Characteristics of in peripheral blood of 70 hospitalized patients and 8 diarrhea patients with COVID-19.

Objective: To analyze the blood test indicators of patients after infection of COVID-19 in Chongqing and analyze the clinical indicators of 8 patients with diarrhea. Materials and Methods: From January 26, 2019 to February 13, 2020, 70 patients diagnosed with 2019-nCoV according to the World Health Organization interim guidance for NCP and divided into diarrhea and non-diarrhea groups. The laboratory tests liver and kidney function, blood routine, coagulation function, and immune status. Results: The study population included 70 hospitalized patients with confirmed CONV-2019. NCP patients (43males and 27 females) with a mean age of 48.57±17.80 (9~82) years and only 4.3% of patients have lung-related diseases. The positive rate of ESR, CRP, PT, IL6, lymphocyte count, GGT, Prealbumin and CD4 was more than 50%. We further analyzed the differences between 8 diarrhea patients and 62 non-diarrhea patients. Among these indicators, only Lymphocyte, CRP, Prealbumin and Cystatin C positive rate is more than 50%. Although there is no statistical difference in GGT, 100% of the 7 patients tested decreased. Conclusion: Our data recommended that the ESR, CRP, PT, IL6, lymphocyte count, GGT, prealbumin and CD4 have important value in the diagnosis of COVID-19, and the decrease of GGT may be an important indicator for judging the intestinal dysfunction of patients.

Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs.

CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.

Targeting ninjurin 2 by miR-764 regulates hydrogen peroxide (H2O2)-induced neuronal cell death.

Ninjurin 2 (NINJ2) is a novel adhesion molecule expressed in neurons and glial cells. The current study determined if specific microRNA (miRNA) can regulate NINJ2 expression in human neuronal cells. Sequence analysis of NINJ2 mRNA 3'-untranslated region (3'-UTR) revealed that microRNA-764 (miR-764) putatively targets NINJ2. In SH-SY5Y/SK-N-BE neuronal cells and primary human neurons, lentivirus-mediated overexpression of miR-764 conferred significant repression on NINJ2 3'-UTR luciferase activity and downregulation of NINJ2 mRNA/protein. Conversely, transfection of the miR-764 inhibitor increased NINJ2 mRNA and protein expression in the neuronal cells. Function studies show that NINJ2 downregulation by miR-764 induced significant viability reduction and apoptosis in the neuronal cells. Further, CRISPR/Cas9-mediated NINJ2 knockout mimicked and abolished miR-764-induced actions in SH-SY5Y cells. Conversely, lentivirus-mediated NINJ2 overexpression or transfection of miR-764 inhibitor protected neuronal cells from hydrogen peroxide (H2O2)-induced cell death and apoptosis. Collectively, these results show that NINJ2 is a pro-survival factor in human neuronal cells. miR-764 regulates NINJ2 expression and neuron functions.

Esm1 modulates endothelial tip cell behavior and vascular permeability by enhancing VEGF bioavailability.

RATIONALE: Endothelial cell-specific molecule 1 (Esm1) is a secreted protein thought to play a role in angiogenesis and inflammation. However, there is currently no direct in vivo evidence supporting a function of Esm1 in either of these processes. OBJECTIVE: To determine the role of Esm1 in vivo and the underlying molecular mechanisms. METHODS AND RESULTS: We generated and analyzed Esm1 knockout (Esm1(KO)) mice to study its role in angiogenesis and inflammation. Esm1 expression is induced by the vascular endothelial growth factor A (VEGF-A) in endothelial tip cells of the mouse retina. Esm1(KO) mice showed delayed vascular outgrowth and reduced filopodia extension, which are both VEGF-A-dependent processes. Impairment of Esm1 function led to a decrease in phosphorylated Erk1/2 (extracellular-signal regulated kinases 1/2) in sprouting vessels. We also found that Esm1(KO) mice displayed a 40% decrease in leukocyte transmigration. Moreover, VEGF-induced vascular permeability was decreased by 30% in Esm1(KO) mice and specifically on stimulation with VEGF-A165 but not VEGF-A121. Accordingly, cerebral edema attributable to ischemic stroke-induced vascular permeability was reduced by 50% in the absence of Esm1. Mechanistically, we show that Esm1 binds directly to fibronectin and thereby displaces fibronectin-bound VEGF-A165 leading to increased bioavailability of VEGF-A165 and subsequently enhanced levels of VEGF-A signaling. CONCLUSIONS: Esm1 is simultaneously a target and modulator of VEGF signaling in endothelial cells, playing a role in angiogenesis, inflammation, and vascular permeability, which might be of potential interest for therapeutic applications.

