Risk factors for cardiovascular adverse events from immune checkpoint inhibitors.

Immune-related adverse events (irAEs), including skin injury, liver and kidney injury, colitis, as well as cardiovascular adverse events, are a series of complications arising during the treatment of immune checkpoint inhibitors (ICIs). Cardiovascular events are the most urgent and the most critical, as they can end life in a short period of time. With the widespread use of ICIs, the number of immune-related cardiovascular adverse events (irACEs) induced by ICIs has increased. More attention has been paid to irACEs, especially regarding cardiotoxicity, the pathogenic mechanism, diagnosis and treatment. This review aims to assess the risk factors for irACEs, to raise awareness and help with the risk assessment of irACEs at an early stage.

Predictive value of small dense low-density lipoprotein cholesterol for cardiovascular events in Chinese elder diabetes mellitus patients.

BACKGROUND: As a subcomponent of low-density lipoprotein cholesterol (LDL-C), small dense LDL-C (sdLDL-C) has been suggested to be a better predictor of cardiovascular diseases (CVD). The aim of this research was to evaluate the predictive value of the sdLDL-C in cardiovascular events (CVs) in Chinese elderly patients with type 2 diabetes mellitus (DM). METHODS: A total of 386 consecutive type 2 DM patients were included into this study during December 2014 to December 2016. The serum sdLDL-C level of each subject was measured by homogeneous method. During a period of 48-month's follow-up, the occurrence of CVs and associated clinical information were recorded. Receiver operating characteristic (ROC) curves were used to assess the predictive value of serum sdLDL-C to occurrence of major CVs. RESULTS: A total of 92 CVs occurred during the study period. The ROC curve analysis manifested that sdLDL-C in the study population had a matchable discriminatory power (AUC for sdLDL-C was 0.7366, P = 0.003). In addition, Kaplan-Meier event-free survival curves displayed an obvious increase of CVs risk for sdLDL-C ≧ 26 mg/dL (log-rank = 9.10, P = 0.003). This phenomenon had analogous results in patients who received statins at baseline (log rank = 7.336, P = 0.007). Cox regression analysis revealed that the increase in HbA1c, glucose, LDL-C, sdLDL-C, non-high-density lipoprotein cholesterol (non-HDL-C) and apolipoprotein B (ApoB) and the decrease in apolipoprotein AI (ApoAI) were obviously interrelated with heightened CVs risk. Multiple Cox regression demonstrated that the increase of sdLDL-C and hemoglobin A1c (HbA1c) was significantly correlated with CVs. The results of the study indicated that high sdLDL-C level (> 10 mg/dL) was a risk factor for CVs in the multivariate model (HR 1.281, 95% CI 1.225-16.032; P < 0.01). CONCLUSION: sdLDL-C level could be an effective predictor in predicting the future CVs for Chinese elderly patients with type 2 DM and dyslipidemia.

Effects of statin therapy on chronic kidney disease patients with coronary artery disease.

BACKGROUND: Long-term persistence of statin therapy provided an ongoing reduction in mortality among patients with and without a known history of CVD, and renoprotective effect on CKD patients. Until now, very few reports are available from China to address the effects of statin therapy in CKD + CAD patients. METHODS: We compared the effects of long-term statin therapy (follow-up time 4 years) in terms of cardiovascular events, all-cause death, and cardiac death among 254 CKD patients with or without CAD. RESULTS: Long-term statin therapy was much more effective in the CKD + CAD patients compared with CKD patients. In the CAD + CKD patients, long-term statins showed a 22.2% reduction in the CVs rate (P = 0.012). With regard to the all-cause and cardiac deaths, long-term statins had significant treatment effects on the CAD + CKD patients (reduction of about 28.1% in mortality rates, P < 0.001). In contrast, long-term statin therapy exerted no significant influence on the clinical outcomes of the CKD-only patients. CONCLUSION: Long-term statin therapy more dramatically reduced the CVs and mortality rates of the CKD patients with concomitant CAD. In contrast, CKD-only patients had a good prognosis and did not appear to require statin treatment.

Genotype distribution characteristics of multiple human papillomavirus in women from the Taihu River Basin, on the coast of eastern China.

