[Clinical effects of pedicled omental flap transplantation in repairing secondary rejection wounds after brain pacemaker implantation].

Objective: To explore the clinical effects of pedicled omental flap transplantation in repairing secondary rejection wounds after brain pacemaker implantation. Methods: A retrospective observational study was conducted. From January to August 2021, 5 patients with secondary rejection wounds after brain pacemaker implantation who met the inclusion criteria were admitted to the Wound Repair Center of Ruijin Hospital of Shanghai Jiao Tong University School of Medicine, including 3 males and 2 females, aged 56-69 years, with the wound developed at the pulse generator implantation site in the chest in 2 cases, at the connection site of the wire and electrode behind the ear in 2 cases, and at both the chest and the back of the ear in 1 case. All the wounds were repaired by pedicled omental flap transplantation. The wound area after debridement was 2-15 cm2. After operation, the wound healing and related complications (pain, infection, incisional hernia, omental flap necrosis, etc.) were observed. During follow-up, the recurrence of the wound was observed. Results: The wounds of all 5 patients healed within 2 weeks after operation, without related complications. During follow up of 12-18 months, 1 patient got a recurrence of rejection wound behind the left ear 4 months after surgery and eventually had the brain pacemaker removed; the other 4 patients had no recurrence of wounds. Conclusions: Pedicled omental flap transplantation can repair the secondary rejection wounds after brain pacemaker implantation safely and effectively, with few postoperative complications.

[Receptor pathways of glycated basic fibroblast growth factor affecting the proliferation and vascularization of human dermal microvascular endothelial cells].

Objective: To investigate the receptor pathways of glycated basic fibroblast growth factor (bFGF) on proliferation and vascularization of human dermal microvascular endothelial cells (HDMECs). Methods: The experimental research method was used. Glycated bFGF stimulating solution was prepared with glucose and bFGF. HDMECs of the third to sixth passages were used in the experiment. Cells were divided into small interfering RNA (siRNA)-positive control group, siRNA-negative control group, siRNA-receptor for advanced glycation end product (RAGE) group, and siRNA-receptor for fibroblast growth factor (FGFR) group and transfected with siRNA-positive control glyceraldehyde-3-phosphate dehydrogenase, siRNA-negative control, siRNA-RAGE, and siRNA-FGFR for 4 to 6 hours, and then were added into HDMEC culture medium for routine culture. The transfection effect of siRNA was identified by reverse transcription polymerase chain reaction. The cells were divided into normal control group, glycated bFGF alone group, siRNA-RAGE alone group, and siRNA-RAGE+ glycated bFGF group, and seeded into 96-well plate and 6-well plate. Cells in siRNA-RAGE alone group and siRNA-RAGE+ glycated bFGF group were transfected with siRNA-RAGE and then were added into HDMEC culture medium for routine culture. After two days, the original HDMEC culture medium was discarded, and cells in siRNA-RAGE alone group were routinely cultured in HDMEC culture medium, cells in siRNA-RAGE+ glycated bFGF group were routinely cultured in glycated bFGF stimulating solution. Cells in normal control group were routinely cultured in HDMEC culture medium, and cells in glycated bFGF alone group were routinely cultured in glycated bFGF stimulating solution. After transfection with siRNA-RAGE, cells were seeded into 48-well plate and divided into siRNA-RAGE alone group and siRNA-RAGE+ glycated bFGF group. Another cells were directly seeded into 48-well plate without transfection and divided into normal control group and glycated bFGF alone group. Cells in the 4 groups were conducted with the corresponding treatment as above. Cells were divided into normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group and seeded into 96-, 6-, and 48-well plates, respectively, with the corresponding treatment the same as above. Only siRNA-RAGE was replaced by siRNA-FGFR. Cell counting kit 8 method was used to determine the proliferation of cells after 2 days of culture (sample number was 6), flow cytometry was used to detect the apoptosis of cells after 2 days of culture (sample number was 3), tube forming test was used to detect the angiogenesis of cells after 6 hours of culture (sample number was 4). Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: At the 200 bp band, there were no target genes in siRNA-positive control group, siRNA-RAGE group, or siRNA-FGFR group, but target genes were detected in siRNA-negative control group, indicating the success of siRNA transfection. After 2 days of culture, the absorbance value of cells in glycated bFGF alone group was significantly lower than that of normal control group (t=2.359, P<0.05); absorbance value of cells in siRNA-RAGE+ glycated bFGF group was significantly higher than that of glycated bFGF alone group (t=3.858, P<0.01), which was similar to that of siRNA-RAGE alone group (t=2.148, P>0.05). The absorbance value of cells in siRNA-FGFR+ glycated bFGF group was similar to that of glycated bFGF alone group (t=0.805, P>0.05), but significantly lower than that of siRNA-FGFR alone group (t=4.201, P<0.01). After 2 days of culture, the apoptotic rate of cells in glycated bFGF alone group was significantly higher than that of normal control group (t=2.416, P<0.05). The apoptotic rate of cells in siRNA-RAGE+ glycated bFGF group was significantly lower than the rates in glycated bFGF alone group and siRNA-RAGE alone group (t=3.861, 2.724, P<0.05 or P<0.01). There were no statistically significant differences in apoptosis rate of cells among normal control group, glycated bFGF alone group, siRNA-FGFR alone group, and siRNA-FGFR+ glycated bFGF group (F=2.218, P>0.05). After 6 hours of culture, the number of tubules of cells in normal control group (636±5) was significantly more than that of glycated bFGF alone group (580±8, t=10.825, P<0.01), and the number of tubules of cells in siRNA-RAGE+ glycated bFGF group (647±10) was significantly more than those of glycated bFGF alone group and siRNA-RAGE alone group (628±4, t=13.040, 3.641, P<0.01). After 6 hours of culture, the number of tubules of cells in siRNA-FGFR+ glycated bFGF group (619±5) was more than that of glycated bFGF alone group (t=9.000, P<0.01), but less than that of siRNA-FGFR alone group (632±3, t=2.814, P<0.05). Conclusions: Glycated bFGF affects the proliferation and angiogenesis of HDMEC through RAGE pathway, which may be one of the reasons for impaired wound healing of diabetic skin.

