[Effects of Dapagliflozin on contrast-induced acute kidney injury in patients with type 2 diabetes mellitus underwent percutaneous coronary intervention].

Objective: To investigate the effect of Dapagliflozin, sodium-glucose cotransporter 2 inhibitor (SGLT2i), on contrast-induced acute kidney injury (CIAKI) in patients with type 2 diabetes mellitus (T2DM) after percutaneous coronary intervention(PCI). Methods: A cohort study. The clinical data of 366 patients with coronary heart disease combined with T2DM who underwent PCI in the Department of Cardiology, Tianjin University Chest Hospital, from June 2021 to June 2022 were retrospectively analyzed, including 218 males and 148 females, aged (64.6±11.0) years old. According to whether the patients had used Dapagliflozin or not, the selected patients were divided into SGLT2i group(n=124) and control group(n=242). The changes in cardiac indicators, renal function, and inflammatory response indicators before and 72 hours after PCI treatment were analyzed and compared between the two groups. The incidence rate of CIAKI in the two groups was analyzed, and the influencing factors of CIAKI were analyzed by multivariate logistic regression. The major adverse cardiac events (MACE) were recorded during the follow-up period of the two groups, and Kaplan-Meier survival analysis and log-rank test were used to compare the differences in MACE occurrence between the two group. Results: The left ventricular ejection fraction (LVEF) of the SGLT2i group was lower than that of the control group, and the proportion of patients with LVEF<45% and CIAKI risk score were higher than those of the control group, with statistical significance (all P<0.05). 72 h after PCI treatment, ß-2 Microglobulin(ß-2MG), cystatin-C(Cys-C), and neutrophil gelatinase-associated lipocalin (NGAL) in both groups were all increased compared to those before PCI treatment, with statistical significance (all P<0.05).ß-2MG, Cys-C, and NGAL in SGLT2i group were all lower than those in the control group, with statistical significance(all P<0.05).The levels of interleukin-6(IL-6), hypersensitive C-reactive protein (hs-CRP), and malondialdehyde in both groups of patients increased compared to preoperative levels, while the levels of superoxide dismutase (SOD) decreased compared to preoperative levels, with statistical significance (all P<0.05). The levels of IL-6, hs-CRP, and malondialdehyde in the SGLT2i group were lower than those in the control group, while SOD was higher than that in the control group, with statistical significance (all P<0.05). Among all patients included, 34 cases experienced CIAKI (9.8%), and the incidence of CIAKI in the SGLT2i group was lower than that in the control group [4.8% (6/124) vs 11.6% (28/242),P=0.037]. Multivariate logistic regression analysis showed that the use of dapagliflozin was a protective factor for CIAKI in T2DM patients receiving PCI treatment (OR=0.321, 95%CI: 0.127-0.816, P=0.017). After a follow-up of 14.0 (12.0, 16.2) months, the incidence of MACE in SGLT2i group was lower than that in the control group (7.3% vs 12.8%, P=0.048). Conclusions: Dapagliflozin may reduce the risk of CIAKI and MACE in T2DM patients after PCI treatment. Its mechanism may be related to the anti-inflammatory and antioxidant effects of SGLT2i.

Glycosylated hemoglobin levels and the risk for contrast-induced nephropathy in diabetic patients undergoing coronary arteriography/percutaneous coronary intervention.

