[Preclinical study of T cell receptor specifically reactive with KRAS G12V mutation in the treatment of malignant tumors].

OBJECTIVE: KRAS gene is one of the most common mutations of proto-oncogenes in human tumors, G12V is one of the most common mutation types for KRAS. It's challenging to chemically acquire the targeted drug for this mutation. Recent studies reported that this mutation peptides can form a neoepitope for T cell recognition. Our study aims to clone the T cell receptor (TCR) which specifically recognizes the neoepitope for KRAS G12V mutation and constructs TCR engineered T cells (TCR-T), and to investigate if TCR-Ts have strong antitumor response ability. METHODS: In this study, tumor infiltrating lymphocytes were obtained from one colorectal cancer patient carrying KRAS G12V mutation. Tumor-reactive TCR was obtained by single-cell RT-5' rapid-amplification of cDNA ends PCR analysis and introduced into peripheral blood lymphocytes to generate TCR-Ts. RESULTS: We obtained a high-affinity TCR sequence that specifically recognized the HLA-A*11:01-restricted KRAS G12V8-16 epitope: KVA11-01. KVA11-01 TCR-T could significantly kill various tumor cells such as PANC-1, SW480 and HeLa (overexpressing HLA-A*11:01 and KRAS G12V), and secreting high levels of interferon-γ (IFN-γ). Non-specific killing experiments suggested KVA11-01 specifically recognized tumor cells expressing both mutant KRAS G12V and HLA-A*11:01. In vivo assay, tumor inhibition experiments demonstrated that infusion of approximately 1E7 KVA11-01 TCR-T could significantly inhibit the growth of subcuta-neously transplanted tumors of PANC-1 and HeLa (overexpressing HLA-A*11:01 and KRAS G12V) cells in nude mice. No destruction of the morphologies of the liver, spleen and brain were observed. We also found that KVA11-01 TCR-T could significantly infiltrate into tumor tissue and had a better homing ability. CONCLUSION: KVA11-01 TCR-T cells can effectively target a variety of malignant tumor cells carrying KRAS G12V mutation through in vitro and in vivo assay. KVA11-01 TCR-T cells have excellent biological activity, high specificity of target antigen and homing ability into solid tumor tissue. KVA11-01 TCR-T is expected to be an effective treatment for patients with KRAS G12V mutant solid malignancies.

[Analysis on the concept and clinical practice of patient-controlled analgesia in the treatment of cancer pain by Chinese medical providers].

Objective: To investigated the concept and clinical practice of patient-controlled analgesia (PCA) in the treatment of cancer pain. Methods: Doctors, nurses, pharmacists from the oncology department, pain department, or hospice department were investigated using an electronic questionnaire from December 1 to December 31, 2021. In addition to the basic information, there were 26 questions were collected, including the current situation of cancer pain treatment, the concept of medical staff on PCA treatment of cancer pain and the clinical practice of PCA. Results: Questionnaires from 2 872 medical staff were collected from 993 hospitals in 30 provincial administrative units. Only 34.8% (955/2 748) of medical staff considered that the satisfaction rate of cancer pain control was over 75%, and 27.9% (548/1 968) of medical staff convinced that the satisfaction rate of breakthrough pain control was less than 50%. 97.1% (2 439/2 513) of medical staff considered that PCA could be effectively used for cancer pain treatment. The proportion of medical staff in secondary and tertiary hospitals who thought that PCA was applicable to cancer pain that could not be effectively alleviated by standardized non-invasive drug administration was 64.6% (319/494) and 69.1% (1 262/1 826) respectively, which was higher than that in primary hospitals [57.0% (110/193)] (P=0.002). In different occupations, the proportion of nurses who convinced PCA treatment of cancer pain increased the risk of addiction and drug overdose was 62.8% (431/686) and 76.1% (522/686), respectively, which was higher than doctors [39.2% (670/1709) and 58.2% (995/1709), respectively] and pharmacists [49.2% (58/118) and 65.3% (77/118), respectively] (all P<0.001). There was no significant difference in type of pump, route of administration, mode of infusion, protocol for PCA administration and selection of common medication in PCA treatment of cancer pain among different hospitals (all P>0.05). The calculation of continuous infusion dose and rescue dose of PCA was not uniform among different hospitals. After initiation of PCA, 71.7% (1 226/1 709) of hospitals had insufficient analgesia and most of them needed to be adjusted for 1-3 times to achieve satisfactory analgesia. Conclusion: Medical staff have insufficient cognition of PCA treatment of cancer pain and there is a lack of unified guidance in clinical practice. Therefore, it is an urgent need to develop an expert consensus on PCA treatment of cancer pain.