[Preliminary evaluation and mechanism of adipose-derived stem cell transplantation from allogenic diabetic rats in the treatment of diabetic rat wounds].

Objective: To investigate whether adipose-derived stem cells (ASCs) from allogeneic diabetic rats can promote wound healing in diabetic rats or not and the mechanism. Methods: (1) Fifty-six male Wistar rats aged 12-16 weeks were divided into diabetic group and healthy group according to the random number table (the same grouping method below), with 28 rats in each group. Rats in healthy group were not treated with any treatment. Rats in diabetic group were injected with 10 g/L streptozotocin 60 mg/kg intraperitoneally in one time to establish the diabetic model. Four rats in diabetic group and 4 rats in healthy group were selected according to the random number table, and the adipose tissue in the inguinal region was taken to culture and purify ASCs, so as to obtain healthy rat-derived ASCs (hereinafter referred to as nASCs) and diabetic rat-derived ASCs (hereinafter referred to as dASCs). The third passage of nASCs (n=3) and dASCs (n=3) were taken, and the positive expression rates of cell surface differentiation antigens CD105, CD31, CD34, and CD44 were detected with flow cytometer for defining ASCs purity. (2) The rest 24 rats in healthy group and 24 rats in diabetic group were used to make three round full-thickness skin defect wounds with a diameter of 12 mm on the back of each rat. Immediately after injury, phosphate buffer saline (PBS), nASCs of 2×10(7)/mL, and dASCs of 2×10(7)/mL each in the volume of 0.5 mL were subcutaneously injected into three wounds and their margins of each rat, respectively. On post injury day (PID) 1, 3, 7, and 12, 6 rats in each group were selected according to the random number table to calculate the wound area, and the wound tissue was stained with hematoxylin-eosin to observe the histological morphology of the wound. (3) Human ASCs (hASCs) were subcultured, and the 4th to 7th passage of cells were used for the subsequent experiments. The hASCs were divided into 7 groups, with 12 samples in each group. Cells in blank control group were cultured with mesenchymal stem cell culture medium, and cells in simple advanced glycation end products (AGEs) group, simple protein group, simple high glucose group, simple high osmotic pressure group, AGEs-high glucose combination group, and protein-high osmotic pressure combination group were cultured with mesenchymal stem cell culture medium containing a final mass concentration of 100 mg/L AGEs, 100 mg/L bovine serum albumin (BSA), 28 mmol/L D-glucose, 28 mmol/L mannitol, 100 mg/L AGEs+ 28 mmol/L D-glucose, 100 mg/L BSA+ 28 mmol/L mannitol, respectively. Cell proliferation was detected by cell counting kit 8 at post culture hour (PCH) 2 and on post culture day (PCD) 2, 4 and 6. (4) The hASCs were divided into blank control group, simple AGE group, simple high glucose group, and AGE-high glucose combination group, with 12 samples in each group, which were treated the same as corresponding groups in experiment (3). On PCD 0, 2, 4, and 6, the positive expression rates of cell surface differentiation antigens CD105, CD44, and CD45 were detected by flow cytometer to estimate their homeostasis. (5) The hASCs were divided into AGE-high glucose combination group and protein-high osmotic pressure combination group, with 9 samples in each group, which were treated the same as corresponding groups in experiment (3). On PCD 2, 4, and 6, the expression of intracellular protein was detected by cyanine 3-streptavidin double-antibody sandwich technique. Data were processed with analysis of variance for factorial design, least significant difference test, and Bonferroni correction. Results: (1) The positive expression rates of CD44 in nASCs and dASCs were both higher than 96%, the positive expression rates of CD31 and CD34 were low, and the positive expression rates of CD105 were about 40%, which basically met the purity requirements. (2) The areas of wounds treated by three methods in rats of healthy group and diabetic group were similar on PID 1 (P>0.05). In healthy group, compared with (0.682 1±0.078 9), (0.314 3±0.113 7), and (0.064 3±0.002 1) cm(2) of the PBS-treated wounds in rats, the area of nASCs-treated wounds in rats decreased significantly on PID 3, 7, and 12 [(0.464 1±0.092 6), (0.223 9±0.072 7), and (0.034 3±0.012 5) cm(2), P<0.05], the area of dASCs-treated wounds in rats decreased significantly on PID 3 and 12 [(0.514 1±0.124 1) and (0.043 7±0.032 8) cm(2), P<0.05] but was not obviously changed on PID 7 [(0.274 2±0.062 