Near-Infrared II Photobiomodulation Preconditioning Ameliorates Stroke Injury via Phosphorylation of eNOS.

BACKGROUND: The current management of patients with stroke with intravenous thrombolysis and endovascular thrombectomy is effective only when it is timely performed on an appropriately selected but minor fraction of patients. The development of novel adjunctive therapy is highly desired to reduce morbidity and mortality with stroke. Since endothelial dysfunction is implicated in the pathogenesis of stroke and is featured with suppressed endothelial nitric oxide synthase (eNOS) with concomitant nitric oxide deficiency, restoring endothelial nitric oxide represents a promising approach to treating stroke injury. METHODS: This is a preclinical proof-of-concept study to determine the therapeutic effect of transcranial treatment with a low-power near-infrared laser in a mouse model of ischemic stroke. The laser treatment was performed before the middle cerebral artery occlusion with a filament. To determine the involvement of eNOS phosphorylation, unphosphorylatable eNOS S1176A knock-in mice were used. Each measurement was analyzed by a 2-way ANOVA to assess the effect of the treatment on cerebral blood flow with laser Doppler flowmetry, eNOS phosphorylation by immunoblot analysis, and stroke outcomes by infarct volumes and neurological deficits. RESULTS: Pretreatment with a 1064-nm laser at an irradiance of 50 mW/cm2 improved cerebral blood flow, eNOS phosphorylation, and stroke outcomes. CONCLUSIONS: Near-infrared II photobiomodulation could offer a noninvasive and low-risk adjunctive therapy for stroke injury. This new modality using a physical parameter merits further consideration to develop innovative therapies to prevent and treat a wide array of cardiovascular diseases.

Neuroprotection Mediated by Upregulation of Endothelial Nitric Oxide Synthase in Rho-Associated, Coiled-Coil-Containing Kinase 2 Deficient Mice.

BACKGROUND: Rho-associated kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton and endothelial nitric oxide synthase (eNOS). Because the phosphorylation of eukaryotic elongation factor-1A1 (eEF1A1) by ROCK2 is critical for eNOS expression, we hypothesized that this molecular pathway may play a critical role in neuroprotection following focal cerebral ischemia.Methods and Results:Adult male wild-type (WT) and mutant ROCK2 and eNOS-/-mice were subjected to middle cerebral artery occlusion (MCAO), and cerebral infarct size, neurological deficit and absolute cerebral blood flow were measured. In addition, aortic endothelium-dependent response to acetylcholine, NG-nitro-L-arginine methyl ester (L-NAME) and sodium nitroprusside were assessed ex vivo. Endothelial cells from mouse brain or heart were used to measure eNOS and eEF1A activity, as well as NO production and eNOS mRNA half-life. In global hemizygous ROCK2+/-and endothelial-specific EC-ROCK2-/-mice, eNOS mRNA stability and eNOS expression were increased, which correlated with enhanced endothelium-dependent relaxation and neuroprotection following focal cerebral ischemia. Indeed, when ROCK2+/-mice were place on an eNOS-/-background, the neuroprotective effects observed in ROCK2+/-mice were abolished. CONCLUSIONS: These findings indicate that the phosphorylation of eEF1A1 by ROCK2 is physiologically important for eNOS expression and NO-mediated neuroprotection, and suggest that targeting endothelial ROCK2 and eEF1A may have therapeutic benefits in ischemic stroke and cardiovascular disease.

Basic Science of Neuroinflammation and Involvement of the Inflammatory Response in Disorders of the Nervous System.

Neuroinflammation is a key immune response observed in many neurologic diseases. Although an appropriate immune response can be beneficial, aberrant activation of this response recruits excessive proinflammatory cells to cause damage. Because the central nervous system is separated from the periphery by the blood-brain barrier (BBB) that creates an immune-privileged site, it has its own unique immune cells and immune response. Moreover, neuroinflammation can compromise the BBB causing an influx of peripheral immune cells and factors. Recent advances have brought a deeper understanding of neuroinflammation that can be leveraged to develop more potent therapies and improve patient selection.