BACKGROUND: There is limited data on the genotype distribution of human papillomavirus (HPV) in the Taihu River Basin, home to 1.29 million people on the coast of eastern China. This study evaluated the prevalence and genotypes among different female age groups in this region. METHODS: Twenty-six HPV strains (low-risk HPV 6, 11, 40, 42, 44, 61, 73 and high-risk HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 82, and 83) were detected using Tellgenplex™ HPV technology in samples obtained from three clinical hospitals located in different regions of the Taihu Lake Basin. RESULTS: The results showed that 1855 samples (20.97% of all samples) were found to be HPV-positive. Of these, 1375 samples (15.55% of all samples) were found to have a single HPV infection. Age-specific prevalence showed two peaks, one that corresponded to the group of 21-30 year-old women and the other peak that corresponded to the group of women over 51 years old. The three most prevalent genotypes were HPV52 (19.95%, 370/1855), HPV16 (13.48%, 150/1855), and HPV58 (11.32%, 210/1855). Mixed strains HPV58 + HPV33 and HPV58 + HPV52 were most commonly found in females infected with multiple HPV types. CONCLUSIONS: This investigation reveals that HPV infection in the Taihu River Basin varied significantly among different age groups. The most prevalent genotypes are included in the nonavalent vaccine, V503, however this vaccine is not licensed for use in mainland China. The most frequently occurring genotypes should be considered in the development of next-generation HPV vaccines for optimal protection of public health.

Small dense low-density lipoprotein cholesterol was associated with future cardiovascular events in chronic kidney disease patients.

BACKGROUND: Cardiovascular disease (CVD) is often comorbid with chronic kidney disease (CKD). Small low-density lipoprotein cholesterol (sdLDL-C) has also been found to increase risk for CVD. The goal of the present study was to determine the nature of the relationship between sdLDL-C concentration and CVD in patients with CKD. METHODS: One-hundred and forty-five subjects (113 men and 32 women) with CKD (Stage 3 and Stage 4) participated this retrospective study. The concentration of sdLDL-C was measured in samples from 145 CKD patients between 2010 and 2012 during a four-year follow-up period. RESULTS: A total of eight fatal cardiovascular events (CVs) and 46 nonfatal CVs were registered in the four-year follow-up period. Multivariate Cox regression analysis showed that sdLDL-C [1.278, 95 % (1.019-1.598)] and sdLDL-C/LDL-C [2.456, 95 % (1.421-15.784)], at final observation, were independent risks of CVs. A Kaplan-Meier survival analysis showed that patients with sdLDL-C >38 mg/dl (logrank: 4.375, P = 0.037), and sdLDL-C/LDL-C ratio >0.3 levels (logrank: 11.94, P = 0.018) were at increased risk for CVs. CONCLUSION: The results of this study indicated that for patients suffering CKD, a significant relationship exists between an elevated sdLDL-C concentration and the risk of cardiovascular disease.

Albumin Binding Function: The Potential Earliest Indicator for Liver Function Damage.

Background. Currently there is no indicator that can evaluate actual liver lesion for early stages of viral hepatitis, nonalcoholic fatty liver disease (NAFLD), and cirrhosis. Aim of this study was to investigate if albumin binding function could better reflect liver function in these liver diseases. Methods. An observational study was performed on 193 patients with early NAFLD, viral hepatitis, and cirrhosis. Cirrhosis patients were separated according to Child-Pugh score into A, B, and C subgroup. Albumin metal ion binding capacity (Ischemia-modified albumin transformed, IMAT) and fatty acid binding capacity (total binding sites, TBS) were detected. Results. Both IMAT and TBS were significantly decreased in patients with NAFLD and early hepatitis. In hepatitis group, they declined prior to changes of liver enzymes. IMAT was significantly higher in cirrhosis Child-Pugh class A group than hepatitis patients and decreased in Child-Pugh class B and class C patients. Both IMAT/albumin and TBS/albumin decreased significantly in hepatitis and NAFLD group patients. Conclusions. This is the first study to discover changes of albumin metal ion and fatty acid binding capacities prior to conventional biomarkers for liver damage in early stage of liver diseases. They may become potential earliest sensitive indicators for liver function evaluation.

Correlation between small dense low-density lipoprotein cholesterol and carotid artery intima-media thickness in a healthy Chinese population.