BACKGROUNDS: Diabetes mellitus is an independent risk factor for Contrast-induced nephropathy (CIN) in patients undergoing Coronary arteriography (CAG)/percutaneous coronary intervention (PCI). Glycosylated hemoglobin (HbA1c) is the gold standard to measure blood glucose control, which has important clinical significance for evaluating blood glucose control in diabetic patients in the past 3 months. This study aimed to assess whether preoperative HbA1c levels in diabetic patients who received CAG/PCI impacted the occurrence of postoperative CIN. METHODS: We reviewed the incidence of preoperative HbA1c and postoperative CIN in 670 patients with CAG/PCI from January 1, 2020 to October 30, 2020 and divided the preoperative HbA1c levels into 5 groups. Blood samples were collected at admission, 48 h and 72 h after operation to measure the Scr value of patients. Categorical variables were compared using a chi-square test, and continuous variables were compared using an analysis of variance. Fisher's exact test was used to compare the percentages when the expected frequency was less than 5. Univariable and multivariable logistic regression analysis was used to exclude the influence of confounding factors, and P for trend was used to analyze the trend between HbA1c levels and the increased risk of CIN. RESULTS: Patients with elevated HbA1c had higher BMI, FBG, and LDL-C, and they were more often on therapy with hypoglycemic agents, Insulin and PCI. They also had higher basal, 48 h and 72 h Scr. The incidence of CIN in the 5 groups of patients were: 9.8, 11.9, 15.2, 25.3, 48.1%. (p < 0.0001) The multivariate analysis confirmed that in the main high-risk subgroup, patients with elevated HbA1C levels (≥8.8%) had a higher risk of CIN disease. Trend test showed the change of OR (1.000,1.248,1.553,2.625,5.829). CONCLUSIONS: Studies have shown that in diabetic patients undergoing CAG/PCI, elevated HbA1c is independently associated with the risk of CIN, and when HbA1c > 9.5%, the incidence of CIN trends increase. Therefore, we should attach great importance to patients with elevated HbA1c at admission and take more active measures to prevent CIN.

Comparative efficacy of adjuvant trastuzumab-containing chemotherapies for patients with early HER2-positive primary breast cancer: a network meta-analysis.

BACKGROUND: Trastuzumab (H) with chemotherapy benefits patients with HER2+ breast cancer (BC); however, we lack head-to-head pairwise assessment of survival or cardiotoxicity for specific combinations. We sought to identify optimal combinations. METHODS: We searched PubMed, updated October 2017, using keywords "Breast Neoplasms/drug therapy," "Trastuzumab," and "Clinical Trial" and searched Cochrane Library. Our search included randomized trials of adjuvant H plus chemotherapy for early-stage HER2+ BC, and excluding trials of neoadjuvant therapy or without data to obtain hazard ratios (HRs) for outcomes. Following PRISMA guidelines, one investigator did initial search; two others independently confirmed and extracted information; and consensus with another investigator resolved disagreements. Before gathering data, we set outcomes of overall survival (OS), event-free survival (EFS), and severe cardiac adverse events (SCAEs). Analyzing 6 trials and 13,621 patients, we made direct and indirect comparisons using network meta-analysis on HR for OS or EFS and on odds ratio (OR) for SCAE; ranked therapy was done based on outcomes using p scores. RESULTS: Compared with anthracycline-cyclophosphamide with taxane (ACT), ACT with concurrent H (ACT+H) showed best OS (HR 0.63, 95% confidence interval [CI] 0.55, 0.72), followed by taxane and carboplatin (TC) with concurrent H (TC+H) (HR 0.77, 95% CI 0.59, 1) and ACT with sequential H (ACT-H) (HR 0.85, 95% CI 0.68, 1.05). Pairwise comparisons showed statistically significant OS benefit for ACT+H over others; similar results for EFS. TC+H showed statistically significant lower SCAE risk compared to ACT+H (OR 0.13, 95% CI 0.03, 0.61). CONCLUSIONS: Concurrent H with ACT or TC showed most clinical benefit for early-stage HER2+ BC; TC+H had lowest cardiotoxicity.

[Effect of probucol on preventing contrast-induced nephropathy in patients undergoing percutaneous coronary intervention].