Treatment for appendicitis in cancer patients on chemotherapy: a retrospective cohort study.

Purpose: Whether to perform surgery or conservatively manage appendicitis in immunosuppressed patients is a concern for clinicians. This study aimed to compare the outcomes of these 2 treatment options for appendicitis in patients with cancer undergoing chemotherapy. Methods: This retrospective study included 206 patients with cancer who were diagnosed with acute appendicitis between August 2001 and December 2021. Among them, patients who received chemotherapy within 1 month were divided into surgical and conservative groups. We evaluated the outcomes, including treatment success within 1 year, 1-year recurrence, and the number of days from the diagnosis of appendicitis to chemotherapy restart, between the 2 groups. Results: Among the 206 patients with cancer who were diagnosed with acute appendicitis, 78 received chemotherapy within 1 month. The patients were divided into surgery (n = 63) and conservative (n = 15) groups. In the surgery group, the duration of antibiotic therapy (7.0 days vs. 16.0 days, P < 0.001) and length of hospital stay (8.0 days vs. 27.5 days, P = 0.002) were significantly shorter than conservative groups. The duration from the diagnosis of appendicitis to the restart of chemotherapy was shorter in the surgery group (20.8 ± 15.1 days vs. 35.2 ± 28.2 days, P = 0.028). The treatment success rate within 1 year was higher in the surgery group (100% vs. 33.3%, P < 0.001). Conclusion: Surgical treatment showed a significantly higher success rate than conservative treatment for appendicitis in patients less than 1 month after chemotherapy. Further prospective studies will be needed to clinically determine treatment options.

Striatal neurons expressing full-length mutant huntingtin exhibit decreased N-cadherin and altered neuritogenesis.

The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in Hdh(Q111) striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdh(Q111) striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdh(Q111) striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell-substratum adhesion, and primary Hdh(Q111) striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.

Role of combined BI-RADS assessment using mammography and sonography for evaluation of incidental hypermetabolic lesions in the breast on 18F-FDG PET-CT.

BACKGROUND: PET-CT is widely used for evaluation and follow-up of malignancy. Incidental hypermetabolic lesions are often found on PET-CT, some of which are confirmed to be malignant. PURPOSE: To estimate the role of combined Breast Imaging-Reporting and Data System (BI-RADS) assessment using mammography and sonography for evaluation of incidental hypermetabolic lesions on 18F-FDG PET-CT and to determine an appropriate next step. MATERIAL AND METHODS: This study included incidental hypermetabolic lesions found in the breasts of 7594 women who underwent PET-CT at three university-affiliated hospitals between January 2006 and December 2011. We reviewed the maximum standardized uptake value (SUVmax) of incidental lesions, combined BI-RADS assessment of mammography and sonography, and final results. We analyzed the negative predictive values of the probably benign (categories 1-3) group and the sensitivity of suspicious (categories 4 and 5) groups according to combined BI-RADS assessment. RESULTS: Forty-three patients (0.6%) had 49 incidental hypermetabolic lesions in the breast. Histologic diagnosis in 17 patients confirmed nine breast cancers (27.3%). Sixteen patients underwent imaging follow-up for at least 2 years; no breast cancer was detected. Thirteen patients were lost to follow-up and were excluded. For the suspicious (n = 14) and probably benign (n = 19) groups according to combined BI-RADS assessment, both the sensitivity and negative predictive values were 100%. Using an optimal diagnostic cut-off value of 2.15, the malignancy rate was not significantly different (16.7% vs. 45.5%, respectively, in the group with SUVmax < 2.15 and the group with SUVmax ≥ 2.15; P > 0.05). The SUVmax of the confirmed malignant and assumed benign groups were not significantly different (3.1% vs. 2.2%, respectively; P > 0.05). CONCLUSION: Both mammography and sonography should be considered the next step to evaluate incidental hypermetabolic lesions on 18F-FDG PET-CT because combined BI-RADS assessment provides an excellent negative predictive value for excluding malignancy.

Fingolimod provides long-term protection in rodent models of cerebral ischemia.