Relationship between sleep disorders and lymphocyte subsets and cytokines in patients with lung cancer.

This study aimed to investigate the relationship between sleep disorders and lymphocyte subsets and cytokines in patients with lung cancer undergoing radiotherapy, and to establish a theoretical foundation for predicting sleep disorders and preventing interventions in radiotherapy in lung cancer patients. Ninety-two patients with lung cancer requiring radiotherapy were selected as the study subjects. The patients' demographic data and disease-related conditions were investigated. Their quality of sleep was measured before radiotherapy, after two and four weeks of radiotherapy, and at the end of radiotherapy. According to the Pittsburgh Sleep Quality Index Number Table (PSQI), patients with PSQI score> 7 points were put into a sleep disorder group, and patients with PSQI score 0-7 were put into a normal sleep group. Lymphocyte subsets were enumerated and cytokine levels (IL-6, IL-1b) were measured during these four periods. The difference in sleep disorders at four weeks between patients with or without synchronous chemotherapy was statistically significant (P less than 0.05). The levels of lymphocyte subsets in the sleep disorder group and the control sleep group showed no difference in the index of lymphocyte subsets before radiotherapy. In the sleep disorder group, CD4+ cells were lower after two weeks of radiotherapy (P less than 0.05). After four weeks of radiotherapy, CD3+, CD4+, and CD16+56+ subsets were lower (P less than 0.05). At the end of radiotherapy, there was no difference in each index. There was no significant difference in IL-6 levels between the two groups before radiotherapy, after two weeks, or after four weeks (P greater than 0.05). At the end of radiotherapy, IL-6 levels in the sleep disorder group were higher than those in the control sleep group (P less than 0.05). There was no significant difference in IL-1b between the two groups (P greater than 0.05). In conclusion, monitoring of T-lymphocyte subsets and IL-6 levels in patients is enhanced during radiotherapy. Clinically effective programs of radiotherapy for lung cancer improve the body's immune status.

Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4.

The feather is a valuable by-product with a huge annual yield produced by the poultry industry. Degradation of feathers by microorganisms is a prerequisite to utilize this insoluble protein resource. To improve the degrading efficiency of feathers, mutagenesis of the bacterium Bacillus subtilis S1-4 was performed. By combining ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine treatment for mutagenesis, a high protease-producing mutant (UMU4) of B. subtilis S1-4 was selected, which exhibited 2.5-fold higher extracellular caseinolytic activity than did the wild-type strain. UMU4 degraded chicken feathers more efficiently, particularly for the release of soluble proteins from the feathers, compared to the wild-type strain. Furthermore, an extracellular protease with a molecular weight of 45 kDa, as determined by SDS-PAGE, was purified from UMU4. Biochemical characterization indicated that the caseinolytic activity of the protease was largely inhibited by phenylmethanesulfonyl fluoride, suggesting that the purified enzyme is a serine protease. This protease was highly active over a wide range of pHs (6.0 to 12.0) and temperatures (50° to 75°C) with an optimal pH and temperature of 8.0 and 65°C, respectively. The purified enzyme exhibited good thermostability with a 72.2 min half-life of thermal denaturation at 60°C. In addition, this protease was not sensitive to heavy metal ions, surfactants, or oxidative reagents. In conclusion, strain improvement for protease production can serve as an alternative strategy to promote feather degradation. The UMU4 mutant of B. subtilis and its serine protease could be potentially used in various industries.