5) cm(2), P>0.05]. Compared with those of the dASCs-treated wounds of rats within the same group, the area of the nASCs-treated wounds of rats in healthy group decreased significantly on PID 3 and 7 (P<0.05) but was not obviously changed on PID 12 (P>0.05). In diabetic group, compared with (0.853 5±0.204 8), (0.670 5±0.164 8), and (0.131 4±0.074 4) cm(2) of the PBS-treated wounds in rats, the area of nASCs-treated wounds in rats decreased significantly on PID 3, 7, and 12 [(0.633 4±0.132 5), (0.331 8±0.023 5), and (0.074 2±0.003 8) cm(2), P<0.05], the area of dASCs-treated wounds in rats decreased significantly on PID 3 [(0.773 6±0.182 2) cm(2), P<0.05] but was not obviously changed on PID 7 and 12 [(0.510 6±0.192 2) and (0.114 4±0.003 1) cm(2), P>0.05]. Compared with the dASCs-treated wounds of rats within the same group, the area of the nASCs-treated wounds of rats in diabetic group was not obviously changed on PID 3 and 7 (P>0.05) but decreased significantly on PID 12 (P<0.05). There was no obvious difference in histological morphology of the wounds treated with three methods in rats of each group on PID 1. On PID 3, a small amount of microvessels were formed in the wounds treated with nASCs and dASCs of rats in both groups, but microvessel formation was almost undetected in the PBS-treated wounds. On PID 7, more small blood vessels and fibroblasts (Fbs) were observed in the wounds treated with nASCs and dASCs of rats in both groups, but the small blood vessels and Fbs were slightly less in the PBS-treated wounds. On PID 12, the wounds treated with nASCs and dASCs of rats in the two groups were covered by epithelial tissue, the granulation tissue in the PBS-treated wounds of rats in healthy group was not obvious, and the PBS-treated wounds of rats in diabetic group were not completely epithelialized. (3) Compared with those of blank control group, the cell number of hASCs in simple AGEs group decreased significantly on PCD 2, 4, and 6 (P<0.05), which increased significantly on PCD 2 and 4 in simple high glucose group (P<0.05), and that in AGEs-high glucose combination group decreased significantly on PCD 4 and 6 (P<0.05). (4) Compared with that on PCD 4 within the same group, the positive expression rate of CD105 in hASCs decreased significantly in blank control group, simple AGEs group, and AGEs-high glucose combination group on PCD 6 (P<0.05). The positive expression rate of CD44 was higher than 95%, and that of CD45 was less than 2% in hASCs of each group at each time point. (5) Detection values of 7 proteins were located in the confidence interval. The expression levels of basic fibroblast growth factor and tissue inhibitor of metalloproteinase-1 in hASCs of AGEs-high glucose combination group and protein-high osmotic pressure combination group showed increasing trend with the prolongation of culture time. The expression level of human monocyte chemoattractant protein 1 (MCP-1) in hASCs of AGEs-high glucose combination group showed increasing trend with the prolongation of culture time, while the expression level of growth-regulated oncogene (GRO) on PCD 6 was significantly higher than that on PCD 4 within the same group (P<0.05); the expression levels of MCP-1 and GRO in hASCs of protein-high osmotic pressure combination group showed decreasing trend with the prolongation of culture time. The expression level of follistatin in hASCs of protein-high osmotic pressure combination group decreased obviously on PCD 4, while that in hASCs of AGEs-high glucose combination group was significantly lower on PCD 6 than that on PCD 4 (P<0.05). The expression level of vascular endothelial growth factor (VEGF) in hASCs of protein-high osmotic pressure combination group decreased gradually with the prolongation of culture time, while that in hASCs of AGEs-high glucose combination group on PCD 4 decreased significantly as compared with that on PCD 2 (P<0.05). The expression level of urokinase-type plasminogen activator receptor in hASCs of protein-high osmotic pressure combination group on PCD 6 was significantly higher than that on PCD 4 within the same group (P<0.05) and that of AGEs-high glucose combination group on PCD 6 (P<0.05). Conclusions: Both nASCs and dASCs can promote wound healing in rats with simple defect injury, but dASCs have no significant effect on wound healing in rats with diabetes mellitus, which may be related to the inhibition of ASCs proliferation and the influence of high glucose and AGEs intervention on their homeostasis and secretory function.

[Thoughts and principles of diagnosis and treatment of chronic refractory wounds in China].