OBJECTIVE: The sphingosine-1-phosphate (S1P) receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver, and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species. METHODS: We used rodent models of middle cerebral artery occlusion and cell-culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod. RESULTS: In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema, and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by tumor necrosis factor alpha. INTERPRETATION: These findings suggest that anti-inflammatory mechanisms, and possibly vasculoprotection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.

Estimating the malaria transmission of Plasmodium vivax based on serodiagnosis.

BACKGROUND: Plasmodium vivax re-emerged in 1993 and has now become a major public health problem during the summer season in South Korea. The aim of this study was to interpret and understand the meaning of seroepidemiological studies for developing the best malaria control programme in South Korea. METHODS: Blood samples were collected in Gimpo city, Paju city, Yeoncheon County, Cheorwon County and Goseong County of high risk area in South Korea. Microscopy was performed to identify patients infected with P. vivax. Antibody detection for P. vivax was performed using indirect fluorescent antibody test (IFAT). RESULTS: A total of 1,574 blood samples was collected from participants in the study areas and evaluated against three parameters: IFAT positive rate, annual antibody positive index (AAPI), and annual parasite index (API). The IFAT positive rate was 7.24% (n = 114). Of the five study areas, Gimpo had the highest IFAT positive rate (13.68%) and AAPI (4.63). Yeongcheon had the highest API in 2005 (2.06) while Gimpo had the highest API in 2006 (5.00). No correlation was observed between any of the three parameters and study sites' distance from the demilitarized zone (DMZ). CONCLUSIONS: These results showed that P. vivax antibody levels could provide useful information about the prevalence of malaria in endemic areas. Furthermore, AAPI results for each year showed a closer relationship to API the following year than the API of the same year and thus could be helpful in predicting malaria transmission risks.

Genetic variation of aldolase from Korean isolates of Plasmodium vivax and its usefulness in serodiagnosis.

BACKGROUND: The malaria aldolase is widely used as rapid diagnostic test (RDT), but the efficacy in aspect of its serological effectiveness in diagnosis is not known. The genetic variation of Korean isolates was analysed and recombinant aldolase was evaluated as a serological antigen in Plasmodium vivax malaria. METHODS: Genomic DNA was purified and the aldolase gene of P. vivax from 25 patients' blood samples was amplified. The samples came from 5 epidemic areas; Bucheon-si, Gimpo-si, Paju-si of Gyeonggido, Gangwha-gun of Incheon metropolitan city, and Cheorwon of Gangwon-do, South Korea. The antigenicity of the recombinant aldolase was tested by western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: Sequence analysis of 25 Korean isolates of P. vivax showed that the open reading frame (ORF) of 1,110 nucleotides encoded a deduced protein of 369 amino acids (aa). This ORF showed 100% homology with the P. vivax Sal I strain (XM_00165894) and P. vivax WDK strain (AF247063), 87.4% homology with Plasmodium falciparum (AF179421), 90.6% homology with Plasmodium chabaudi (AF247060), 89.5% homology with Plasmodium vinckei (AF247061), and 96.7% homology with Plasmodium knowlesi. A single nucleotide polymorphism (SNP) at nucleotide 180 (G to A, n = 5) was also observed in the isolates. The expressed recombinant protein had a molecular weight of approximately 31 kDa (monomeric form) and 62 kDa (dimeric form) as analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Among 109 P. vivax patients, 32 (29.4%) had positive in an enzyme-linked absorbance assay (ELISA). This result showed significant correlation between ELISA and an indirect fluorescent antibody test (IFAT) (P < 0.0001). CONCLUSIONS: The aldolase gene from Korean isolates of P. vivax showed one SNP at nucleotide position 180; this SNP mutant was discovered in only the western part of Han River, and included the regions of Ganghwa, Gimpo, and Bucheon. Based on the results, the relationship between antibody production against aldolase and the pattern of disease onset should be more investigated before using aldolase for serodiagnosis.

Comparison of the antibody responses to Plasmodium vivax and Plasmodium falciparum antigens in residents of Mandalay, Myanmar.