High-powered microwave ablation of larger hepatocellular carcinoma: evaluation of recurrence rate and factors related to recurrence.

AIM: To evaluate the safety and efficacy of high-powered (80-100 W) percutaneous microwave ablation (MWA) at a frequency of 2450±10 MHz for treating larger hepatocellular carcinoma (HCC) and to predict the risk factors of local recurrence after high-powered MWA. MATERIALS AND METHODS: The study was approved by the Institutional Review Board, and informed consent was waived because of the retrospective study design. Forty-five patients with a total of 60 lesions received high-power (80-100 W) MWA at a frequency of 2450±10 MHz through a percutaneous approach that was guided by ultrasound. Of the 60 lesions with a maximum tumour measuring 3-8 cm, 46 lesions were 3-5 cm and 14 were 5-8 cm. The complete ablation rates, local recurrence rates, complications, and short-term survival were analysed. Ten possible risk factors for local recurrence were analysed. RESULTS: The complete ablation rates were 82.61% for the first ablation and 100% for the second ablation for 3-5 cm lesions. The complete ablation rates were 64.29% (82.61% versus 64.29%, p=0.037) for the first ablation and 85.71% (100% versus 85.71%, p=0.055) for the second ablation for 5-8 cm lesions. Local recurrence was observed in 11 out of the 45 (24.44%) successfully treated patients. The 1-year and 2-year survival rates were 95.56% (43/45) and 86.67% (39/45), respectively. No procedure-related mortality was observed and no major bleeding, liver rupture, or liver abscesses occurred. Univariate analysis showed that a positive correlation existed between the number of lesions (p=0.022), proximity to the risk area (p=0.001), pre-ablation alpha-fetoprotein (AFP) levels (p=0.025), hepatitis B virus (HBV)-DNA replication (p=0.027) and local recurrence. Multivariate analysis identified HBV-DNA (p=0.031) and proximity to the risk area (p=0.039) as the independent prognosis factors causing postoperative HCC local recurrence. CONCLUSION: High-powered MWA of larger hepatocellular carcinomas appears to be a safe and effective treatment. HBV-DNA and proximity to the risk area appear to be independent predictors of local tumour recurrence.

[Clinical study of the cytokine panel in the diagnosis of ocular chronic graft-versus-host disease].

Objective: To investigate the association between cytokines and ocular chronic graft-versus-host disease (cGVHD) and identify specific biomarkers for ocular cGVHD to enhance clinical diagnosis, treatment, and evaluation. Methods: A mouse model of cGVHD was established to explore the correlation between cGVHD and serum cytokines. Based on the findings from the animal experiments and literature review, a panel of 16 cytokine combinations was identified. Enzyme-linked immunosorbent assay (ELISA) was used to compare the cytokine concentrations in the serum and tear samples from patients who underwent allogeneic hematopoietic stem cell transplantation from June 2017 to March 2022 at the Medical Center of Hematology, Xinqiao Hospital, Army Medical University. Results: ① Compared with the control group, mice with cGVHD exhibited elevated serum IL-1ß, IL-6, IL-8, IL-17, IFN-γ, CX3CL1, CXCL11, CXCL13, CCL11, and CCL19 concentrations (all P<0.05). ② Analysis of the cytokine profiles of the serum and tear samples revealed that compared with patients without ocular cGVHD, those with ocular cGVHD exhibited increased serum IL-8 [P=0.032, area under the curve (AUC) =0.678]; decreased serum IL-10 (P=0.030, AUC=0.701) ; elevated IL-8, IFN-γ, CXCL9, and CCL17 in tear samples; and lower IL-10 and CCL19 in tear samples (all P<0.05, all AUC>0.7). Moreover, cytokines in tear samples showed correlations with ocular surface parameters related to ocular cGVHD. Conclusions: Tear fluid demonstrates greater specificity and sensitivity as a biomarker for diagnosing ocular cGVHD than serum biomarkers. Among the identified cytokines in tear samples, IL-8, IL-10, IFN-γ, CXCL9, CCL17, and CCL19 serve as diagnostic biomarkers for ocular cGVHD post-transplantation, offering practical reference value for diagnosis.