The correct thoughts and principles of diagnosis and treatment of chronic refractory wounds need to be formulated. Through the relevant domestic and international consensus and based on clinical experience, the Thoughts and principles of diagnosis and treatment of chronic refractory wounds in China is proposed. It is considered that in the diagnosis and treatment of chronic refractory wounds, in the case of fully understanding the patient's medical history, the following thoughts and principles should be complied in order. (1) Pay attention to the cleanliness of the wound after being cleaned. (2) Reasonably perform debridement to avoid being " excessive" or " not thorough". (3) Reasonably perform examination, diagnosis, and differential diagnosis of pathogenic factors. (4) Treat according to etiology. (5) Find comorbidities and prevent adverse outcomes. (6) Select the correct wound treatment method reasonably and timely. When the conservative wound care treatment is considered, pay attention to embodying the concept of etiological treatment, treat the wound according to the principles of safety, phase, selectivity, and effectiveness, and make a reasonable choice of continuing conservative treatment or surgical treatment in time after completing the preparation of the wound bed. When surgical treatment is considered, pay attention to the selection of reasonable surgical method and donor site, pay attention to the healing rate of surgical wound site and the outcome of donor site, and give reasonable protection to the wound site after surgery. (7) Carry out rehabilitation treatment after wound healing and related health education.

[Effects of denatured collagen type â on differentiation of human fibroblasts into myofibroblasts].

Objective: To investigate the effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts. Methods: A small amount of normal skin donated by burn patients undergoing scar surgery was collected. Human fibroblasts were obtained by method of explant culture and then sub-cultured. The fourth passage of cells were used in the following experiments. (1) Fibroblasts were divided into normal collagen group and denatured collagen group according to the random number table, with 10 wells in each group. Fibroblasts in normal collagen group were cultured on normal collagen type Ⅰ coated coverslips. Fibroblasts in denatured collagen group were cultured on denatured type Ⅰ collagen coated coverslips. Expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical method, and the percentage of PCNA positive cells was calculated. (2) Another batch of fibroblasts were grouped and treated as in (1), with 12 wells in each group. Proliferation activity of cells was determined with methyl-thiazolyl-tetrazolium colorimetry method. (3) Another batch of fibroblasts were grouped and treated as in (1), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of fibroblasts were grouped and treated as in (1). Expression of α smooth muscle actin (α-SMA) of cells was detected by immunohistochemical method, and expression of OB-cadherin of cells was detected by immunofluorescence method. (5) Another batch of fibroblasts were divided into normal collagen, denatured collagen, and common coverslips groups according to the random number table, with 6 wells in each group. Fibroblasts in normal collagen and denatured collagen groups were treated as in (1), while fibroblasts in common coverslips group were cultured on coverslips without collagen coating. Expressions of α-SMA and OB-cadherin of cells were determined with Western blotting. (6) Another batch of fibroblasts were grouped and treated as in (5), and then the mRNA expressions of collagen type Ⅰ, collagen type Ⅲ, and α-SMA of cells were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with t test, one way analysis of variance, and least-significant difference test. Results: (1) The percentage of PCNA positive cells in denatured collagen group was (83±9)%, significantly higher than (29±9)% in normal collagen group (t=13.53, P<0.01). (2) The proliferation activity of fibroblasts in denatured collagen group was 0.32±0.06, significantly higher than 0.25±0.05 in normal collagen group (t=3.06, P<0.01). (3) The microfilament of fibroblasts in normal collagen group was arranged vertically and in parallel way, paralleling the long axis of cells. The microfilament of fibroblasts in denatured collagen group was denser and thicker. (4) Most fibroblasts in normal collagen group showed long shuttle-like shape typically. Morphology of fibroblasts in denatured collagen group changed, and cells were obviously spreading. Expressions of α-SMA and OB-cadherin of fibroblasts in denatured collagen group were stronger than those in normal collagen group. (5) Expressions of α-SMA of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 1.69±0.41, 0.89±0.27, and 1.46±0.42. Expression of α-SMA of fibroblasts in denatured collagen group was significantly higher than that in normal collagen group (P<0.01). Expressions of OB-cadherin of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 5.17±0.28, 2.21±0.10, and 4.01±0.56. Expression of OB-cadherin of fibroblasts in denatured group was significantly higher than that in normal collagen group (P<0.01). (6) There was no significant difference in mRNA expression of collagen type Ⅰ of fibroblasts in denatured collagen, normal collagen, and common coverslips groups (F=2.71, P>0.05). The mRNA expressions of collagen type Ⅲ and α-SMA of fibroblasts in normal collagen group were significantly lower than those in denatured collagen group (P<0.01). Conclusions: Denatured collagen type Ⅰ may influence the activity of fibroblasts, thus inducing fibroblasts differentiating into myofibroblasts.

MiR-133a inhibits fracture healing via targeting RUNX2/BMP2.