BACKGROUND: The aim of this study was to investigate the profile of antibodies against several antigens of Plasmodium vivax and Plasmodium falciparum in Mandalay, Myanmar. METHODS: Malaria parasites were identified by microscopic examination. To test the antibodies against P. vivax and P. falciparum in sera, an indirect immunofluorescence antibody test (IFAT) was performed using asexual blood parasite antigens. An enzyme-linked immunosorbent assay (ELISA) was performed with circumsporozoite protein (CSP), Pvs25 and Pvs28 recombinant proteins of transmission-blocking vaccine candidates for P. vivax, and liver stage specific antigen-1 and -3 (PfLSA-1, PfLSA-3) for P. falciparum. RESULTS: Fourteen patients among 112 were found to be infected with P. vivax and 26 with P. falciparum by thick smear examination. Twenty-three patients were found to be infected with P. vivax, 19 with P. falciparum and five with both by thin smear examination. Blood samples were divided into two groups: Group I consisted of patients who were positive for infection by microscopic examination, and Group II consisted of those who showed symptoms, but were negative in microscopic examination. In P. falciparum, IgG against the blood stage antigen in Group I (80.8%) was higher than in Group II (70.0%). In P. vivax, IgG against the blood stage antigen in Group I (53.8%) was higher than in Group II (41.7%). However, the positivity rate of the PvCSP VK210 subtype in Group II (40.0%) was higher than in Group I (23.1%). Similarly for the PvCSP VK247 subtype, Group II (21.7%) was higher than that for Group I (9.6%). A similar pattern was observed in the ELISA using Pvs25 and Pvs28: positive rates of Group II were higher than those for Group I. However, those differences were not shown significant in statistics. CONCLUSIONS: The positive rates for blood stage antigens of P. falciparum were higher in Group I than in Group II, but the positive rates for antigens of other stages (PfLSA-1 and -3) showed opposite results. Similar to P. falciparum, the positive rate of pre-blood stage (CSP VK210 and 247 subtype) and post-blood stage (Pvs25 and 28) antigens of P. vivax were higher in Group II than in Group I. Therefore, sero-diagnosis is not helpful to discriminate between malaria patients and symptomatic individuals during the epidemic season in Myanmar.

Detection of an antibody against Plasmodium vivax in residents of Gimpo-si, South Korea, using an indirect fluorescent antibody test.

BACKGROUND: First reemerged malaria case was reported in 1993 after two decades absent in South Korea. Thereafter, Plasmodium vivax spreads out near demilitarized zone (DMZ). This study investigated the prevalence of P. vivax after the malaria transmission season in Gimpo-si where adjacent to DMZ of South Korea. An indirect fluorescent antibody test (IFAT) was performed to evaluate anti-malaria antibodies in blood samples. METHODS: Microscopic examinations were performed to identify the presence of malaria parasites. Antibodies against P. vivax were detected using IFAT, and blood samples from antibody-positive cases were tested using a polymerase chain reaction (PCR) assay that detects malaria parasites. RESULTS: A total of 5,797 blood samples were collected from residents in Gimpo-si. The positivity rate by IFAT was 2.16% (n = 125). Yangchon-myeon (3.28%) had the highest positivity rate of the seven administrative districts tested. Positivity rates increased with age (P < 0.05). Sixteen of the IFAT positive samples (12.80%, n = 125) were positive for malaria DNA according to PCR. Blood samples with an antibody titer over 1:256 had high positivity rates in the PCR analysis (P < 0.05). CONCLUSIONS: These results indicate that antibody titers obtained using IFAT may provide useful information about the prevalence of P. vivax in low endemic areas and could be used to detect asymptomatic patients. Finding asymptomatic patients is important in eliminating vivax malaria in South Korea.

Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus.

BACKGROUND: To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope. METHODS: A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot. RESULTS: The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n=38) and a clinical specificity of 100% (n=24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. CONCLUSIONS: The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.

The role of Pvs28 in sporozoite development in Anopheles sinensis and its longevity in BALB/c mice.

