TIMP1 preserves the blood-brain barrier through interacting with CD63/integrin 1 complex and regulating downstream FAK/RhoA signaling

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability are hallmark features of several neurological disorders, including traumatic brain injury (TBI). However, there is no viable therapeutic strategy to rescue BBB function. Tissue inhibitor of metalloproteinase-1 (TIMP1) has been considered to be beneficial for vascular integrity, but the molecular mechanisms underlying the functions of TIMP1 remain elusive. Here, we report that TIMP1 executes a protective role on neuroprotective function ameliorating BBB disruption in mice with experimental TBI. In human brain microvessel endothelial cells (HBMECs) exposed to hypoxia and inflammation injury, the recombinant TIMP1 (rTIMP1) treatment maintained integrity of junctional proteins and trans-endothelial tightness. Mechanistically, TIMP1 interacts with CD63/integrin 1 complex and activates downstream FAK signaling, leading to attenuation of RhoA activation and F-actin depolymerization for endothelial cells structure stabilization. Notably, these effects depend on CD63/integrin 1 complex, instead of the MMP-inhibitory function. Together, our results identified a novel MMP-independent function of TIMP1 in regulating endothelial barrier integrity. Therapeutic interventions targeting TIMP1 and its downstream signaling may be beneficial to protect BBB function following brain injury and neurological disorders.

Effect of autologous whole blood injections on expression of FcεRI and CD63 on basophils in patients with refractory chronic spontaneous urticaria and positive autologous serum skin test / 中华皮肤科杂志

Objective To evaluate the clinical efficacy of autologous whole blood injections (AWBI) combined with antihistamines for the treatment of patients with refractory chronic spontaneous urticaria and positive autologous serum skin test (ASST),to evaluate its effect on the expression of the high-affinity IgE receptor (FcεR Ⅰ) and CD63 on basophils,and to analyze the possible mechanism underlying the treatment of ASST-positive chronic urticaria with AWBI.Methods Eighty patients with ASST-positive chronic intractable urticaria were enrolled from Department of Dermatology,The First Hospital Affiliated to Army Medical University between November 2017 and June 2018,and randomly and equally divided into two groups by a random number table:AWBI group and control group were both conventionally treated with oral loratadine and ebastine,and AWBI group were additionally treated with AWBI once a week for 12 sessions.Before the treatment and after 12-week treatment,urticaria activity score of 7 days (UAS7) and dermatology life quality index (DLQI) in the two groups were evaluated.Among 30 patients in the AWBI group,flow cytometry was performed to determine the expression of FcεRⅠ and CD63 on the basophils in the peripheral blood at the baseline,weeks 4,8 and 12 after the initial treatment.Statistical analysis was carried out with GraphPad Prism 7.00 software by t test for the comparison of UAS7 or DLQI scores,Mann-Whitney U test for the comparison of FcεR Ⅰ α expression,paired Wilcoxon signed rank test for comparing FceR Ⅰ α or CD63 expression between two different time points,and Spearman correlation analysis for analyzing the correlation between FcεR Ⅰ α and CD63 expression.Results Before the treatment,no significant differences in UAS7 or DLQI scores were observed between the AWBI group and control group (UAS7:27.15 ± 4.53 vs.26.90 ± 5.22;DLQI:16.88 ± 6.01 vs.17.08 ± 6.79;both P ＞ 0.05).After 12-week treatment,UAS7 and DLQI scores both significantly decreased in the two groups compared with those before the treatment (all P ＜ 0.01),and were significantly lower in the AWBI group than in the control group (UAS7:14.25 ± 7.56 vs.19.93 ± 6.32;DLQI:8.48 ± 4.15 vs.13.93 ± 5.43;both P ＜ 0.01).At the baseline,weeks 4,8 and 12 after the initial treatment,the fluorescence intensities of FcεR Ⅰα on basophils (M [P25,P75]) in the AWBI group were 22 532 (16 740,29 220),16 911 (10 240,21 816),13 282 (7 600,16 848) and 11 466 (7 161,14 578) respectively,and the proportions of CD63+ basophils induced by ASST-positive serum (M [P25,P75]) in the AWBI group were 35.25％ (26.75％,49.13％),25.95％ (19.37％,37.54％),13.57％ (7.79％,19.57％) and 9.87％ (6.43％,16.52％) respectively.At week 4 after the initial treatment,the expression of FcεR Ⅰα and CD63 on basophils in the AWBI group both significantly decreased compared with those at the baseline (both P ＜ 0.01),but significantly increased compared with those at week 8 (both P ＜ 0.01).The changes in FcεR Ⅰ α expression from baseline to week 4,from week 4 to week 8,and from week 8 to week 12 were positively correlated with the changes in CD63 expression induced by ASST-positive serum (r =0.364,0.422,0.455,respectively,all P ＜ 0.05).Conclusion AWBI combined with antihistamines can improve the clinical symptoms of ASST-positive refractory chronic urticaria,likely by affecting the expression of FcεR Ⅰ and CD63 on basophils.