OBJECTIVE: To analyze the mechanism of miR-133a in inhibiting fracture healing through regulating runt-related transcription factor 2 (RUNX2) signaling pathway. PATIENTS AND METHODS: A total of 80 patients with fracture admitted to our hospital from January 2016 to January 2017 were divided into 2 groups according to nonunion fracture or healing fracture: nonunion fracture group (n = 40) and control group (n= 40). After admission, patients underwent the surgery, respectively, and the bone tissues were taken for stand-by application. The expression level of bone morphogenetic protein 2 (BMP2) was detected using the immunohistochemical method, the expression level of RUNX2 protein was detected by Western blotting, and the expression level of micro ribonucleic acid (miR)-133a was detected by quantitative polymerase chain reaction (qPCR). Moreover, the bioinformatics method was used to predict the target gene of miR-133a, and the luciferase reporter gene was used to detect the binding of miR-133a to RUNX2. RESULTS: Compared with those in control group, the expression of BMP2 and the relative expressions of RUNX2 protein and miR-133a were significantly decreased; the differences were statistically significant (p < 0.05). Pearson correlation analysis showed that miR-133a was negatively correlated with RUNX2. After overexpression of miR-133a, the expression level of RUNX2 was decreased, but it was increased significantly after interference in miR-133a. Besides, it was found in dual-luciferase reporter assay that miR-133a bound to RUNX2. CONCLUSIONS: MiR-133a inhibits the bone formation through inhibiting the RUNX2/BMP2 signaling pathway, thereby negatively regulating the fracture healing.

Overexpression of PIK3CA in murine head and neck epithelium drives tumor invasion and metastasis through PDK1 and enhanced TGFß signaling.

Head and neck squamous cell carcinoma (HNSCC) patients have a poor prognosis, with invasion and metastasis as major causes of mortality. The phosphatidylinositol 3-kinase (PI3K) pathway regulates a wide range of cellular processes crucial for tumorigenesis, and PIK3CA amplification and mutation are among the most common genetic alterations in human HNSCC. Compared with the well-documented roles of the PI3K pathway in cell growth and survival, the roles of the PI3K pathway in tumor invasion and metastasis have not been well delineated. We generated a PIK3CA genetically engineered mouse model (PIK3CA-GEMM) in which wild-type PIK3CA is overexpressed in head and neck epithelium. Although PIK3CA overexpression alone was not sufficient to initiate HNSCC formation, it significantly increased tumor susceptibility in an oral carcinogenesis mouse model. PIK3CA overexpression in mouse oral epithelium increased tumor invasiveness and metastasis by increasing epithelial-mesenchymal transition and by enriching a cancer stem cell phenotype in tumor epithelial cells. In addition to these epithelial alterations, we also observed marked inflammation in tumor stroma. AKT is a central signaling mediator of the PI3K pathway. However, molecular analysis suggested that progression of PIK3CA-driven HNSCC is facilitated by 3-phosphoinositide-dependent protein kinase (PDK1) and enhanced transforming growth factor ß (TGFß) signaling rather than by AKT. Examination of human HNSCC clinical samples revealed that both PIK3CA and PDK1 protein levels correlated with tumor progression, highlighting the significance of this pathway. In summary, our results offer significant insight into how PIK3CA overexpression drives HNSCC invasion and metastasis, providing a rationale for targeting PI3K/PDK1 and TGFß signaling in advanced HNSCC patients with PIK3CA amplification.

Redox-dependent Brca1 transcriptional regulation by an NADH-sensor CtBP1.

C-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor and metabolic sensory protein, which often represses tumor suppressor genes. Hence, we sought to determine if CtBP1 affects expression of the tumor suppressor Brca1 in head and neck tissue, as downregulation of Brca1 begins at the early stages of head and neck squamous cell carcinomas (HNSCCs). We found that CtBP1 represses Brca1 transcription by binding to the E2F4 site of the Brca1 promoter. Additionally, the recruitment of CtBP1 to the Brca1 promoter is redox-dependent, that is, increased at high NADH levels in hypoxic conditions. Further, immunostaining using a human HNSCC tissue array revealed that nuclear CtBP1 staining began to accumulate in hyperplasic lesions and HNSCCs, this staining correlated with Brca1 downregulation in these lesions. Pharmacological disruption of CtBP1 binding to Brca1 promoter by the antioxidant Tempol, which reduces NADH levels, relieved CtBP1-mediated repression of Brca1, leading to increased DNA repair in HNSCC cells. As tumor cells are generally hypoxic with increased NADH levels, the dynamic control of Brca1 by a 'metabolic switch' found in this study not only provides an important link between tumor metabolism and tumor suppressor expression but also suggests a potential chemo preventative or therapeutic strategy for HNSCC by blocking NADH-dependent CtBP1 activity at early stages of HNSCC carcinogenesis.

Inhibition of group A streptococcal infection by Melaleuca alternifolia (tea tree) oil concentrate in the murine model.

