of DNA synthesis and 8.7 and 11.36 mumol.L-1 for proliferation. These results suggest that PKC inhibitors H-7 and quercetin protect BPAEC from TNF induced injuries and PKC play an important role in EC activation by TNF.

The avian eggshell extracellular matrix as a model for biomineralization.

The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest that the dermatan sulfate proteoglycans may be important in modulating crystal morphology in the hen eggshell and correlate with mineralization-modulating biomolecules from other calcified tissue, which are generally anionic.

Protein kinase C inhibitor H-7 blocks effects of tumor necrosis factor on bone cells.

AIM: To study the effects of tumor necrosis factor (TNF) on the neonatal mouse osteoblast-enriched calvarial cells and effects of protein kinase C (PK C) inhibitor, 1-(5-isoquinolinesulfonyl)-2 -methylpiperazine (H-7) on the TNF actions. METHODS: [3H]TdR uptake by the osteoblasts was used to measure cell proliferation. Cellular alkaline phosphatase (AIP) and tartrate resistant acid phosphatase (trAcP) activities were determined spectrophotometrically. RESULTS: TNF (1-100 kU . L-1) inhibited both proliferation and expression of AIP activity, but stimulated trAcP activity. These TNF-induced actions were blocked by simultaneous addition of H-7 (5-20 mumol . L-1). CONCLUSION: TNF has potent effects on the osteoblasts, and the blockade of TNF actions by H-7 suggests that TNF exert its effects through PK C.

Crystallization studies on avian eggshell membranes: implications for the molecular factors controlling eggshell formation.

The avian eggshell is a natural biopolymer and mineral composite. It is a very useful model for biomimetic mineralization, since it is among the fastest forming hard tissues known. Isolated eggshell membranes, which were demineralized in vitro, were used to investigate the in vitro modulation of CaCO3 crystal deposition by organic matrix materials. Crystallization on the demineralized eggshell membrane occurred almost exclusively at the peripheries of residual calcium reserve assemblies, which contain a high concentration of sulfur. Similar structures are observed for eggshell membranes after natural demineralization. The characteristic rhombohedral crystal morphologies of the calcite crystals grown in this in vitro system are much less regular when grown in the presence of organic matrix or partially purified dermatan sulfate proteoglycans obtained from the eggshell. The effect of these macromolecules on the morphology and size of CaCO3 crystals is concentration-dependent. These studies indicate the complexity of the molecular and ionic interactions involved in the initiation and formation of the eggshell, with the focus on the role of the organic matrix.

Mastoparan B, synthesis and its physical and biological properties.

Mastoparan B, a tetradecapeptide toxin (LKLKSIVSWAKKVL) isolated from the hornet (Vespa basalis) venom, was synthesized chemically. The physical and biological properties of both the native and synthetic peptides were studied and proved to be identical. Mastoparan B was found to have a potent antibacterial activity to both Gram negative and Gram positive bacteria.