To develop a vivax malaria vaccine for blocking malarial transmission, the ookinete surface protein Pvs28 was cloned from Korean malaria patients using polymerase chain reaction. The Pvs28 gene consists of 726bp and encodes 241 amino acids. It was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein, rPvs28, has a molecular weight of about 28 kDa in SDS-PAGE analysis. A monoclonal antibody against rPvs28 was produced using BALB/c mice. It inhibited sporozoite development in Anopheles sinensis mosquitoes (n = 81) which is one of the malaria vectors in Korea, with relatively high antibody titer against rPv28 persisting for more than 6 months. These results indicate that rPvs28 induces an immune response in mice that effectively blocks sporozoite development in mosquitoes. Therefore it could be a vaccine candidate for preventing vivax malaria in Korea.

Linking Notch signaling to ischemic stroke.

Vascular smooth muscle cells (SMCs) have been implicated in the pathophysiology of stroke, the third most common cause of death and the leading cause of long-term neurological disability in the world. However, there is little insight into the underlying cellular pathways that link SMC function to brain ischemia susceptibility. Using a hitherto uncharacterized knockout mouse model of Notch 3, a Notch signaling receptor paralogue highly expressed in vascular SMCs, we uncover a striking susceptibility to ischemic stroke upon challenge. Cellular and molecular analyses of vascular SMCs derived from these animals associate Notch 3 activity to the expression of specific gene targets, whereas genetic rescue experiments unambiguously link Notch 3 function in vessels to the ischemic phenotype.

Molecular cloning and expression of the VK247 circumsporozoite protein for serodiagnosis of variant form Plasmodium vivax.

Plasmodium vivax is classified into two serotypes, VK210 [the dominant form-GDRA(D/A)GQPA repeats] and VK247 [the variant form-ANGA(G/D)(N/D)QPG repeats], based on sequence variation of the repeat region of the circumsporozoite (CS) protein gene. Genomic DNA for the variant CS protein gene was obtained from field isolate strains in Myanmar. The repetitive region has highly 19 immunogenic repeats flanked by non-repeat stretches of amino acids. The sequence including this region (717 bp) was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein has a molecular weight of about 50 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Anti-VK247 antibodies were found in malaria patients who have been exposed to variant form of P. vivax in western blot analysis. Therefore, this recombinant protein might be a useful tool in serodiagnosis of malaria patients who have been infected with variant form of P. vivax.

Vascular Stiffening Mediated by Rho-Associated Coiled-Coil Containing Kinase Isoforms.

Background The pathogenesis of vascular stiffening and hypertension is marked by non-compliance of vessel wall because of deposition of collagen fibers, loss of elastin fibers, and increased vascular thickening. Rho/Rho-associated coiled-coil containing kinases 1 and 2 (ROCK1 and ROCK2) have been shown to regulate cellular contraction and vascular remodeling. However, the role of ROCK isoforms in mediating pathogenesis of vascular stiffening and hypertension is not known. Methods and Results Hemizygous Rock mice (Rock1+/- and Rock2+/-) were used to determine the role of ROCK1 and ROCK2 in age-related vascular dysfunction. Both ROCK activity and aortic stiffness increased to a greater extent with age in wild-type mice compared with that of Rock1+/- and Rock2+/- mice. As a model for age-related vascular stiffening, we administered angiotensin II (500 ng/kg per minute) combined with nitric oxide synthase inhibitor, L-Nω-nitroarginine methyl ester (0.5 g/L) for 4 weeks to 12-week-old male Rock1+/- and Rock2+/- mice. Similar to advancing age, angiotensin II/L-Nω-nitroarginine methyl ester caused increased blood pressure, aortic stiffening, and vascular remodeling, which were attenuated in Rock2+/-, and to a lesser extent, Rock1+/- mice. The reduction of aortic stiffening in Rock2+/- mice was accompanied by decreased collagen deposition, relatively preserved elastin content, and less aortic wall hypertrophy. Indeed, the upregulation of collagen I by transforming growth factor-ß1 or angiotensin II was greatly attenuated in Rock2-/- mouse embryonic fibroblasts. Conclusions These findings indicate that ROCK1 and ROCK2 mediate both age-related and pharmacologically induced aortic stiffening, and suggest that inhibition of ROCK2, and to a lesser extent ROCK1, may have therapeutic benefits in preventing age-related vascular stiffening.