AIMS: To investigate the effect of a water-soluble Melaleuca alternifolia concentrate (MAC) on group A streptococcus (GAS; Streptococcus pyogenes)-induced necrotizing fasciitis. METHODS AND RESULTS: MAC pretreatment (1% and 2% v/v) was able to protect mice from GAS infection in an air pouch model. GAS-induced mouse death and skin injury were inhibited dose dependently by MAC. Administration of MAC at 6 h post-GAS infection partially delayed mouse death. Surveys of the exudates of the air pouch of MAC-treated mice revealed that the survival of infiltrating cells was prolonged, the bacteria were eliminated, and the production of inflammatory cytokines was inhibited. MAC could directly inhibit the growth of GAS in vitro, and the minimal inhibitory concentration (MIC) of MAC for GAS was determined as 0.05% v/v using the time-kill assay. Furthermore, a sub-MIC dose of MAC not only enhanced the bactericidal activity of RAW264.7 macrophage cells against GAS but also increased susceptibility of GAS for blood clearance. CONCLUSIONS: These results suggest that MAC may inhibit GAS-induced skin damage and mouse death by directly inhibiting GAS growth and enhancing the bactericidal activity of macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide scientific data on the use of MAC for the treatment of GAS-induced necrotizing fasciitis in the murine model.

In situ RT-PCR detection of hepatitis C virus genotypes in Chinese patients with hepatocellular carcinoma.

The correlation of hepatitis C virus genotypes and the development of hepatocellular carcinoma is still controversial. The most pertinent studies are conducted by seroepidemiological methods. It has been suggested that tissue RT-PCR may be more efficient than serological testing for hepatitis C virus RNA. The purpose of this study was to investigate the infectious status of hepatitis C virus genotypes in hepatocellular carcinoma tissue for revealing the role of hepatitis C virus genotypes in carcinogenesis of hepatocellular carcinoma. Hepatitis C virus genotypes in cancerous and noncancerous liver tissues of 95 Chinese patients with hepatocellular carcinoma were analyzed by a modified type-specific in situ reverse transcription polymerase chain reaction (RT-PCR) method. Hepatitis C virus genotypes were simultaneously examined by Okamoto's method using extracts from hepatocellular carcinoma tissues. The hepatitis C virus genotypes in hepatocellular carcinoma tissues were found including 1b, 2a, mixed type (1b+2a) and 3a in 13, 23, 2 and 4 samples respectively. There was no significant difference in either the positivities between 1b (13.7%, 13/95) and 2a (24.2%, 23/95) (P>0.05) or the staining intensity and distribution of positive cells between 1b and 2a (p>0.05). No statistically significant difference was found between the two virus genotypes in main clinico-pathologic parameters of hepatocellular carcinoma. The localizations of hepatitis C virus RNA-positive signals were mainly cytoplasmic (22/36) in noncancerous regions, but nucleocytoplasmic (19/36) or nuclear (3/36) in cancerous regions. The positive nucleocytoplasmic/nuclear signals of genotype 1b (11/13) in hepatocellular carcinoma were seen more frequently than those of genotype 2a (11/23) (P<0.05). The discordant hepatitis C virus 2a and 1b positivities in serum samples and in cancerous tissues of hepatocellular carcinoma patients were also found, suggesting that the detection of serum hepatitis C virus genotypes in the patients with hepatocellular carcinoma may not reflect the actual status of hepatitis C virus genotypes in cancerous tissue. The pathogenetic significance of existence of hepatitis C virus gene in the nucleus of hepatocytes and malignant cells needs further investigation.

Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype.

ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Atm-DeltaSRI, was designed as a model of one of the most common deletion mutations (7636del9) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556-2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-DeltaSRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm(-/-) mice. The life span of Atm-DeltaSRI mice was significantly longer than that of Atm(-/-) mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-DeltaSRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-DeltaSRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm(-/-) mice were observed in older Atm-DeltaSRI animals. Thus, expression of mutant protein in Atm-DeltaSRI knock-in mice gives rise to a discernibly different phenotype to Atm(-/-) mice, which may account for the heterogeneity seen in A-T patients with different mutations.

Synthesis of 2-amido-3-hydroxypyridin-4(1H)-ones: novel iron chelators with enhanced pFe3+ values.

The synthesis of a range of 2-amido-3-hydroxypyridin-4-ones as bidentate iron(III) chelators with potential for oral administration is described. The pKa values of the ligands together with the stability constants of their iron(III) complexes have been determined. Results indicate that the introduction of an amido substituent at the 2-position leads to an appreciable enhancement of the pFe3+ values. The ability of these novel 3-hydroxypyridin-4-ones to facilitate the iron excretion in bile was investigated using a 59Fe-ferritin loaded rat model. The optimal effect was observed with the N-methyl amido derivative 15b, which has an associated pFe3+ value of 21.7, more than two orders of magnitude higher than that of deferiprone (1,2-dimethyl-3-hydroxypyridin-4-one) 1a (pFe3+ = 19.4). Dose response studies suggest that chelators with high pFe3+ values scavenge iron more effectively at lower doses when compared with simple dialkyl substituted hydroxypyridinones.

Design, synthesis, and biological evaluation of aromatic ester prodrugs of 1-(3'-hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41) as orally active iron chelators.