[Evaluation of differences in quality of life in patients with chronic graft-versus-host disease].

Objective: To evaluate the status of, differences in, and factors influencing quality of life (QoL) in patients with chronic graft-versus-host disease (GVHD). Methods: From September 2021 to February 2023, a cross-sectional study of 140 patients with chronic GVHD was conducted at our center. Symptom burden was assessed by the Lee Symptomatology Scale (LSS), and QoL was assessed by the Medical Outcome Study 36-Item Short-Form Health Survey (SF-36) (version 1) and five-level EuroQoL five-dimensional questionnaire (EQ-5D-5L). Results: Data from 140 respondents, including 32 (22.9%) with mild chronic GVHD, 87 (62.1%) with moderate chronic GVHD, and 21 (15.0%) with severe chronic GVHD, were analyzed. Of the respondents, 61.4% were male, and the median transplantation age was 34 (15-68) years. The primary diagnoses were acute myeloid leukemia (50.0%), acute lymphoblastic leukemia (20.0%), and myelodysplastic syndrome (15.0%). The common chronic GVHD-affected organs included the skin in 74 patients (52.9%), the eyes in 57 patients (40.7%), and the liver in 50 patients (35.7%). Among the whole cohort, the eye (20.48±23.75), psychological (16.13±17.00), and oral (13.66±20.55) scores were highest in the LSS group. The physiological function (36.07±11.13), social function (36.10±10.68), and role-emotional functioning (38.36±11.88) scores were lowest in the SF-36 group. The EQ-5D index was 0.764. The total LSS scores for mild, moderate, and severe chronic GVHD were 6.51±6.15, 10.07±5.61, and 20.90±10.09, respectively. The SF-36 physical component scores (PCSs) were 43.12±6.38, 40.73±7.14, and 36.97±6.97, respectively, and the mental component scores (MCSs) were 43.00±8.47, 38.90±9.52, and 28.96±9.63, respectively. The EQ-5D values were 0.810±0.124, 0.762±0.179, and 0.702±0.198, respectively. The multivariate analysis showed that the overall symptom burden (ß=-0.517), oral symptom burden (ß=-0.456), National Institute of Health (NIH) criteria for the eyes (ß=-0.376), and nutrition-related symptom burden (ß=-0.211) were significantly negatively correlated with the PCS. The NIH score (ß=-0.260) was negatively correlated with the MCS score. Oral symptom burden (ß=-0.400), joint/fascia NIH criteria (ß=-0.332), number of involved systems (ß=-0.253), overall NIH criteria (ß=-0.205), and number of immunosuppressants taken (ß=-0.171) were significantly negatively correlated with the EQ-5D score (all P<0.05). Medium to strong correlations were found between the EQ-5D score and the SF-36 score (|r|=0.384-0.571, P<0.001). Conclusions: The QoL of patients with chronic GVHD is impaired, and the more severe the disease, the poorer the QoL. Overall symptom burden, severity of eyes, and oral symptom burden were the most important factors affecting QoL.

The efficacy and safety of flow-diverting device and coil embolization for intracranial aneurysms: a meta-analysis.