Objective: To investigate the preventive effect, possible mechanism and safety of probucol on contrast-induced nephropathy (CIN) after percutaneous coronary intervention (PCI) in patients with coronary heart disease (CHD). Methods: A total of 641 patients with coronary heart disease were consecutively enrolled from Department of Cardiology, in Tianjin Chest Hospital, Tianjin TEDA International Cardiovascular Hospital, Tianjin First Central Hospital, Tianjin Fourth Central Hospital. They were randomly divided into probucol group (n=321) and control group (n=320). The probucol group was given oral probucol 500 mg twice daily for day 0 to day 3 after PCI; the control group was given only conventional therapy. All patients were given intravenous drip 0.9% sodium chloride solution before 12 to 24 hours of operation. The levels of serum creatinine (Scr), blood urea nitrogen (BUN), evaluate glomerular filtration rate (eGFR), cystatin C (Cys-C), and high-sensitivity C-reactive protein (hs-CRP), neutrophil gelatinase associated lipocalin (NGAL), superoxide dismutase (SOD) and glutathione (GSH) were measured before and 72 h after the PCI operation in both groups. The incidence rates of CIN, the adverse events during hospitalization and postoperative 14-day follow-up were recorded in two groups. Results: There was no statistically significantly difference in the levels of Scr, BUN, eGFR, Cys-C, hs-CRP, NGAL, SOD and GSH between the two groups before PCI (P>0.05). The levels of serum Scr, BUN, Cys-C, hs-CRP, NGAL, SOD and GSH after operation in the two groups were higher than those before the operation (P<0.05). The levels of hs-CRP and NGAL in the probucol group were lower than those in the control group [(10±4) vs (11±4)mg/L, (25±8)vs (34±7)U/ml, P<0.05]. The levels of eGFR, SOD and GSH in probucol group were higher than those in control group [(80±27) vs (72±26) ml·min(-1)·1.73 m(-2,) (67±9) vs (58±8)U/ml, (4.6±0.9) vs (3.9±0.8)U/ml, P<0.05]. The incidence of CIN was 4.0% in the probucol group and 10.9% in the control group, and the difference was statistically significant (P<0.05, χ(2)=-3.31). Multivariate Logistic regression analysis showed that probucol was an independent protective factor for CIN (OR=0.334, 95%CI 0.172-0.648, P=0.001). There were no adverse events such as myasthenia gravis, abnormal liver function and cardiovascular events during the hospitalization and 14-day follow-up. Conclusions: Probucol can reduce the incidence of contrast-induced nephropathy after PCI. The protection mechanism is related with its anti-inflammatory and anti-oxidative stress effects, and it has good safety.

[An interpretation of the AASLD practice guideline on the diagnosis and management of nonalcoholic fatty liver disease in 2017].

The American Association for the Study of Liver Diseases (AASLD) updated and published the Practice Guidance for the Diagnosis and Management of Nonalcoholic Fatty Liver Disease (NAFLD) in July 2017, which provides recommendations for the accurate diagnosis, treatment, and effective prevention of NAFLD. Related metabolic diseases should be considered during the initial evaluation of patients suspected of NAFLD. Noninvasive diagnostic techniques including transient elastography, magnetic resonance elastography, and serum biochemical models should be used to evaluate the development and progression of liver fibrosis in patients with NAFLD. Clinical liver pathology report should clearly differentiate between nonalcoholic fatty liver (NAFL), NAFL with inflammation, and nonalcoholic steatohepatitis (NASH) and identify the presence or absence of liver fibrosis and its degree. Early medication for NAFLD can only be used in patients with pathologically confirmed NASH and liver fibrosis, and it is not recommended to use pioglitazone and vitamin E as the first-line drugs for patients with NASH which has not been proven by biopsy or non-diabetic NASH patients. Foregut bariatric surgery can be considered for obese patients with NAFLD/NASH who meet related indications. It is emphasized that the risk factors for cardiovascular disease should be eliminated for NAFLD patients. Statins can be used for the treatment of dyslipidemia in patients with NAFLD/NASH, but they cannot be used in patients with decompensated liver cirrhosis. Routine screening or hepatocellular carcinoma surveillance is not recommended for NASH patients without liver cirrhosis. Cardiovascular disease should be taken seriously during liver transplantation evaluation. There is still no adequate clinical evidence for the treatment of NAFLD in children and adolescents, and intensive lifestyle intervention is recommended as the first-line therapy for such patients.

Overexpression of cofilin 1 in prostate cancer and the corresponding clinical implications.