In order to improve chelation efficacy and to minimise toxicity, eleven aromatic ester prodrugs of 1-(3'-hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41) have been synthesised. The distribution coefficients of these ester prodrugs between 1-octanol and MOPS buffer pH 7.4 were measured together with their rates of hydrolysis at pH 2 and pH 7.4, in rat blood and liver homogenate. The biliary metabolic profiles of selected ester prodrugs were investigated in rats. The in vivo iron mobilisation efficacy of these ester prodrugs has been compared with that of the parent drug using a 59Fe-ferritin loaded rat model. The hydrolytic rates of these esters vary appreciably, esters with heteroaromatic acid moieties being less stable than the corresponding benzoyl analogues. Many prodrugs were found to enhance the ability of the parent hydroxypyridinone to facilitate 59Fe excretion, the optimal effect being observed with the 4-methylbenzoyl ester derivative 8d. However, not all prodrugs provide an increased efficacy, indicating that lipophilicity is not the only factor which influences drug efficacy. Furthermore no clear correlation between lipophilicity, susceptibility towards hydrolysis and efficacy was detected.

Hydrolytic and metabolic characteristics of the esters of 1-(3'-hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41), potentially useful iron chelators.

1-(3'-Hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41) has been extensively investigated as an orally effective iron chelator. In order to improve the pharmacokinetic and metabolic properties of CP41, eleven aromatic esters have been synthesised and tested as potential prodrugs. In the present study, the hydrolytic rates of these CP41 esters in phosphate buffer (pH2.0 and pH 7.4), rat blood and rat liver homogenate have been determined and found to cover a wide range. Generally, they possessed relatively slow hydrolytic rates in phosphate buffer (0-50 nmol/ml/hr at pH 2.0 and 0-140 nmol/ml/hr at pH 7.4). The hydrolytic rates in rat blood fell in the range of 9-5766 nmol/ml blood/hr and in rat liver homogenate 1-800 micromol/g liver tissue/hr. All esters possess a higher lipophilicity than that of the parent compound CP41. Although no apparent relationship was observed between the lipophilicities and hydrolytic rates, the esters with relatively higher hydrolytic rates in liver homogenate tend to possess higher iron scavenging efficacies. Further investigation of the metabolism of selected CP41 esters indicates that metabolism is a key factor influencing the efficacy of CP41 esters, as some esters can be metabolically inactivated in the liver in preference to undergoing ester hydrolysis. Ester design, combined with a knowledge of the prodrug metabolism, is a useful strategy for the production of 3-hydroxypyridin-4-ones with enhanced iron scavenging efficacy.

Design, synthesis and evaluation of N-basic substituted 3-hydroxypyridin-4-ones: orally active iron chelators with lysosomotrophic potential.

To investigate the possibility of targeting chelators into the lysosomal iron pool, nine bidentate 3-hydroxypyridin-4-ones with basic chains have been synthesized. As the turnover of ferritin iron is centred in the lysosome, such strategy is predicted to increase chelator efficacy of bidentate ligands. The pKa values of the ligands together with their distribution coefficients between 1-octanol and 4-morpholinepropane sulphonic acid (MOPS) buffer pH 7.4 have been determined. The in-vivo iron mobilization efficacy of these basic 3-hydroxypyridin-4-ones has been investigated in a 59Fe-ferritin-loaded rat model. No obvious correlation was observed between efficacy and the pKa value of the side chain, although those with pKa > 7.0 tended to be more efficient than those with pKa < 7.0. The imidazole-containing molecules are much less effective than the tertiary amine derivatives. A dose-response study suggested that basic pyridinones are relatively more effective at lower doses when compared with N-alkyl hydroxypyridinones. Optimal effects were observed with the piperidine derivatives 4h and 4i. The derivative 4i at a dose of 150 micromol kg(-1) was more effective than 450 micromol kg(-1) deferiprone, the widely adopted clinical dose.

Synthesis, physicochemical characterization, and biological evaluation of 2-(1'-hydroxyalkyl)-3-hydroxypyridin-4-ones: novel iron chelators with enhanced pFe(3+) values.

The synthesis of a range of 2-(1'-hydroxyalkyl)-3-hydroxypyridin-4-ones as bidentate iron(III) chelators with potential for oral administration is described. The pK(a) values of the ligands and the stability constants of their iron(III) complexes have been determined. Results indicate that the introduction of a 1'-hydroxyalkyl group at the 2-position leads to a significant improvement in the pFe(3+) values. Such an effect was found to be greater with the hydroxyethyl substituent than with the hydroxymethyl substituent, particularly in the cases of 1-ethyl-2-(1'-hydroxyethyl)-3-hydroxypyridin-4-one (pFe(3+) = 21.4) and 1,6-dimethyl-2-(1'-hydroxyethyl)-3-hydroxypyridin-4-one (pFe(3+) = 21.5) where an enhancement on pFe(3+) values in the region of two orders of magnitude is observed, as compared with Deferiprone (1, 2-dimethyl-3-hydroxypyridin-4-one) (pFe(3+) = 19.4). The ability of these novel 3-hydroxypyridin-4-ones to facilitate the iron excretion in bile was investigated using a [(59)Fe]ferritin-loaded rat model. Chelators and prodrug chelators possessing high pFe(3+) values show great promise in their ability to remove iron under in vivo conditions.

Gradient ion-pair high-performance liquid chromatographic method for analysis of 3-hydroxypyridin-4-one iron chelators.