Cutting edge: Endothelial-specific gene ablation of CD99L2 impairs leukocyte extravasation in vivo.

CD99-like 2 (CD99L2) is a membrane protein with moderate sequence homology to CD99, which initiates cell aggregation of transfected cells and that is strongly expressed on endothelial cells, neutrophils, and lymphocytes. We showed recently that Abs against CD99L2 inhibit neutrophil, but not T lymphocyte, recruitment into inflamed tissues. In this study, we have generated conditional gene-deficient mice for CD99L2 and show by analyzing them in various inflammation models several results. First, gene ablation of CD99L2 impairs neutrophil recruitment into inflamed cremaster and peritoneum. Second, despite the strong expression of CD99L2 on peripheral neutrophils, only gene ablation on endothelial cells but not on myeloid cells affects neutrophil extravasation. Third, in contrast to our previous Ab-based results, recruitment of activated T cells into inflamed skin was impaired in mice lacking CD99L2 on endothelial cells. We conclude that CD99L2 is an essential endothelial Ag for leukocyte extravasation, which does not require homophilic interactions with CD99L2 on leukocytes.

Treatment of osteomyelitis by liposomal gentamicin-impregnated calcium sulfate.

OBJECTIVES: Traditional therapy of staphylococcal osteomyelitis is ineffective in producing complete sterilization of infected bones due to the formation of the Staphylococcus aureus biofilms. The aim of this study was to develop a new drug-delivery system of antibiotics for treatment of chronic experimental osteomyelitis. METHODS: In the current work, cationic liposomal gentamicin was prepared and impregnated in calcium sulfate (CS), and tested for anti-biofilm activities in vitro and in vivo. RESULTS AND CONCLUSIONS: The combination of liposomal gentamicin and CS showed initial burst-release of active liposomal gentamicin and had continuous-release (12 days). Liposomal gentamicin released from CS had the same anti-biofilm activity with the liposomal gentamicin prepared freshly. Meanwhile, both agents were more effective relative to free gentamicin at low drug concentration. Therapeutic trials with antibiotics given intravenously revealed that free gentamicin for 14 days was ineffective in sterilizing bone. Treatment with liposomal gentamicin for 14 days resulted in recovery of 33.3% of treated animals, which was the lower slightly than the result treated with implantation of gentamicin-impregnated CS (66.7%). Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group of liposomal gentamicin-impregnated CS treated for 14 days. The new drug-delivery system was effective in preventing biofilm infection in a contaminated defect, and it could also be used clinically for bacterial infections in the conditions like plaque formation or in arresting biofilm formation in the implanted devices or dead bone of osteomyelitis.

HS1 deficiency impairs neutrophil recruitment in vivo and activation of the small GTPases Rac1 and Rap1.