BACKGROUND: Small dense low-density lipoprotein cholesterol (sdLDL-C) concentration was useful in the assessment of the presence of cardiovascular diseases (CVD) and its severity. We examined whether SdLDL-C is more closely associated with carotid artery intima-media thickness (CA-IMT), a surrogate measure of atherosclerosis, than LDL-C and traditional CVD risk factors in Chinese healthy subjects. METHODS: We measured CA-IMT, blood pressure (BP), sdLDL-C, glucose metabolism and lipid in 183 native Chinese healthy subjects. CA-IMT was assessed by ultrasonography, and sdLDL-C concentrations were measured by a homogenous assay. Pearson's correlation coefficient analyses and Multiple regression analyses were used to examine the relationships between CA-IMT values and other clinical variables. RESULTS: The sdLDL-C level was significantly higher in males than in females (p <0.05) and there was an age effect on sdLDL-C (p <0.05). When the effects of age, gender and other traditional CVD risk factors were adjusted using multiple regression analysis. CA-IMT remained significantly associated with sdLDL-C(ß = 0.437, p <0.001). CONCLUSIONS: There are gender and age differences in sdLDL-C levels among a healthy Chinese population. Moreover, we found adjusted traditional CVD risk factors such as higher age, male sex, and other traditional CVD risk factors, the association between CA-IMT and SdLDL-C remained significant. sdLDL-C is may be a useful predictor in the assessment of CA-IMT in Chinese population.

Anti-Sp17 monoclonal antibody-doxorubicin conjugates as molecularly targeted chemotherapy for ovarian carcinoma.

Sperm protein 17 (Sp17) is selectively overexpressed in several human malignancies including ovarian carcinoma, but is absent or expressed at low levels in most normal tissues. Previous work from our group characterized an anti-Sp17 monoclonal antibody (clone 3C12) and showed that it specifically targeted tumor cells. In this report, we investigated whether a novel immunoconjugate containing 3C12 linked to the chemotherapeutic agent doxorubicin [(DOX) Adriamycin] had antitumor activity against ovarian cancer cell lines and tumor models. DOX was conjugated to 3C12 using a linker, and the specificity of 3C12-DOX was examined in Sp17-positive SKOV3 and Sp17-negative COC2 ovarian cancer cells using cell-based ELISA and internalization assays. The cytotoxicity of 3C12-DOX was assessed with the MTT assay, and its therapeutic effectiveness was evaluated in immunodeficient mice bearing SKOV3 cells. In vitro, the 3C12-DOX immunoconjugate specifically bound to and was internalized by Sp17-positive SKOV3 cells but did not bind to Sp17-negative cells. Treatment with 3C12-DOX (0.001 to 10 µg/mL) decreased the viability of SKOV3 cells in a Sp17-specific manner. In vivo, 3C12-DOX (3 mg/kg) induced the regression of established SKOV3 xenograft tumors in BALB/c mice compared with control treatment. The antitumor effects of 3C12-DOX were significantly associated with the induction of apoptosis in tumor cells. In addition, 3C12-DOX showed no observable adverse effects or toxicity when compared with DOX alone in mice bearing ovarian tumor xenografts. Our findings suggest that 3C12-DOX may be a potential antibody-drug conjugate for clinical development.

Diagnostic value of immunoglobulin G antibodies against Candida enolase and fructose-bisphosphate aldolase for candidemia.

BACKGROUND: The yeast Candida is one of the most frequent pathogens isolated from bloodstream infections and is associated with significant morbidity and mortality. Problems with clinical and microbiological diagnosis of invasive candidiasis (IC) have prompted the development of non-culture-based laboratory methods. Previous reports suggest that serological detection of antibodies might be useful for diagnosing systemic candidiasis. METHODS: Diagnosis of IC using antibodies against recombinant Candida albicans enolase (Eno) and fructose-bisphosphate aldolase (Fba1) was evaluated. Using recombinant Eno and Fba1 as coating antigens, enzyme-linked immunosorbent assays (ELISAs) were used to analyze sera from patients with candidemia (n = 101), Candida colonization (n = 50), bacteremia (n = 84), invasive aspergillosis (n = 40); and from healthy controls (n = 200). RESULTS: The results demonstrated that ELISA detection of anti-Eno and anti-Fba1 IgG distinguished IC from other pathogenic infections in patients and healthy individuals. The sensitivity, specificity, and positive and negative predictive values were 72.3%, 94.7%, 78.5% and 93% for anti-Eno, and 87.1%, 92.8%, 76.5% and 96.4% for anti-Fba1 antibodies, respectively. Combining these two tests improved sensitivity up to 90.1% and negative predictive value up to 97.1%, with specificity and positive predictive values of 90.6% and 72.2%. The tests were specific to the Candida genus and antibody titers were higher for candidemia patients than for controls. Positive antibody tests were obtained before blood culture results for 42.2% of patients for anti-Eno and 51.1% for anti-Fba1. CONCLUSION: These data suggest that tests that detect IgG antibodies against Candida enolase and fructose-bisphosphate aldolase, especially when used in combination, could be a powerful tool for diagnosing IC.