A gradient ion-pair HPLC separation of highly hydrophilic 3-hydroxypyridin-4-one (HPO) iron chelators is described. The separation of HPOs was performed using a reversed-phase polymer HPLC column (PLRP-S 100 A, 15x0.46 cm ID, 5 microm). The ion-pair buffer contained 1-heptanesulfonic acid (sodium salt) (5 mM) and the pH was adjusted to 2.0 using HCl. The gradient was 2%-35% CH3CN in 20 min and post-run was followed for 5 min using 2% CH3CN and 98% buffer. The flow-rate was 1 ml/min and the analytes were monitored at 280 nm. The retention times of 30 hydrophilic HPOs fell in the range of 10-18 min with sharp peak shapes, although these iron chelators possess various functional groups and distribution coefficients. The application of this HPLC method in the analysis of HPO chelators and their metabolites in rat bile and urine is described.

Synthesis, physicochemical properties and biological evaluation of aromatic ester prodrugs of 1-(2'-hydroxyethyl)-2-ethyl-3-hydroxypyridin-4-one (CP102): orally active iron chelators with clinical potential.

The synthesis of seven aromatic ester derivatives of 1-(2'-hydroxyethyl)-2-ethyl-3-hydroxypyridin-4-one is described. These ester prodrugs have been designed to target iron chelators to the liver, the major iron storage organ. In principle this should improve chelation efficacy and minimize toxicity. The distribution coefficients of these ester prodrugs between 1-octanol and MOPS buffer pH 7.4 were measured together with their rates of hydrolysis at pH 2 and pH 7.4, in rat blood and liver homogenate. Esters with heteroaromatic acid moieties were found to be less stable than benzoyl analogues. The in-vivo iron mobilisation efficacy of these ester prodrugs has been compared with that of the parent drug using a 59Fe-ferritin loaded rat model. Many prodrugs were found to enhance the ability of the parent hydroxypyridinone to facilitate 59Fe excretion. However, not all prodrugs provided increased efficacy, demonstrating that lipophilicity is not the only factor which influences drug efficacy. Furthermore, no clear correlation between efficacy and susceptibility to hydrolysis was detected. The picolinic and nicotinic ester derivatives appear to offer the best potential as prodrugs as they have a relatively low LogP value and yet lead to enhanced efficacy over the parent hydroxypyridinone.

Predominant germ-line mutation of the hMSH2 gene in Japanese hereditary non-polyposis colorectal cancer kindreds.

By means of PCR-SSCP and direct sequencing, we detected 12 germ-line mutations of hMSH2 or hMLH1 in 37 Japanese hereditary non-polyposis colorectal cancer (HNPCC) kindreds, of whom 15 satisfied the Amsterdam and 22 the Japanese criteria. The germ-line mutation detection rate of hMSH2 was much higher than that of hMLH1 (11/37 vs. 1/37). The total mutation detection rate of hMSH2 and hMLH1 in the Amsterdam criteria group was significantly higher than that in the Japanese criteria group (9/15 vs. 3/22). Furthermore, the mean age of the HNPCC patients in the mutation-positive group was lower than that in the mutation-negative one; the rates of both vertical transmission and multiplicity of tumors in the mutation-positive group were higher than those in the mutation-negative one. In addition, the number of patients with microsatellite instability-positive cancers in the mutation-positive group was higher than that in the mutation-negative one. Our results suggest firstly that the hMSH2 gene plays a much more important role than hMLH1 in the carcinogenesis of Japanese HNPCC patients, secondly that the rate of hMSH2 and hMLH1 mutations is high in the kindreds satisfying the Amsterdam criteria and thirdly that both the clinical phenotypes (early onset, vertical transmission and multiplicity of tumors) and the microsatellite instability status are important for the genetic screening of HNPCC.

Two divergent signaling pathways for TGF-beta separated by a mutation of its type II receptor gene.

Transforming growth factor beta (TGF-beta) can inhibit epithelial cell growth and induce extracellular matrix formation through signal transduction via its two receptors and its downstream intracellular Smad proteins. We recently reported a germline mutation, i.e., substitution of methionine for threonine at codon 315 in the kinase subdomain IV, of the TGF-beta type II receptor gene in a kindred of hereditary nonpolyposis colorectal cancer without microsatellite instability and found that the mutant receptor abolished the signal transduction for growth inhibition by TGF-beta. In this study, we performed further functional analysis of this mutant receptor. The results showed that, in contrast to its failure to mediate growth inhibition by TGF-beta, the mutant receptor still retained the ability to induce one of the extracellular matrix proteins, plasminogen activator inhibitor type 1, upon TGF-beta treatment. However, coincident with its failure to mediate growth inhibition by TGF-beta, the mutant receptor failed to transcriptionally upregulate one of the cyclin-dependent kinase inhibitors, p15(INK4B), in response to TGF-beta. These data suggest that threonine 315 of the TGF-beta type II receptor is dispensable for extracellular matrix protein production, but is essential for the growth inhibition by TGF-beta, and that the lack of growth inhibition due to the mutant receptor is possibly mediated through its failure to upregulate p15(INK4B).