Neutrophil extravasation is a critical step of the innate immune system's response to inflammation. This multistep process is tightly regulated by adhesion and signaling molecules in the endothelium and neutrophils. Activation of the ß2 integrin LFA-1 is critical for adhesion of leukocytes to postcapillary venules. This step requires coordinated activation of signaling pathways in chemokine-stimulated neutrophils, including GTPase activation and cytoskeletal remodeling, leading to conformational changes in LFA-1. Hematopoietic cell-specific lyn substrate 1 (HS1) is a cortactin-related and leukocyte-specific actin-binding protein (ABP) that regulates several processes in various immune cells. It has been shown in vitro that HS1 is important for neutrophil chemotaxis and transendothelial migration of NK cells, but its role in neutrophil extravasation in vivo has not been investigated yet. Intravital microscopy of CXCL1-stimulated cremaster venules revealed an increased rolling velocity and reduced neutrophil adhesion and transmigration in HS1 knockout (KO) mice. CXCL1-induced rapid neutrophil arrest in vivo and adhesion under flow conditions in vitro were also reduced significantly. Whereas random motility of neutrophils was unaffected, chemotaxis toward a CXCL1 gradient was reduced in the absence of HS1. Further analysis of the underlying mechanisms demonstrated that HS1 controls CXCL1-induced activation of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap1), thus supporting LFA-1-mediated neutrophil adhesion. Importantly, with the use of Rac1 KO neutrophils, we could show that Rac1 acts upstream of Rap1. Our results establish HS1 as an important regulator of proper Rac1 and Rap1 activation and neutrophil extravasation.

Marked prevention of tumor growth and metastasis by a novel immunosuppressive agent, FTY720, in mouse breast cancer models.

FTY720 is a unique immunosuppressive agent that exerts its activity by inducing apoptosis in lymphocytes. We conducted the present study to investigate the effects of FTY720 on cancer growth and metastasis, as well as its mechanism of action. In vitro treatment with FTY720 induced dramatic cancer cell apoptosis in a mouse breast cancer cell line, JygMC(A). Electron microscopy revealed distinct changes on the cell surface with decreased filopodias and microvilli in cancer cells treated with FTY720 at 2 microM and clear evidence of apoptosis at 10 microM. Interestingly, the effect of FTY720 was significantly less in the normal fibroblasts than in the cancer cells, indicating greater susceptibility of cancer cells to the agent. We then tested the in vivo effect of FTY720 in a mouse breast cancer model created by inoculating JygMC(A) cells (s.c.) in the flank region of BALB/c-nu/nu mice at three different dosages (2, 5, and 10 mg/kg/day; n = 30/group). Tumor growth was markedly suppressed at a dosage of 5 mg/kg or more without notable side effects. In addition, tumor metastasis, which was dramatically evident in control mice, was significantly prevented even at a low dose (2 mg/kg/day), resulting in a significant prolongation of animal survival. These data led us to additionally investigate the mechanism of action, especially the prevention of metastasis at a low dose. FTY720 treatment at 2 microM caused a remarkable cytoskeletal change with deformed and decreased filopodias in cancer cells. In addition, it significantly decreased the ability of cancer cells to adhere and migrate to extracellular matrix components, and markedly reduced the expression of integrins on the cancer cell surface. These results indicate that FTY720 is a potent anticancer agent that induces cancer cell apoptosis and is markedly effective for prevention of metastasis. The changes of cellular structure with reduction of integrin expression may be one of its underlying mechanisms of action.

The expression difference of insulin-like growth factor 1 receptor in breast cancers with or without diabetes.