Effect of electroacupuncture on glial fibrillary acidic protein and nerve growth factor in the hippocampus of rats with hyperlipidemia and middle cerebral artery thrombus.

Electroacupuncture (EA) has been shown to reduce blood lipid level and improve cerebral ischemia in rats with hyperlipemia complicated by cerebral ischemia. However, there are few studies on the results and mechanism of the effect of EA in reducing blood lipid level or promoting neural repair after stroke in hyperlipidemic subjects. In this study, EA was applied to a rat model of hyperlipidemia and middle cerebral artery thrombosis and the condition of neurons and astrocytes after hippocampal injury was assessed. Except for the normal group, rats in other groups were fed a high-fat diet throughout the whole experiment. Hyperlipidemia models were established in rats fed a high-fat diet for 6 weeks. Middle cerebral artery thrombus models were induced by pasting 50% FeCl3 filter paper on the left middle cerebral artery for 20 minutes on day 50 as the model group. EA1 group rats received EA at bilateral ST40 (Fenglong) for 7 days before the thrombosis. Rats in the EA1 and EA2 groups received EA at GV20 (Baihui) and bilateral ST40 for 14 days after model establishment. Neuronal health was assessed by hematoxylin-eosin staining in the brain. Hyperlipidemia was assessed by biochemical methods that measured total cholesterol, triglyceride, low-density lipoprotein and high-density lipoprotein in blood sera. Behavioral analysis was used to confirm the establishment of the model. Immunohistochemical methods were used to detect the expression of glial fibrillary acidic protein and nerve growth factor in the hippocampal CA1 region. The results demonstrated that, compared with the model group, blood lipid levels significantly decreased, glial fibrillary acidic protein immunoreactivity was significantly weakened and nerve growth factor immunoreactivity was significantly enhanced in the EA1 and EA2 groups. The repair effect was superior in the EA1 group than in the EA2 group. These findings confirm that EA can reduce blood lipid, inhibit glial fibrillary acidic protein expression and promote nerve growth factor expression in the hippocampal CA1 region after hyperlipidemia and middle cerebral artery thrombosis. All experimental procedures and protocols were approved by the Animal Use and Management Committee of Beijing University of Chinese Medicine, China (approval No. BUCM-3-2018022802-1002) on April 12, 2018.

Regulation of endothelial nitric oxide synthase and postnatal angiogenesis by Rac1.

Diminished bioavailability of nitric oxide is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis. Because Rac1, a Rho family member, mediates cellular motility and generation of reactive oxygen species, it could be involved in the regulation of endothelial nitric oxide production. However, the pathophysiological consequences of postnatal endothelial Rac1 deletion on endothelial function have not been determined. We generated endothelial-specific Rac1 haploinsufficient mice (EC-Rac1(+/-)) using Cre-loxP technology. The EC-Rac1(+/-) mice have decreased expression and activity of endothelial nitric oxide synthase (eNOS), impaired endothelium-dependent vasorelaxation, and mild hypertension compared with control (Rac1(+/flox)) mice. Hind limb ischemia model and aortic capillary sprouting assay showed that eNOS activity and angiogenesis was impaired in EC-Rac1(+/-) mice. Indeed, Rac1 promotes eNOS gene transcription through p21-activated kinase but not NADPH oxidase, increases eNOS mRNA stability, and enhances eNOS activity by promoting endothelial uptake of l-arginine. These findings indicate that endothelial Rac1 is essential for endothelium-dependent vasomotor response and ischemia-induced angiogenesis. These effects of Rac1 on endothelial function are largely due to the upregulation of eNOS through multiple mechanisms that are mediated, in part, by p21-activated kinase. Therapeutic strategies to enhance Rac1 function, therefore, may be important for preventing endothelial dysfunction.