OBJECTIVE: To compare the clinical efficacy and safety of flow-diverting device (FDD) and coil embolization therapy (CET) in the treatment of intracranial aneurysms through a meta-analysis. MATERIALS AND METHODS: We comprehensively searched in PubMed, Embase, Cochrane Library, CNKI, Wan Fang, VIP databases, and China Biology Medicine disc (CBM) for eligible literature. Odds ratio (SMD) and 95% confidence intervals (CIs) were considered as effect measures. Statistical heterogeneity was tested by Cochran's Q statistic and I2 tests, and sensitivity analysis was used to evaluate the stability of research results. Publication bias was detected by funnel diagrams. RESULTS: A total of 888 patients from 9 studies were finally enrolled in our analysis. Through meta-analysis, the results showed that the aneurysm occlusion rate in the FDD group was significantly higher than that in the CET group (OR, 95% CI=1.68, 1.20 to 2.36, p=0.002), and the retreatment rate after aneurysm operation in the FDD group was significantly lower than that in the FDD group (OR, 95% CI=0.40, 0.22 to 0.74, p=0.003). There was no significant difference in the proportion of mRS score (0-2) between the two groups during postoperative follow-up (OR, 95% CI=0.63, 0.20 to 1.94, p=0.43). In terms of safety, there was no significant difference in the incidence of postoperative complications (OR, 95% CI=1.11, 0.68 to 1.81, p=0.67) and mortality (OR, 95% CI=1.35, 0.53 to 3.42) between the two groups. CONCLUSIONS: Compared with CET, FDD has achieved satisfactory results in increasing the rate of aneurysm occlusion and reducing the rate of retreatment of intracranial aneurysms. There is no significant difference in security between FDD and CET, though. These findings are reported in this paper, but because of the limitations of the included study, they need to be further verified by well-designed multicenter randomized controlled trials (RCT).

Evaluation of multiple sclerosis diagnostic criteria in Suzhou, China--risk of under-diagnosis in a low prevalence area.

OBJECTIVE: To evaluate the discharge diagnosis of demyelinating diseases in the central nervous system (CNS) and analyze the predictive value of the new diagnostic criteria in Suzhou, China. MATERIALS AND METHODS: We collected clinical information and data of laboratory examinations for all cases with a diagnosis of various demyelinating diseases in the CNS. All data were reviewed individually by four senior neurologists, and a diagnosis was finally given to each patient according to the McDonald criteria and the Poser criteria for multiple sclerosis (MS). RESULTS: In the analysis, 176 patients with a diagnosis of demyelinating diseases in the CNS at discharge were included. In 82 patients with a diagnosis of MS at discharge, the MS diagnosis was confirmed for 74 patients according to the McDonald criteria for MS, and the positive predictive value for the discharge diagnosis of MS was 90.2% (74/82). According to the Poser criteria, 61 patients were diagnosed as MS. The consistency of the two diagnostic criteria for MS was 78.4%, based on the results of the evaluation. CONCLUSIONS: Under-diagnosis of MS could be one of the explanations for the low prevalence of MS in China. Compared to the Poser criteria, the McDonald criteria had a higher sensitivity for the diagnosis of MS.

[Progress in research of burden of disease attributed to population ageing].

Objective: To summarize recent progress in research of burden of disease attributed to population ageing and provide reference for relevant research in the future. Methods: We conducted a systematic literature review of quantitative studies about the impact of population ageing on burden of disease published from 2009 to 2019 according to the inclusion and exclusion criteria through PubMed, Web of Science, Embase, Cochrane Library, Wangfang database and China National Knowledge Infrastructure (CNKI) databases and extracted basic information and key results of the searched literature. Results: A total of 65 studies were included in the analysis, in which 29 (44.6%) studies used death number or mortality rate as outcome measures, 43 (66.2%) studies focused on a single country, such as China, United Kingdom and United States, 39 (60.0%) studies quantified the impact of population ageing on single disease, such as diabetes, lung cancer and coronary heart disease, 44 (67.7%) studies used decomposition methods to quantify the impact of population ageing, and 10 (15.4%) studies evaluated the effect of interventions on alleviating the impact of population ageing. Most studies found that population ageing increased the burden of some diseases, such as cancer, cardiovascular disease and dementia, while a few studies reported that population ageing decreased the burden of some diseases, such as neonatal disease and malaria. Various decomposition methods were adopted in 65 included studies, but several common methods were sensitive to the preconditions that were assumed, the decomposition order of three factors (population size, age structure, and age-specific rate) and the choice of control group, resulting incomparable and unstable results. Conclusions: The published decomposition studies adopted various methods and only evaluated the impact of population ageing in very limited countries and for several diseases. Therefore, a systematic evaluation using robust decomposition method is very necessary to evaluate the impact of population aging on disease burden across countries and diseases.