CONTEXT: The insulin-like growth factor (IGF) and insulin receptors' (IR) axes play important roles in both breast cancer and diabetes mellitus. AIM: We tend to explore the expression characteristics of proteins in IGF/IR axis in breast cancer with type 2 diabetes mellitus (T2DM). SETTINGS AND DESIGN: We conducted a case-control investigation of T2DM and non-diabetes (n = 40, 1:1) in breast cancer patients. MATERIALS AND METHODS: Some important molecules of IGF/IR axis were detected in breast cancer tissues by immunohistochemical staining. The multivariable analyses of the relationship of clinicopathological characters with the significant molecules were also detected. STATISTICAL ANALYSIS USED: The results were statistically evaluated by Statistical Package for the Social Sciences (SPSS version 17.0) software. Chi-square test and logistic regression are used. RESULTS: Higher expression of IGF 1 receptor (IGF1R) was found in breast cancers of patients with T2DM, compared those without diabetes (P = 0.044). Negative expression of human epidermal growth factor receptor 2 (Her2) was found to be associated with higher expression of IGF1R in the breast cancers of patients with T2DM. There were no differences found in the expression of proteins of IGF-1, IGF-2, IGF-binding protein 3 (IGFBP3), IR, insulin receptor substrate (IRS)-1, IRS-2 and mammalian target of rapamycin (mTOR) between T2DM group and non-diabetes group. CONCLUSION: Our study found that breast cancer with T2DM had a higher expression of IGF1R, and the higher IGF1R was associated with negative Her2 expression.

Genetic study of a large Chinese kindred with von Hippel-Lindau disease.

BACKGROUND: Von Hippel-Lindau (VHL) disease is a heraditary cancer syndrome caused by germline mutations of the VHL tumor on the suppressor gene. This study was to show the clinical characteristics of a large Chinese kindred with von Hippel-Lindau disease and to evaluate the role of the genetic test of VHL disease in the diagnosis of VHL disease and clinical screening of members of the VHL disease family. METHODS: DNA extracted from peripheral blood was amplified by PCR to three exons of the VHL gene in 27 members of a large kindred with VHL disease. PCR products were directly sequenced. The involvements of multi-organs in the kindred with VHL disease were confirmed by history taking and radiography. RESULTS: Of 47 members in the four generations of the kindred, 18 members were diagnosed as having VHL disease. Clinical manifestations of 18 patients included: central nervous system (CNS) hemangioblastoma (5), renal cell carcinoma and CNS hemangioblastoma (3), renal cell carcinoma and retinal angioma (3), renal cell carcinoma and multiple pancreatic cysts (1), renal cell carcinoma and retinal angioma and multiple pancreatic cysts (2), renal cell carcinoma and CNS hemangioblastomas and multiple pancreatic cysts (1), and multiple pancreatic cysts and multiple renal cysts (1), multiple pancreatic cysts (2). The common lesions of the 18 patients were renal cell carcinoma (55.6%), CNS hemangioblastoma (50.0%), retinal angioma (27.8%), and multiple pancreatic cysts (38.9%). Among the 27 members who volunteered for genetic analysis, 15 members including 9 affected family patients and 2 asymptomatic patients and 4 carriers, who are still alive, presented a codon 78 from Asn to Ser change at nucleotide 446 (A-->G) in exon 1. Four members were carriers with the same VHL gene mutation. Two asymptomatic patients were initially diagnosed by genetic testing and subsequently confirmed radiologically and surgically. Members without gene mutation had no clinical evidence of VHL disease. CONCLUSIONS: The large Chinese kindred with VHL disease was classified as type I. The main characteristics in the kindred were higher incidence of renal cell carcinoma and lower incidence of retinal angioma. Genetic test plays an important role in early detecting asymptomatic patients and the carriers in clinical screening of members of the families with VHL disease. It is also important to prevent the transmission of VHL disease to their offsprings in the kindred.

[Familial and genetic study in a large Chinese kindred with von Hippel-Lindau disease and gene mutation analysis].