Antibody specific to thioredoxin reductase as a new biomarker for serodiagnosis of invasive aspergillosis in non-neutropenic patients.

BACKGROUND: Invasive aspergillosis (IA) is an important cause of mortality in critically ill patients, but the diagnosis is difficult as clinical and radiological signs are neither sensitive nor specific. Serum galactomannan (GM) is a useful marker for IA, but exhibits low sensitivity in non-neutropenic patients. In our previous work, strong antibody reactivity to thioredoxin reductase of Aspergillus fumigatus was found in non-neutropenic IA patients. METHODS AND RESULTS: Using recombinant thioredoxin reductase GliT (TR), an antigenic protein secreted by A. fumigatus, as the coating antigen, an enzyme-linked immunosorbent assay (ELISA) for detecting anti-TR antibodies was developed. The antibody response to TR in IA animal models and 42 non-neutropenic patients with culture- and/or histology-documented IA was investigated. The results showed that anti-TR antibody was detectable in rabbit serum 7-9 days after exposure to the fungus. The sensitivity and specificity of the anti-TR antibody assay in patients were 80.9% and 96%, respectively, while the sensitivity of GM in this group of patients was only 52.3%. The specificity of the assay was confirmed by testing the sera from patients infected with other pathogenic fungal species and bacteria.

Immunoproteomics based identification of thioredoxin reductase GliT and novel Aspergillus fumigatus antigens for serologic diagnosis of invasive aspergillosis.

BACKGROUND: There has been a rising incidence of invasive aspergillosis (IA) in critically ill patients, even in the absence of an apparent predisposing immunodeficiency. The diagnosis of IA is difficult because clinical signs are not sensitive and specific, and serum galactomannan has relatively low sensitivity in this group of patients. Therefore, more prompt and accurate disease markers for early diagnosis are needed. To establish disease markers demands a thorough knowledge of fungal antigens which may be detected in the serum or other body fluids of patients. Herein we report novel immunodominant antigens identified from extracellular proteins of Aspergillus fumigatus. RESULTS: Extracellular proteins of A. fumigatus were separated by two-dimensional electrophoresis (2-DE) and probed with the sera from critically ill patients with proven IA. The immunoreactive protein spots were identified by MALDI-TOF mass spectrometry (MALDI-TOF -MS). Forty spots from 2DE gels were detected and 17 different proteins were identified as immunogenic in humans. Function annotation revealed that most of these proteins were metabolic enzymes involved in carbohydrate, fatty acid, amino acid, and energy metabolism. One of the proteins, thioredoxin reductase GliT (TR), which showed the best immunoactivity, was analyzed further for secretory signals, protein localization, and homology. The results indicated that TR is a secretory protein with a signal sequence exhibiting a high probability for secretion. Furthermore, TR did not match any human proteins, and had low homology with most other fungi. The recombinant TR was recognized by the sera of all proven IA patients with different underlying diseases in this study. CONCLUSIONS: The immunoreactive proteins identified in this study may be helpful for the diagnosis of IA in critically ill patients. Our results indicate that TR and other immunodominant antigens have potential as biomarkers for the serologic diagnosis of invasive aspergillosis.

Toxic effects of chlortetracycline on maize growth, reactive oxygen species generation and the antioxidant response.

The toxicity of chlortetracycline (CTC) on maize (Zea mays L.) growth and reactive oxygen species (ROS) generation was studied. The root and shoot lengths and fresh weights of maize seedlings were inhibited by CTC treatment (p < 0.05). Root length was more sensitive than other parameters with the EC10 value of 0.064 mg/L. The spin trapping technique followed by electron paramagnetic resonance (EPR) analysis was used to quantify the ROS production. The ROS generated in maize roots after exposure to CTC was identified as hydroxyl radical (*OH). The EPR signal intensity correlated positively with the logarithm of CTC concentrations exposed (p < 0.05). The dynamic changes of malondialdehyde (MDA) contents and the antioxidative enzyme activities in maize roots were also determined. As compared to the control group, CTC was found to significantly increase MDA content. Treatment of maize roots with the *OH scavenger sodium benzoate (SB) reduced the MDA content and enhanced the antioxidative enzyme activities. The results demonstrated the harmfulness of CTC at high dose to maize in the early developmental stage, and clarified that the inducement of *OH is one of the mechanisms of CTC toxicity.