MicroRNA-132 improves myocardial remodeling after myocardial infarction.

OBJECTIVE: To elucidate the potential role of microRNA-132 in myocardial infarction (MI) and its underlying mechanism. MATERIALS AND METHODS: The myocardial infarction model was established in WT and microRNA-132 KO mice using the LAD ligation method. WT mice were assigned into the control group (LAD ligation for MI) and sham group. After animal procedures, infarct size was calculated using hematoxylin and eosin (HE) staining and cardiac function was evaluated using echocardiography, respectively. By analyzing differentially expressed microRNAs relative to MI in a microarray, microRNA-132 was screened out and further verified by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Hemodynamic parameters and cardiac function indexes in mice were accessed, including scar length/LV length, FS, dp/dtmax, dp/dtmin, ESV, EDV, EF and Tau_w. RESULTS: QRT-PCR data showed a gradual decrease in microRNA-132 expression in the infarction zone, border zone and remote zone within 7 days after MI. Compared with mice in the control group, microRNA-132 KO mice showed a higher percentage of scar length/LV length at postoperative day 14 and day 28. MicroRNA-132 KO mice showed decreased FS, dp/dtmax and EF, but increased dp/dtmin, ESV and EDV. The injection of different concentrations of microRNA-132 mimics into mice (8 mg/kg, 16 mg/kg and 32 mg/kg) could reduce LVIDD, LVIDs, ESV, EDV, dp/dtmin and Tau_w. However, FS, EF and dp/dtmax increased by the injection of microRNA-132 mimics at postoperative day 28. The injection of 16 mg/kg microRNA-132 mimics significantly reduced the percentage of scar length/LV length in microRNA-132 KO mice than the control group and miR-CO group. After injection of 16 mg/kg microRNA-132 mimics, LVIDD and LVIDs markedly decreased at postoperative day 14 and day 28 compared with the control group and miR-CO group. However, FS was elevated by microRNA-132 mimics. CONCLUSIONS: MicroRNA-132 is involved in the development of myocardial infarction. The MicroRNA-132 expression is upregulated after myocardial infarction, influencing infarct size and cardiac function.

Relationship between LAPTM4B gene polymorphism and susceptibility of colorectal and esophageal cancers.

BACKGROUND: Lysosome-associated protein transmembrane 4 beta (LAPTM4B) is a novel gene of the mammalian LAPTM family and has been shown to be overexpressed in human hepatocellular carcinoma. There are two alleles, LAPTM4B*1 and *2, which share the same sequence except for one segment of 19 bp in the 5' untranslated region of the exon 1. LAPTM4B*1 has one 19 bp segment, while LAPTM4B*2 has two tight tandem segments. The current case-control study was aimed to identify relationship between the gene polymorphism of LAPTM4B and the susceptibility of colorectal and esophageal cancers. PATIENTS AND METHODS: Blood samples were collected from patients with colon, rectal or esophageal cancers and control subjects. Genotypes of LAPTM4B were determined by PCR to detect differences between cancer cases (n = 701) and healthy controls (n = 350). Association between the LAPTM4B polymorphism and the risk of cancer was calculated by unconditional logistic regression models. RESULTS: We found that there was a significant difference (P = 0.0016) in allelic frequencies of LAPTM4B*2 between colon cancer cases (33.2%) and controls (24.1%). The risk of colon cancer was elevated significantly in cases with *1/2 genotype [odds ratio (OR) = 1.474; 95% confidence interval (CI) = 1.037-2.095] and *2/2 genotype (OR = 2.531; 95% CI = 1.316-4.868) when compared with the *1/1 genotype. No significant difference was observed for LAPTM4B*2 between the rectal or esophageal cancer cases when compared with the controls. The polymorphism in LAPTM4B was associated with increased risk of colon cancer but not of rectal and esophageal cancers. CONCLUSIONS: These results indicate that the genetic polymorphism of LAPTM4B is a potential risk factor for the development of colon cancer.