OBJECTIVE: To report the clinical characterization of a large Chinese kindred with von Hippel-Lindau (VHL) disease and to evaluate the role of VHL genetic testing in diagnosis of VHL disease and clinical screening for members in VHL disease family. METHODS: A large kindred with VHL disease was studied. DNA extracted from peripheral blood was amplified by PCR to three exons of VHL gene in 27 members. PCR products were directly sequenced. The data on involvement of multi-organs in the VHL disease kindred were obtained by medical history taking and radiography. RESULTS: There were 47 members in the four generations of the Chinese VHL kindred; among them, 18 members were patients with diagnostically proven VHL disease. Their clinical manifestations included: central nervous system(CNS) hemangioblastoma (n=5), renal cell carcinomas and CNS hemangioblastoma (n=3), renal cell carcinomas and retinal angiomas (n=3), renal cell carcinomas and multiple pancreatic cysts (n=1), renal cell carcinomas and retinal angiomas and multiple pancreatic cysts (n=2), renal cell carcinomas and CNS hemangioblastomas and multiple pancreatic cysts (n=1), and multiple pancreatic cysts and multiple renal cysts (n=1), and multiple pancreatic cysts (n=2). The common lesions of 18 patients in the large kindred were: renal cell carcinomas (56%), CNS hemangioblastomas(50%),retinal angiomas(28%), and multiple pancreatic cysts(39%). Of the 27 members who volunteered for genetic analysis, all 11 affected family patients who are still alive, including 9 affected family patients and 2 asymptomatic patients, presented a codon 78 from Asn to Ser change at nucleotide 446(A to G) in exon 1. Four members were carriers with the same VHL gene mutation. Two asymptomatic cases were initially diagnosed by genetic testing and subsequently confirmed by radiological imaging and surgery. Members not having the gene mutation had no clinical evidence of VHL disease. CONCLUSION: The large Chinese kindred with VHL disease was classified as type . The main characteristics of the kindred are higher incidence of renal cell carcinomas and lower incidence of retinal angiomas. The genetic testing played an important role in early detecting asymptomatic patients and the carriers in clinical screening for members in the VHL families. Also, it is important to prevent the transmission of VHL disease to the offspring in the kindred.

Effect of Diazoxide Preconditioning on Cultured Rat Myocardium Microvascular Endothelial Cells against Apoptosis and Relation of PI3K/Akt Pathway.

BACKGROUND: Anti-apoptotic mechanism for cell protection on reperfusion may provide a new method to reduce reperfusion injury. AIMS: The aim of the present study is to explore the effect of mitochondrial ATP sensitive potassium channel (Mito-KATP) opener diazoxide (DZ) preconditioning on hypoxia/reoxygen (H/R) injury of rat myocardium microvascular endothelial cells (MMECs) against apoptosis and relation of PI3K/Akt pathway. STUDY DESIGN: Animal experimentation. METHODS: The rat MMECs were cultivated, and H/R model was made to imitate ischemia-reperfusion injury. The cells were seeds in 96-wellplates (100µL/hole) or in 6cm diameter dishes (2 mL/dish) with the density of 1×106/mL and randomly divided into 4 groups (n=6 each): control group (Group N), hypoxia-regoxygen group (Group H/R), Diazoxide preconditioning+H/R group (Group DZ) and Diazoxide preconditioning +mitochondrial KATP blocker 5-hydroxydecanoate (5-HD) + H/R group (Group DZ+5-HD). The cells were exposed to 2h hypoxia followed by 2h reoxygenation. Diazoxide 100µmol/L and diazoxide 100µmol/L+ 5-HD100µmol/L were added to the culture medium 2h before hypoxia in DZ and DZ+5-HD groups respectively. Each group was observed the proliferation in MTT, apoptotic rate in Annexin V-FITC/PI double standard, cell structure of Hoechst staining, and the levels of PI3K, Akt and p53 mRNA by RT-qPCR. RESULTS: Compared with Group N, apoptotic rate of Group H/R increased (p<0.01) and the vitality decreased significantly (p<0.05), and the expression of PI3K, Akt and p53 mRNA elevated in Group H/R (p<0.05). Compared with Group H/R, apoptotic rate and p53 mRNA level of Group DZ depressed significantly (p<0.01, p<0.05), while the vitality, PI3K and Akt mRNA levels increased (p<0.05). Compared with Group DZ, apoptotic rate and p53 mRNA level of Group DZ+5-HD increased significantly (p<0.01, p<0.05), but the vitality, PI3K and Akt mRNA levels decreased (p<0.05). CONCLUSION: Under the condition of H/R, mito-KATP opened by DZ may depend on PI3K/Akt pathway to regulate expression level of the downstream p53 mRNA to inhibit apoptosis and improve viability of MMECs at the same time.