Regulation of influenza virus-caused oxidative stress by Kegan Liyan oral prescription, as monitored by ascorbyl radical ESR signals.

To study the oxidative stress level of the influenza virus A FM1 subset-infected mouse in intranasal inhalation as a model, we employ an ascorbyl radical's ESR (electron spin resonance) spectrum as an oxidative stress biomarker. These infected mice were pretreated with Ribavirin, ascorbic acid, superoxide dismutase (SOD) or Kegan Liyan oral prescription (KGLY, proprietary Chinese medicine for influenza and common cold) in the stomach tube for 3 days, and then followed by the virus-infecting for 4 days. On the 4th day, samples were collected. It is recognized the strength of ascorbyl radical's ESR signal (A(-.)) (a(H4 = 0.177) Gauss, g = 2.00517) denotes oxidative stress level in vivo and in vitro. The magnitude of ESR spectrum (28.65 +/- 10.71 AU) in mice infected with influenza virus was significantly higher than those of healthy control mice (19.10 +/- 3.61 AU). Serum A(-.) in mice treated with Ribavirin, ascorbic acid, SOD and KGLY declined to 19.70 +/- 6.05, 18.50 +/- 2.93 and 16.25 +/- 3.59, 18.40 +/- 2.14 AU respectively. It is close to A(-.) signal height in healthy controls via down-regulation of the influenza virus-caused oxidative stress level getting decline in the lung index of pneumonia as compare to those of untreated healthy and the influenza virus infected mice pneumonia. It is well known that SOD can prevent the influenza virus pneumonia enhancing mouse survival rate; Ribavirin can treat viral diseases. Data from this study suggested that KGLY may indirectly relieve influenza virus-infected pneumonia via down- regulation of virus caused oxidative stress coupled with a redox reaction cascade as ribavirin, ascorbic acid and SOD.

Copper ions inhibit S-adenosylhomocysteine hydrolase by causing dissociation of NAD+ cofactor.

S-Adenosylhomocysteine hydrolase (SAHH) regulates biomethylation and homocysteine metabolism and thus is an attractive target in drug design studies. SAHH has been shown to be a copper binding protein in vivo; however, the structure and catalytic mechanism of SAHH exclude a role for Cu2+. In the present work, we studied the mechanism of inhibition of SAHH activity by Cu2+. The experimental results showed that Cu2+ inhibited SAHH activity in a noncompetitive manner. Binding of Cu2+ to SAHH resulted in the release of NAD+ cofactors, explaining the loss of the enzymatic activity of SAHH. Further investigation by an ESR probe and computational simulation suggested that Cu2+ could bind at the central channel and interrupt the subunit interactions of SAHH, resulting in a large decrease in affinity to the NAD+ cofactor. This effect of Cu2+ resembled that of enzyme mutations at the C-terminal domain or Asp244 [Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme, J. Biol. Chem. 275, 32147-32156]. The mechanism of action of Cu2+ on SAHH suggested a possible regulative role for Cu2+ on the intracellular activity of SAHH. This could be helpful in understanding the biological effects of copper compounds and suggest a potential coupling mechanism between biomethylation and the redox states of cells.

The mechanism of kinetic inhibition of Cu(II)-induced oxidation of low density lipoprotein by lanthanide ions.

Oral administration of lanthanum chloride (LaCl(3)) was reported to inhibit atherosclerosis in experimental animals, but the mechanism was not clear. In the present work, the effects of La(III) and other lanthanide ions (Ln(III)) on Cu(II)-induced oxidation of isolated low-density lipoprotein (LDL) and the related mechanism were investigated. By monitoring the formation of conjugated dienes (CD), low concentrations of La(III), Gd(III) and Y(III) were found to inhibit Cu(II)-induced LDL oxidation kinetically, as characterized by the prolongation of the lag time, the decrease of the maximal accumulation of CD, and the maximal rate of CD accumulation. Using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin trapping agents, the electron spin resonance (ESR) results showed that La(III) and Gd(III) at low concentrations significantly decreased the level of free radicals, including alkoxyl radical (LO*), alkyl radical (L*), and a transient radical, alkylperoxyl radical (LOO*), generated during LDL oxidation induced by Cu(II). In addition, Fourier-transform infrared spectroscopy (FT-IR) study revealed that La(III) might cause the conformational change and the less aggregation of apolipoprotein B-100 (apoB) in LDL, as demonstrated by the decreasing contents of alpha-helix, intermolecular beta-sheet, unordered structure and beta-turns, and the increasing contents of intramolecular beta-sheet and beta-strands. The inhibitory effect of Ln(III) on Cu(II)-induced LDL oxidation was discussed on the basis of the decreased free radical level and the second structural changes of apoB in LDL.