Cofilin 1 (CFL1) is a cytoskeletal protein and overexpression of the protein has been associated with aggressiveness in certain types of malignancies. The aim of the present study was to investigate the clinical implications of CFL1 expression in prostate cancer (PCa). Immunohistochemical analysis was performed using formalin-fixed paraffin-embedded tissue sections obtained from 111 patients with PCa and 47 patients with benign prostatic hyperplasia (BPH). In total, 78 (70.3%) out of 111 PCa tissues were found to express the CFL1 protein, while no expression was detected in BPH tissues. In addition, CFL1 was also observed to be significantly associated with the Gleason score (GS; <7 vs. ≥7; P<0.0001) and presence of lymph node metastasis (presence vs. absence; P<0.0001). However, there was no association between the expression of CFL1 and other clinicopathological variables, such as age (<69 years vs. ≥69 years; P=0.54), pre-operative prostate specific antigen level (<20 ng/ml vs. ≥20 ng/ml; P=0.45) and pathological stage (T2 vs. ≥T3a; P=0.055). In addition, 35 tissues (31.5%) were observed to possess a CFL1-positive mesenchyme. CFL1 expression was revealed to be an independent predictive factor for a high GS. The status of CFL1 expression in the mesenchyme also found to individually predict extraprostatic extension in PCa patients, based on multivariate analysis. The results of the present study indicated that CFL1 may specifically predict the development of PCa, and that the expression of CFL1 in the mesenchyme may be closely associated with the development of lymph node metastasis.

Tetraploid complementation proves pluripotency of induced pluripotent stem cells derived from adipose tissue.

OBJECTIVES: Recently, pluripotency of induced pluripotent stem (iPS) cells has been displayed after producing adult mice, in tetraploid complementation assays. These studies lead us to the last piece of the puzzle for reprogramming somatic cells into fully pluripotent cells which function as embryonic stem cells in most applications. However, in all of previous studies, skin fibroblasts were used as the starting population for reprogramming, raising questions as to whether the pluripotency of the iPS cells was dependent on the particular starting cell type. MATERIALS AND METHODS: Our iPS cell lines were prepared from murine adipose stem cells (ASCs). Their multi-potency was first tested by teratoma formation in nude mice. Then, tetraploid complementation was performed to generate progeny from them. RESULTS: We succeeded to the birth of viable and fertile adult mice derived entirely from reprogrammed ASC, indicating cell types other than fibroblasts can also be restored to the embryonic level of pluripotency. CONCLUSIONS: We also directed differentiation of iPS cells into chondrocytes, thus adipose-derived iPS cells can be used as models to study chondrogenic differentiation and cartilage regeneration.

Adipogenic differentiation potential of adipose-derived mesenchymal stem cells from ovariectomized mice.

OBJECTIVES: In human post-menopausal osteoporosis, enhanced adipogenesis in bone marrow and enhanced formation of adipose tissue in vivo are observed. These changes correlate with reduced trabecular bone volume and increased adipocyte cell size as well as cell number. However, cellular and molecular mechanisms underlying osteoporosis-related changes in adipocyte cell volume are not known. This study was designed to compare adipogenic potential of adipose tissue-derived stem cells (ADSCs) obtained from ovariectomized mice with that of control ADSCs, and to analyse pathological mechanisms from the point of functional changes of ADSCs. MATERIALS AND METHODS: Healthy female C57BL/6J mice were randomly divided into ovariectomy and sham-surgery groups. Mouse ADSCs were isolated and cultured in vitro up to passage 3. After adipogenic induction, oil red O staining of lipid droplets was used to detect adipogenic ability of ADSCs; real-time PCR and immunofluorescence staining were used to detect expression of adipogenesis-related genes and proteins. RESULTS: As indicated by increased expression of adipogenic and lipogenic genes and proteins, and lipid droplets accumulation shown by oil red-O staining, adipogenic differentiation of ADSCs was significantly enhanced in the ovariectomy group compared to the sham-surgery group (P < 0.05). CONCLUSION: These findings suggest that enhanced adipogenic differentiation of ADSCs is likely to be the important cause for increased adipogenesis in vivo and subsequent obesity-like changes in body mass, in mice, after ovariectomy.