Qualitative and quantitative analysis of four species of Curcuma rhizomes using twice development thin layer chromatography.

The rhizomes of Curcuma phaeocaulis, Curcuma kwangsiensis, Curcuma wenyujin and Curcuma longa are used as Ezhu or Jianghuang in traditional Chinese medicine for a long time. Due to their similar morphological characters, it is difficult to distinguish their origins of raw materials used in clinic. In this study, a simple, rapid and reliable twice development TLC method was developed for qualitative and quantitative analysis of the four species of Curcuma rhizomes. The chromatography was performed on silica gel 60F(254) plate with chloroform-methanol-formic acid (80:4:0.8, v/v/v) and petroleum ether-ethyl acetate (90:10, v/v) as mobile phase for twice development. The TLC markers were colorized with 1% vanillin-H(2)SO(4) solution. The four species of Curcuma were easily discriminated based on their characteristic TLC profiles, and simultaneous quantification of eight compounds, including bisdemethoxycurcumin, demethoxycurcumin, curcumine, curcumenol, curcumol, curdione, furanodienone and curzerene, in Curcuma were also performed densitometrically at lambda(scan)=518nm and lambda(reference)=800 nm. The investigated compounds had good linearity (r(2)>0.9905) within test ranges. Therefore, the developed TLC method can be used for quality control of Curcuma rhizomes.

20-Hydroxyeicosa-tetraenoic acid (20 HETE) activates protein kinase C. Role in regulation of rat renal Na+,K+-ATPase.

It is well documented that the activity of Na+,K+-ATPase can be inhibited by the arachidonic acid metabolite, 20-hydroxyeicosa-tetraenoic acid (20 HETE). Evidence is presented here that this effect is mediated by protein kinase C (PKC). PKC inhibitors abolished 20 HETE inhibition of rat Na+,K+-ATPase in renal tubular cells. 20 HETE caused translocation of PKC alpha from cytoplasm to membrane in COS cells. It also inhibited Na+,K+-ATPase activity in COS cells transfected with rat wild-type renal Na+,K+-ATPase alpha1 subunit, but not in cells transfected with Na+,K+-ATPase alpha1, where the PKC phosphorylation site, serine 23, had been mutated to alanine. PKC-induced phosphorylation of rat renal Na+,K+-ATPase, as well as of histone was strongly enhanced by 20 HETE at the physiologic calcium concentration of 1.3 microM, but not at the calcium concentration of 200 microM. The results indicate that phospholipase A2-arachidonic acid-20 HETE pathway can exert important biological effects via activation of PKC and that this effect may occur in the absence of a rise in intracellular calcium.

Resveratrol decreases the insoluble Aß1-42 level in hippocampus and protects the integrity of the blood-brain barrier in AD rats.

Our previous studies demonstrated resveratrol (Res) administration protected Alzheimer's disease (AD) rats from developing memory decline by anti-oxidation. Beta-amyloid peptide 1-42 (Aß1-42) is not only the primary protein component of senile plaques in AD but also is believed to play an important part in its pathology. Increasing evidence has shown neuroinflammation and the integrity of the blood-brain barrier (BBB) is closely related to the pathogenesis of AD. The aim of the present study is to further elucidate whether Res prevents AD rats from inflammation induced by Aß1-42 and protects the integrity of BBB. Rats were divided into six groups: (1) ovariectomized (OVX)+D-galactose (D-gal) 100mg/kg group (OVX+D-gal); (2-4) OVX, D-gal and Res 20, 40 and 80 mg/kg treated groups; and (5) OVX, D-gal and estradiol valerate 0.8 mg/kg treated group (ET); (6) Sham control group. 12 weeks later, Res 40 and 80 mg/kg treatment exhibited a significant decrease of Aß1-42 compared with the OVX+D-gal rats of hippocampus, which was accompanied by decreased expression of advanced glycation endproducts (RAGE), matrix metalloprotein-9 (MMP-9), nuclear factor kappaB (NF-κB) and the increase of Claudin-5. These results suggest that Res is useful not only in protecting OVX+D-gal rats from neuroinflammation mediated by Aß1-42 by decreasing the expression of NF-κB but also the integrity of BBB by increasing Claudin-5 and decreasing RAGE, MMP-9.