Short-term and long-term influences of family arguments and gender difference on developing psychological well-being in Taiwanese adolescents.

Adolescent mental health is crucial for social competence and accomplishment in later life. The World Health Organization (WHO) estimates that approximately 20% of adolescents suffer from psychological symptoms. However, improving family risk and school environments can largely promote adolescent mental health. A longitudinal survey was conducted to investigate adolescent psychological well-being (PWB) status and associated factors in adolescents 15-20 years of age. Family and school context variables were interviewed and recorded. A total of 2896 participants were included from high, middle, and less urbanized resident areas in Northern Taiwan with completed interview data. Using multivariate regression analysis, factors associated with adolescent PWB at various stages included quarrelsome parents, quarrels with parents, severed friendships, and cigarette and alcohol use. In all three adolescent stages, females yielded higher psychological symptom scores than did males, and diverse weights of risk factors on PWB were observed between genders. Family arguments and cigarette and alcohol use were found to have more pronounced effects on outcomes among females than males. Whereas males are more sensitive to severed friendships than females, cigarette and alcohol use showed more harmful effects on mental health in earlier adolescence than in later life. Moreover, family arguments and severed friendships in earlier adolescence were found to have lasting effects on PWB in later adolescence. In this study, gender differences were observed in the temporal relationship on adolescent mental health. Variables of family arguments and severed friendships exhibited short-term and long-term effects on adolescent mental health across the early to late developmental stages. The family argument environment and regulating cigarette and alcohol use are worthy of focus to promote adolescent mental health.

Quarrelsome family environment as an enhanced factor on child suicidal ideation.

Suicide is a leading cause of death in adolescents, and develops through a process leading from depression to suicidal ideation and self-injury. In this study, we analyzed and compared suicidal ideation among elementary school children from distinct families and school-related backgrounds. We conducted a cross-sectional study to investigate suicidal ideation in elementary school children in Miaoli County of Western Taiwan. Our study included 979 eligible participants and collected data, including suicidal ideation, depression scores, demographic characteristics, and family and school variables. The results revealed that 175 students (17.9%) exhibited depression, and 146 students (14.9%) had contemplated suicide. A quarrelsome family environment was found to be an important independent factor in child suicidal ideation after controlling for depression status. Children living in quarrelsome families showed a 3.7-fold risk of suicidal ideation compared with children in a harmonious family. Among boys living in quarrelsome family environments, suicidal ideation risk was 7.4-fold higher than for girls living in harmonious families. A 27-fold high increased suicidal ideation risk was also observed among the depressed children who living in the quarrelsome family environment, compared with the non-depressed in the harmonious family environment. This study provides novel evidence indicating the enhanced effects of a quarrelsome family environment combined with depression symptoms and among boys on suicidal ideation. These findings suggest of quarrels in a family environment playing an important role on elementary school children's psychological development, and may help parents in improving their mental health.

In vitro study on the feasibility of magnetic stent hyperthermia for the treatment of cardiovascular restenosis.