Electrospun P34HB fibres: a scaffold for tissue engineering.

OBJECTIVES: Amongst the fourth generation of PHAs is bio-plasticpoly3-hydroxybutyrate4-hydroxybutyrate (P34HB); it is thus appropriate to perform novel research on its uses and applications. The main objective of this study was to determine whether electrospun P34HB fibres would accommodate viability, growth and differentiation of mouse adipose-derived stem cells (mASCs). MATERIALS AND METHODS: In the present study, we looked at P34HB in two forms, electrospun P34HB fibres and P34HB film. Morphology of electrospun P34HB fibres and P34HB film were characterized using scanning electron microscopy, fluorescence microscopy and confocal laser scanning microscopy, after cell seeding. Cell adhesion, proliferation and cytotoxicity tests were conducted on both by MTT and CCK-8 assays, respectively. After being cultured with osteogenic induction, expression of adipogenic genes Runx2, OPN and OCN, were examined by real-time PCR. RESULTS: By scanning electron microscopy, light microscopy and confocal laser scanning microscopy, we observed that the mASCs grew well associated with the P34HB materials. After MTT and CCK-8 assay, we concluded that P34HB would, indeed, be a material suitable for further cell adhesion and proliferation studies. More importantly, we found that the P34HB matrices promoted expression of Runx2, OPN and OCN with osteogenic induction. CONCLUSIONS: In this investigation, we can confirm that the electrospun P34HB fibres accommodated survival, proliferation and differentiation of mASCs, and we have been able to draw the conclusion that fibre scaffolds produced by the electrospinning process are promising for application of bone tissue engineering.

Effects of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse adipose-derived stem cells.

OBJECTIVES: As mesenchymal stem cells (MSCs) can be isolated easily from adipose tissues while retaining their self-renewal and multi-potential differentiation capacities, they hold promising possibilities for being applied extensively in tissue engineering. Bone morphogenetic protein (BMP) family members have been reported to provide instructive signals to MSCs for them to differentiate into several different cell lineages. The study described here aims to investigate whether BMP-4 could promote adipose-derived stem cell (ASC) differentiation into adipocytes under various concentrations. MATERIALS AND METHODS: ASCs were isolated from mouse inguinal adipose pads and cultured in vitro. 10 ng/ml and 50 ng/ml BMP-4 were added to adipogenic media for 8 days. Oil red-O staining, reverse transcription/polymerase chain reaction and immunocytofluorescence staining were performed to examine differentiation of the ASCs. RESULTS: As indicated by increased expression of adipogenic and lipogenic genes (PPAR-γ, APN and LPL) and proteins, 50 ng/ml BMP-4 seemed to induce mASCs to differentiate into the adipo-lineage compared to 10 ng/ml BMP-4, and control groups. In addition, lipid droplets accumulated within the adipocytes under 50 ng/ml BMP-4 stimulation, as shown by oil red-O staining. CONCLUSIONS: Our present study suggests that BMP-4, as an adipo-inducing factor, promoted adipogenesis of ASCs at higher concentrations (50 ng/ml) and can perhaps be considered as a candidate for use in adipose tissue engineering.

Low-intensity pulsed ultrasound induced enhanced adipogenesis of adipose-derived stem cells.

OBJECTIVE: The aim of this study was to investigate effects of low-intensity pulsed ultrasound (LIPUS) on differentiation of adipose-derived stem cells (ASCs), in vitro. MATERIALS AND METHODS: Murine ASCs were treated with LIPUS for either three or five days, immediately after adipogenic induction, or delayed for 2 days. Expression of adipogenic genes PPAR-γ1, and APN, was examined by real-time PCR. Immunofluorescence (IF) staining was performed to test for PPAR-γ at the protein level. RESULTS: Our data revealed that specific patterns of LIPUS up-regulated levels of both PPAR-γ1 and APN mRNA, and PPAR-γ protein. CONCLUSIONS: In culture medium containing adipogenic reagents, LIPUS enhanced ASC adipogenesis.