Prevalence of Plasmodium vivax VK210 and VK247 subtype in Myanmar.

BACKGROUND: Plasmodium vivax is divided into two subtypes, a dominant form, VK210 and a variant form, VK247. This division is dependent on the amino acid composition of the circumsporozoite (CS) protein. In this study, the prevalence of the VK247 variant form of P. vivax was investigated in Myanmar. METHODS: The existence of malaria parasites in blood samples was determined by microscopic examination, polymerase chain reaction (PCR) and DNA hybridization assays. To test for antibodies against P. vivax and Plasmodium falciparum in blood samples, an indirect immunofluorescence antibody test (IFAT) was performed using asexual blood antigens. An enzyme-linked immunosorbent assay with synthetic VK210 and VK247 antigens was carried out to discriminate between the P. vivax subtypes. RESULTS: By thick smear examination, 73 (n=100) patients were single infected with P. vivax, one with P. falciparum and 13 with both species. By thin smear, 53 patients were single infected with P. vivax, eight with only P. falciparum and 16 with both. Most of the collected blood samples were shown to be P. vivax positive (n=95) by PCR. All cases that were positive for P. falciparum by PCR (n=43) were also positive for P. vivax. However, 52 cases were single infected with P. vivax. IFAT showed antibody titres from 1:32 to 1:4,096. Additionally, using specific antibodies for VK210 and VK247, ELISA showed that 12 patients had antibodies for only the VK210 subtype, 4 patients had only VK247 subtype antibodies and 21 patients had antibodies for both subtypes. Using a DNA hybridization test, 47 patients were infected with the VK210 type, one patient was infected with VK247 and 23 patients were infected with both subtypes. CONCLUSIONS: The proportion of the VK247 subtype in Myanmar was 43.1% (n=25) among 58 positive cases by serodiagnosis and 25.6% (n=24) among 94 positive cases by genetic diagnosis. In both diagnostic methods, the infection status of malaria patients is highly diverse with respect to malaria species, and multiple clonal infections are prevalent in Myanmar. Therefore, the complexity of the infection should be considered carefully when diagnosing malaria in Myanmar.

Analysis of the dihydrofolate reductase-thymidylate synthase gene sequences in Plasmodium vivax field isolates that failed chloroquine treatment.

BACKGROUND: To use pyrimethamine as an alternative anti-malarial drug for chloroquine-resistant malaria parasites, it was necessary to determine the enzyme's genetic variation in dihydrofolate reductase-thymidylate syntase (DHFR-TS) among Korean strains. METHODS: Genetic variation of dhfr-ts genes of Plasmodium vivax clinical isolates from patients who did not respond to drug treatment (n = 11) in Korea were analysed. The genes were amplified using the polymerase chain reaction (PCR) with genomic DNA as a template. RESULTS: Sequence analysis showed that the open reading frame (ORF) of 1,857 nucleotides encoded a deduced protein of 618 amino acids (aa). Alignment with the DHFR-TS genes of other malaria parasites showed that a 231-residue DHFR domain and a 286-residue TS domain were seperated by a 101-aa linker region. This ORF shows 98.7% homology with the P. vivax Sal I strain (XM001615032) in the DHFR domain, 100% in the linker region and 99% in the TS domain. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed that nine isolates belonged to the sensitive strain, whereas two isolates met the criteria for resistance. In these two isolates, the amino acid at position 117 is changed from serine to asparagine (S117N). Additionally, all Korean isolates showed a deletion mutant of THGGDN in short tandem repetitive sequences between 88 and 106 amino acid. CONCLUSIONS: These results suggest that sequence variations in the DHFR-TS represent the prevalence of antifolate-resistant P. vivax in Korea. Two of 11 isolates have the Ser to Asn mutation in codon 117, which is the major determinant of pyrimethamine resistance in P. vivax. Therefore, the introduction of pyrimethamine for the treatment of chloroquine-resistant vivax malaria as alternative drug in Korea should be seriously considered.