Thermal treatment or hyperthermia has received considerable attention in recent years due to its high efficiency, safety and relatively few side-effects. In this study, we investigated whether it was possible to utilize targeted thermal or instent thermal treatments for the treatment of restenosis following percutaneous transluminal coronary angioplasty (PTCA) through magnetic stent hyperthermia (MSH). A 316L stainless steel stent and rabbit vascular smooth muscle cells (VSMCs) were used in the present study, in which the inductive heating characteristics of the stent under alternative magnetic field (AMF) exposure, as well as the effect of MSH on the proliferation, apoptosis, cell cycle and proliferating cell nuclear antigen (PCNA) expression of the rabbit VSMCs, were evaluated. The results demonstrated that 316L stainless steel coronary stents possess ideal inductive heating characteristics under 300 kHz AMF exposure. The heating properties were shown to be affected by the field intensity of the AMF, as well as the orientation the stent axis. MSH had a significant effect on the proliferation and apoptosis of VSMCs, and the effect was temperature-dependent. While a mild temperature of 43°C demonstrated negligible effects on the growth of VSMCs, MSH treatment above 47°C effectively inhibited the VSMC proliferation and induced apoptosis. Furthermore, a 47°C treatment exhibited a significant and long-term inhibitory effect on VSMC migration. The results strongly suggested that MSH may be potentially applied in the clinic as an alternative approach for the prevention and treatment of restenosis.

Low-dose tacrolimus (FK506)-based immunosuppressive protocol in living donor renal transplantation.

In order to avoid the side effects of tacrolimus (FK506), a lowdose FK506-based regimen was started from 1 June 1991. The dose was adjusted to maintain the FK506 whole blood trough level at 15-20 ng/ml for 7 days postoperatively, at 10-15 ng/ml for 2 months, and under 10 ng/ml thereafter. The graft survival rates at 3 years and 5 years were 87.8 and 82.3% (FK506) vs 86.8 and 86.8% [cyclosporine (CyA)]. The incidence of acute rejection within the first 90 days was 31.6% in the FK506 group which was lower than the 57.1% of the CyA group (P = 0.0585). Grades of acute rejection episodes over IIA in the FK506 group were 20%, which was lower than the 37% in the CyA group. The mean oral dosages of FK506 were 0.061 and 0.04 mg/kg per day at 3 and 5 years, respectively. The incidence of new onset diabetes was 27.8% in the FK506 group and 17.1% in the CyA group. However, insulin therapy was withdrawn in all patients of the FK506 group within 5 months. The percentage of patients who required an antihypertensive agent was 28.6% and 40% in the FK506 group and 73.2% and 88% in the CyA group at 1 and 3 years, respectively (P < 0.05). Nephrotoxicity was seen in 20% of the FK506 group and 14.3% of the CyA group. Hypercholesterolemia was less frequent in the FK506 group than the CyA group. The FK506-based regimen described here is a protocol with the potential to reduce its adverse effects. The Whole blood concentration of FK506 should be monitored and blood levels maintained in the range of 5-10 ng/ml after 90 postoperative days for optimal efficacy and minimal toxicity.

Anticarcinogenic effect of FTY720 in human prostate carcinoma DU145 cells: modulation of mitogenic signaling, FAK, cell-cycle entry and apoptosis.

Despite the high frequency of prostate cancer, therapeutic options for advanced disease are limited to chemotherapy, radiation or hormonal therapy and eventually fail in all patients. Therefore, alternative approaches need to be developed. We previously reported that FTY720, a metabolite from Isaria sinclarii, is a unique antitumor agent for an androgen-independent prostate cancer cell line and requires caspase-3 activation in apoptosis. In our study, we have evaluated the effect of FTY720 on a family of mitogen-activated protein kinases (MAPKs), focal adhesion kinase (FAK), mitochondrial transmembrane potential, caspase-9 and caspase-8 and analyzed the expression of some cell-cycle regulator proteins in DU145 cells in order to understand the various antitumor effects of FTY720. Apoptosis was quantified by phosphatidylserine exposure. Activation of MAPKs, cleavage of caspase-9 and caspase-8, status of cyclin-dependent kinases (CDKs) and Cip1/p21, a cyclin-dependent kinase inhibitor, were evaluated by Western blot analysis, in addition to FAK and phospho-FAK immunoprecipitation and cell-cycle analysis by FACScan. We found that in DU145 cells, 40 microM FTY720 caused activation of p38 MAPK and the upstream kinase MKK3/MKK6 but not SAPK/JNK. Mitochondrial transmembrane potential, FAK and ERK1/2 were reduced while caspase-9 and caspase-8 were cleaved. The p38-specific inhibitor had no effect on apoptosis induced by FTY720, whereas z-VAD.FMK, a broad-spectrum caspase inhibitor, did not inhibit the p38 MAPK activation. An amount of 20 microM FTY720 resulted in G(1) arrest and a decrease of CDK2 as well as CDK4, whereas it induced Cip1/p21. FTY720 may exert anticarcinogenic effects against prostate cancer cells possibly involving modulation of mitogenic signaling, cell-cycle regulators, induction of G(1) arrest and apoptotic death in DU145 cells.