Osteogenic differentiation of adipose-derived stem cells prompted by low-intensity pulsed ultrasound.

OBJECTIVES: Based on in vivo studies, low-intensity pulsed ultrasound (LIPUS) stimulation has been widely used in the clinic for advancing bone growth during healing of non-union alignment, fractures and other osseous defects. In this study, we have investigated osteogenic differentiation of adipose stem cells (ASCs) regulated by LIPUS, and also in a preliminarily manner, we have discussed diverse effects of different duty ratio parameters. MATERIALS AND METHODS: Mouse adipose stem cells were isolated and osteogenically induced. Then they were treated with LIPUS for 10 min/day for 3 days, 5 days and 7 days, respectively. Finally, effects of LIPUS on osteogenic differentiation of the ASCs were analysed by real-time PCR, western blotting and immunofluorescence. RESULTS: Our data indicated that LIPUS promoted mRNA levels of runt-related transcription factor 2, osteopontin and osterix in the presence of osteo-induction medium; moreover, protein levels of runt-related transcription factor 2 and osteopontin were upregulated. CONCLUSIONS: We successfully demonstrated that LIPUS enhanced osteogenesis of ASCs, specially at the duty ratio of 20%.

Characteristics of anaphylaxis-inducing IgG immune complexes triggering murine passive systemic anaphylaxis.

BACKGROUND: With the broad and increasing application of therapeutic monoclonal antibodies (mAbs) in clinical settings, IgG-induced allergic reactions, including passive systemic anaphylaxis (PSA), have attracted significant attention. However, it is not clear which types of IgG mAb-antigen complexes or IgG aggregates formed by antigen binding can trigger PSA, as not all immune complexes (ICs) are capable of triggering PSA. Here, we characterise mAb-antigen complexes capable of inducing murine PSA to evaluate and predict which ICs are able to induce PSA. METHODS: Thirty-six combinatory reactions with eight antigens and 27 corresponding mAbs were used to trigger PSA, which was defined by rectal temperature. Sandwich ELISA, passive cutaneous anaphylaxis (PCA) induction and flow cytometry analysis of CD16/32 (FcγRIII/II) expression were used to characterise the ICs. The dynamic concentrations of antigen in the peripheral blood were measured by ELISA. RESULTS: Only 14 of the 36 ICs could trigger PSA and thus be termed anaphylaxis-inducing immune complexes (Ai-ICs). The Ai-ICs could be characterised by constructing sandwich ELISA, inducing PCA and down-regulating CD16/32 (FcγRIII/II) expression on blood neutrophils in vitro and in vivo. Additionally, the occurrence and severity of PSA was found to be associated with the instantaneous concentration of antigen in the peripheral blood in the presence of antibody. CONCLUSIONS: Only Ai-ICs, not all ICs, could trigger IgG-mediated PSA, which could be characterised by the above simple methods. The occurrence and severity of PSA was associated with the instantaneous concentration of antigen in the peripheral blood in the presence of antibody.

CC chemokine ligand 21 enhances the immunogenicity of the breast cancer cell line MCF-7 upon assistance of TLR2.

CC chemokine ligand 21 (CCL21) is a known attractant for CCR7-positive (CCR7+) cells, but its additional role in the immunogenicity of CCR7+ cells remains poorly understood. This study explored the effects of CCL21-CCR7 ligation on cancer immunogenicity and related antitumor immune response, in the presence and absence of mitomycin C (MMC) treatment. CCL21-CCR7 binding upregulated human leukocyte antigen class I-restricted tumor antigen presentation with increased expression of human leukocyte antigen class I and transporter associated with antigen processing-1. In addition, CCL21 restrained the tumor-derived immunosuppressive factors FasL and transforming growth factor-ß. Consequently, CCL21 facilitated cancer-educated lymphocytes reaction in vitro. In the tumor-bearing mouse, CCL21 inhibited tumor growth and prolonged mouse survival via lymphocytes, especially in CCR7+ cancer cells. Furthermore, Toll-like receptor 2 activation of lymphocytes assisted the tumor-suppression functions of CCL21, in vitro and in vivo. This study implies that CCL21 improved the immunogenicity of the CCR7+ breast cancer cell line even with MMC treatment and triggered antitumor response by lymphocytes. These findings provide a new insight into the research and application of CCL21-associated antitumor response.