Further evidence for dominant inheritance at the chromosome 15q11-13 locus in familial Angelman syndrome.

Eleven patients with Angelman syndrome (AS) and their parents from 5 families have been studied with high resolution chromosome analysis and molecular probes from region 15q11-13 in an attempt to elucidate the mode of inheritance in familial AS. No deletions were detected. All families were informative with a combination of different short arm cytogenetic markers. All sets of sibs inherited the same maternal chromosome 15, but in 3 families sibs inherited different paternal 15s. Analysis of 6 polymorphic DNA markers supported the conclusion that AS sibs inherit the same maternal 15, but often different paternal 15s. These data make autosomal recessive inheritance at a 15q11-13 locus very unlikely and support the hypothesis that familial AS is due to maternal transmission of a mutation within 15q11-13.

Bacterial expression of a neutralizing mouse monoclonal antibody Fab fragment to a 150-kilodalton surface antigen of Entamoeba histolytica.

A mouse monoclonal antibody (MAb) (EH3015, IgG1 with a K light chain) prepared by hybridoma technology recognizes a 150-kD surface antigen of Entamoeba histolytica and inhibits adherence and cytotoxicity of the ameba to mammalian cells. The genes encoding the light chain and the Fd region of the heavy chain of the MAb were cloned and expressed in Escherichia coli. The plasmid used was designed for the expression of Fab with a hexa-histidine tag in the periplasmic space. Recombinant Fab fragments were purified and analyzed by an indirect immunofluorescence antibody test and Western immunoblot. The specificity of the recombinant Fab fragment was comparable with the parent whole IgG. In addition, the Fab fragments significantly inhibited the adherence of E. histolytica to erythrocytes. These results suggest that the production of a neutralizing MAb in Escherichia coli is practical and efficient with this expression system.

Ribodemes of Blastocystis hominis isolated in Japan.

To determine if genetic diversity of Blastocystis hominis exists in Japan, we monitored 64 B. hominis-infected people: 39 asymptomatic people whose infections were detected during routine medical check-ups (32 Japanese and 7 non-Japanese) and 25 patients with gastrointestinal symptoms who visited the outpatient clinics of St. Luke's International Hospital (19 Japanese and 6 non-Japanese). We detected 6 known and 2 new riboprint patterns in isolates from the infected people. There were no differences in the distribution of ribodemes between isolates from Japanese and non-Japanese people, similar to that in other countries. However, we noted a possible relationship between ribodeme type and pathogenicity. The results suggest that ribodemes I, III, and VI may be responsible for gastrointestinal symptoms.

Study on immunopathogenesis of minimal change nephropathy. Effects of mitogens activated lymphocytes on glomerular polyanions in vitro.

Rat spleen lymphocytes (RSL) stimulated by mitogens in vitro were cultured on cryostat sections of rat kidney. After 36 hours glomerular polyanions (GPA) were stained with colloidal iron (CI). The results showed that the RSL stimulated with Con A were able to reduce GPA stainability as that treated with neuraminidase solution, and the effect was dose-dependent on Con A, whereas the supernatant of lymphocytes induced with Con A and the lymphocytes induced with PHA were not able to reduce GPA stainability. The result is in favor of the recent concept that the pathogenesis of MCN is associated with the loss of GPA which results from the dysfunction of the subpopulation of T lymphocytes.