Analysis of allograft biopsy specimens from long-term surviving patients with stable renal function: predictive value of long-term graft prognosis.

Chronic allograft dysfunction is multi-factorial, and histology of long-term renal allograft shows variable findings. It is important to characterize the pathological features of graft kidneys with normal function to understand the natural course of transplants, which in turn would contribute to elucidate the causes of chronic allograft nephropathy (CAN). To address this issue, we performed 'non-episode' biopsies on well-functioning renal allografts, and evaluated the correlation between clinical outcome and histopathological findings. Patients who underwent a non-episode biopsy had a serum creatinine concentration less than 2.0 mg/dL, urinary protein of less than 500 mg/day and a stable clinical course. In total, 90 such biopsies were performed. Mean follow-up period after biopsy was 29 +/- 16 months. We evaluated the histopathological findings and clinical outcome on each finding. Moreover, we compared the findings in the patients on tacrolimus with those of patients taking cyclosporin. Twenty-three biopsy specimens were essentially normal. Graft dysfunction during the follow-up period was recognized more frequently in patients showing more than one pathological process than in those with isolated findings. Graft outcome was not associated with drug-induced nephropathy, but with acute rejection (P = 0.0193) and CAN (P = 0.0032). Patients found to have CAN-b had a worse outcome than those with CAN-a. CAN-b was less common in the tacrolimus group than in the cyclosporin group. Non-episode biopsy has a predictive value of the long-term outcome of a renal allograft. CAN is associated with graft dysfunction; neither is drug-induced nephropathy. Patients treated with tacrolimus had lower rates of CAN-b than did cyclosporin-treated subjects.

Hepatocyte growth factor prevents the development of chronic allograft nephropathy in rats.

Long-term renal isografts in humans and laboratory animals exhibit features similar to those of chronic allograft nephropathy (CAN), indicating that antigen-independent factors, such as acute renal ischemia, are likely to be involved in the development of CAN. Hepatocyte growth factor (HGF) has been demonstrated to play a renotropic role in renal regeneration and protection from acute ischemic injury. This study was thus conducted to investigate the effect of HGF on the development of CAN, using an established rat model. HGF was administered daily (100 microg/d, intravenously) for 4 wk after engraftment. Control animals received saline solution. Allografts from control animals exhibited early evidence of severe structural collapse and necrotic cell death in the proximal tubules and outer medulla, with mononuclear cell infiltration, within 1 wk after engraftment. This was followed by sequential upregulation of adhesion molecules and cytokines, accompanied by dense macrophage infiltration. Fibrogenic events, as indicated by marked increases in transforming growth factor-beta1 expression and the accumulation of smooth muscle alpha-actin, occurred during the same period. Control animals ultimately developed features typical of CAN, with functional deterioration and severe histologic changes; a survival rate of 50.6% by 32 wk was observed. In contrast, remarkably little early injury and no late fibrogenic events were observed for the HGF-treated group. All treated animals survived, with well preserved graft function, during the 32-wk follow-up period. These results indicate that renal protection and recovery from early allograft injury with HGF treatment greatly contribute to a reduction of susceptibility to the subsequent development of CAN in a rat model. The potential application of HGF in the prevention of CAN warrants further attention.