Improvement in reproductive parameters in hypogonadal female mice by regulated gene replacement therapy in the central nervous system.

One of the challenges of gene targeting is to achieve regulated transgene expression in specific target cells. The hypogonadal (hpg) mice are genetically deficient in hypothalamic gonadotropin-releasing hormone (GnRH) production due to a deletion in the GnRH gene, resulting in hypogonadotropic hypogonadism. Here we show an improvement in reproductive parameters of adult female homozygous hpg mice by direct infusion into the hypothalamic preoptic area (POA) of a herpes simplex virus (HSV)-based amplicon vector containing a 13.5 kb genomic fragment encoding the GnRH gene together with its cognate promoter and regulatory elements. Following vector injection, GnRH-expressing neurons were detected in the POA, and pituitary and plasma gonadotropin levels as well as ovarian and uterine weights increased. In addition, a subset of injected hpg mice demonstrated cyclic estrous changes, consistent with regulated control of GnRH production. Administration of kisspeptin-10 resulted in an increase in plasma luteinizing hormone levels, further supporting appropriate regulation of the introduced GnRH transgene. These findings indicate that delivery of the GnRH gene resulted in selective neuronal expression of GnRH and regulated hypothalamic GnRH release. To our knowledge, this is the first example of the correct targeting of a gene under its cognate promoter to neurons resulting in selective and regulated synthesis of a biologically active peptide, and thus may have a wide range of applications in the treatment of human disorders.

Stellate ganglion block inhibits formalin-induced nociceptive responses: mechanism of action.

BACKGROUND AND OBJECTIVE: Stellate ganglion block has been extensively used in clinical practice for the management of painful conditions such as cephalic, facial and upper limb pains yet its mechanism of action and its analgesic efficacy are poorly understood. METHOD: Formalin (3% 0.2 mL) was injected into the plantar region of the right upper limb paw in rabbits and 50 min after this injection, saline or bupivacaine 2.5% 0.5 mL was administered via a chronic implantation catheter near the right stellate ganglion. Behavioural modification, changes in heart rate and plasma norepinephrine release at different time points after formalin and bupivacaine or saline injection were observed. Finally, the cervical spinal cord was harvested and immunostaining for substance P and c-Fos was performed. RESULTS: Formalin caused stress noxious behavioural changes and a significant increase in heart rate and norepinephrine release. These changes were inhibited by bupivacaine stellate ganglion block but not by saline injection. Immunoreactants of substance P were significantly decreased by formalin injection compared with that in controls. However, with bupivacaine injection, substance P levels were restored though not reaching the levels seen in the controls. Formalin injection also caused a significant increase of c-Fos expression in cervical spinal cord. This increase was not affected by stellate ganglion block. CONCLUSION: Stellate ganglion block can effectively alleviate nociceptive responses induced by formalin injection. The mechanism of its action may involve reduction of substance P in the spinal cord and plasma catecholamine release caused by noxious stimuli.

Engineering and functional evaluation of a single-chain antibody against HIV-1 external glycoprotein gp120.