Analysis of the interactions of ribonuclease inhibitor with kanamycin.

The interaction of ribonuclease inhibitor (RI) with kanamycin was studied by molecular modeling. The preliminary binding model was constructed using the Affinity module of the Insight II molecular modeling program and the key residues involved in the combination of RI binding to kanamycin were determined. Meanwhile, we determined relevant surface characteristics determining the interaction behavior. The modeling results indicated that electrostatic interactions and H-bond forces may work as major factors for the molecular interaction between kanamycin and RI. The above results are useful for elucidating the molecular principles upon which the selectivity of a kanamycin is based. The quartz-crystal microbalance (QCM) is a new method usually used to monitor the binding function of macromolecules with samples online in a flow-injection analysis (FIA) system. The experimental results demonstrate that kanamycin has an extraordinary affinity to the basic protein RI, and our result is consistent with the molecular modeling results. These principles can in turn be used to study the molecular recognition mechanism and design a mimic of kanamycin for the development of new RI binders.

Evidence of nuclear localization of neuronal nitric oxide synthase in cultured astrocytes of rats.

With immunocytochemistry, we have observed the nuclear localization of neuronal nitric oxide synthase (nNOS) in cultured cerebral cortical astrocytes of rats. During the early six days in the subcultures of these cells, nNOS-immunoreactivity was mainly distributed in the cytoplasm. However, nNOS-immunoreactivity was mainly distributed in the nucleus at day 7, and this nuclear localization lasted about ten hours. Meanwhile, inducible nitric oxide synthase expression was significantly inhibited in these cells. Thereafter, nNOS-immunoreactivity was mainly distributed in the cytoplasm again. By confocal microscopy and western blot analysis, the phenomenon of nNOS nuclear localization was further confirmed; and the activity of nNOS in nuclear protein extracts from astrocytes of day 7-subculture could be detected using electron spin resonance (ESR) technique. These results may represent a new pathway of nitric oxide/nNOS participating in inducible nitric oxide synthase gene transcription regulation.

Upregulated expression of neuronal nitric oxide synthase by insulin in both neurons and astrocytes.

Both insulin and nitric oxide (NO) play important roles in the brain. However, there are no unequivocal evidences pointing to a direct effect of insulin on nitric oxide pathway in the brain. In the present study, the effects of insulin on the expression and activity of neuronal nitric oxide synthase (nNOS) were investigated in the cultured cerebellum cell line R2, cerebral cortical astrocytes, and neurons of rats by using flow cytometry, in situ hybridization, RT-PCR, and electron spin resonance (ESR) techniques. In astrocytes, the expression of nNOS was significantly stimulated by insulin in a concentration-dependent manner, with a maximal increase of about 47.6% compared with the control values (p<0.05, t test, n=5). Furthermore, in situ hybridization analysis showed that the expression of nNOS was also significantly increased by insulin (0.64 ng/ml, 6 h), reaching 134.2+/-9.6% of the control values (p<0.05, t test, n=3). In addition, by using nNOS specific primers, RT-PCR analysis also demonstrated the same effect of insulin (0.64 ng/ml, 6 h) on nNOS mRNA expression. Similarly, significant increase of the expression of nNOS protein and mRNA were also observed in both R2 cells and neurons of rats after incubation with insulin. In addition, significant increase of the activity of nNOS in R2 cells and astrocytes were also detected after incubation with insulin (0.64 ng/ml, 9 h) by using ESR technique. Overall, our results suggested that exogenous insulin could upregulate the expression and activity of nNOS in R2 cells, cerebral cortical astrocytes, and neurons of rats. The phenomena opened new insights for further investigation of the physical and pathological significances of insulin in the brain.