The HIV-1 envelope glycoprotein surface subunit gp120 is an attractive target for molecular intervention. This is because anti-HIV-1 gp120 neutralizing antibodies display the potential ability to inhibit HIV-1 infection. The present investigation describes the construction of a genetically engineered single chain antibody (scFv102) against HIV-1 gp120, its expression and functional evaluation. The parental hybridoma cell line (102) produces an immunoglobulin directed against the conserved CD4-binding region of gp120. cDNAs encoding the variable regions of the heavy (V(H)) and light (V(L)) chains were prepared by reverse transcription PCR and linked together with an oligonucleotide encoding a linker peptide (Gly(4)Ser)(3) to produce a single chain antibody gene. The resulting DNA construct was cloned into a prokaryotic expression vector (pET28) and recombinant scFv102 was expressed in Eserichia coli as an insoluble protein. The denatured scFv102 was refolded and purified by immobilized metal ion affinity chromatography. Purified scFv102 had the same specificity as the intact IgG in immuno-blotting assays and immuno-fluorescence (IF) detection, but ELISA analyses demonstrated the affinity of scFv102 to be 5-fold lower than that of the parental monoclonal antibody. In neutralization assays, scFv102 at concentrations lower than 40 microg/ml exhibited efficient interference with viral replication and inhibition of viral infection (90%) across a range of primary isolates of subtype B HIV-1. These results suggest that the constructed anti-HIV-1 gp120 scFv102 has good biological activity and can potentially be used for in vitro diagnostic and in vivo therapeutic applications.

Apoptosis induced by isoliquiritigenin in human gastric cancer MGC-803 cells.

Isoliquiritigenin, which is possibly a principal anti-tumor constituent of licorice, a traditional Chinese herb, was examined for apoptosis-inducing activity in human gastric cancer MGC-803 cells. Typical morphological and biochemical features of apoptosis including cell shrinkage, chromatin condensation, DNA ladder formation, and appearance of apoptotic peaks (subG(1)) were observed in MGC-803 cells with isoliquiritigenin treatment. Using Fluo-3 and Rh123 as fluorescent probes, respectively, it was found that the intracellular free calcium concentration increased and the mitochondrial transmembrane potential (Deltapsi(m)) decreased in a dose-dependent manner in apoptotic cells. These results suggest that isoliquiritigenin induced apoptosis of MGC-803 cells through calcium- and Deltapsi(m)-dependent pathways, indicating that it is potentially useful as a natural anti-cancer agent.

Soluble member(s) of the mesothelin/megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma.

mAb OV569 was made by immunizing mice with ovarian carcinoma cells. It binds to cells from ovarian carcinomas and, to a lesser extent, to cells from certain other carcinomas whereas the binding to normal tissues is low to nondetectable. It also binds to soluble molecule(s) in culture supernatants from antigen-positive carcinomas. OV569 recognizes a protein(s) of 42-45 kDa with the same N-terminal amino acid sequence as the membrane-bound portion of mesothelin and megakaryocyte potentiating factor (MPF). Binding assays with fusion proteins comprising either the N-terminal part of mesothelin/MPF (D1Ig), reported to be easily cleaved off, or a noncleavable, membrane-associated part (D2Ig) showed that OV569 only binds to D2hIg. A new member of the mesothelin/MPF family was discovered, which has an 82-bp insert in the membrane-associated part, leading to a frameshift of 212 bp, and whose predicted molecular structure indicates that it is soluble. To test patient sera for soluble tumor antigen, antigen was isolated from cell-free tumor culture supernatants via immunoadsorption with OV569 and used to generate murine mAbs to an epitope different from the one to which OV569 binds, after which mAbs to two different epitopes were used to develop a "sandwich ELISA." Using this assay, the level of circulating antigen was elevated significantly in 23 of 30 sera from patients with ovarian carcinoma, as compared with 0 of 68 sera from healthy controls, 0 of 3 sera from patients with nonneoplastic diseases, and 25 of 75 sera from patients with other tumors. Soluble molecules of the mesothelin/MPF family may provide useful new marker(s) for diagnosis of ovarian carcinoma and/or monitoring its response to therapy.

[Anti-promoting and anti-mutagenic actions of G9315].

G9315, a complex extracted from Glycyrrhizae inflata Bat (III), consists of 6 flavonoids with significant antioxidant effects. At 1 mg dose it inhibited the mouse ear edema induced by croton oil, and showed strong anti-promoting effects on two-stage carcinogenesis in mouse skin induced by DMBA plus croton oil. The TPA enhanced 32Pi-incorporation into phospholipid fraction in HeLa cells was inhibited, and the micronuclei in mouse bone marrow cells induced by cytoxan was also depressed.