Expression of CD63 in Lung Tissue of Guinea Pigs Dying of Anaphylactic Shock / 法医学杂志

Objective To study the protein expression of cluster of differentiation 63 （CD63） in lung tissues of guinea pigs that died of anaphylactic shock and discuss the diagnostic value of CD63 for death from anaphylactic shock. Methods Twenty guinea pigs were randomly divided into control group, anaphylactic shock immediate death group, cold storage group （4 ℃ for 48 h） and frozen group （-20 ℃ for 7 d）. The animal model of guinea pigs that died of anaphylactic shock was established with human mixed serum injection. The expression changes of CD63 protein and CD63 mRNA in lung tissues were detected by hematoxylin-eosin （HE） staining, immunohistochemical staining, Western blotting, enzyme-linked immunosorbent assay （ELISA） and real-time RT-PCR. Results HE staining results showed congestion, and edema of lung tissues, and eosinophil infiltration in the anaphylactic shock groups. Western blotting analysis results showed that the expression of CD63 protein in the lung tissues of guinea pigs that died of anaphylactic shock was significantly higher than that in the control group （P<0.05）. Comparison between the anaphylactic shock groups was made, and the differences had no statistical significance. The results of immunohistochemical staining and real-time RT-PCR were consistent with that of Western blotting. ELISA results showed that CD63 protein expression in the immediate death group was higher than that in the control group （P<0.05）. Conclusion The expression of CD63 protein and CD63 mRNA in the lung tissues of guinea pigs that died of anaphylactic shock is significantly enhanced. Animal carcasses which were put in cold storage for 48 h and frozen for 7 d do not affect the examination of the above indicators. CD63 protein is expected to become an auxiliary diagnostic indicator of death from anaphylactic shock.

Changes and clinical significance of platelet activation and four platelet parameters in diabetic retinopathy / 眼科新进展

Objective To explore the relationship between platelet activating factors and four platelet parameters in diabetic retinopathy (DR).Methods Eighty patients in our hospital were chosen from January 2015 to December 2015,regular ophthalmological examination and fundus fluorescence were performed on all cases.On the basis of international clinical diabetic retinopathy classification standard,the patients were divided into four groups:No DR group with 20 cases,non-proliferative diabetic retinopathy (NPDR) group with 20 cases,and proliferative diabetic retinopathy (PDR) group with 20 cases.20 healthy people who received physical examination during the same period were recruited as the control group.The flow cytometry instrument was used to determinate platelet activating factor CD62p and CD63,automatic blood analyzer was used to determinate platelet parameter which including blood platelet count (PLT),mean platelet volume (MPV),plateletcrit (PCT) and platelet distribution width (PDW),and the changes of platelet activation factor,parameters and its clinical significance in DR were discussed.Results With the development of DR,CD62p,CD63,MVP were numerical gradually increased,and PLT were numerical gradually reduced,the differences were statistically significant (all P ＜ 0.05);Compared with control group,CD62p,CD63,MVP were increased and PLT were reduced in other group,the differences were statistically significant (all P ＜ 0.05);Compared with pure diabetes group,CD62p,CD63,MVP were increased and PLT were reduced in NPDR group and PDR group,the differences were statistically significant (all P ＜ 0.05);Compared with NPDR group,CD62p,CD63,MVP were increased and PLT were reduced in PDR group,the differences were statistically significant(all P ＜ 0.05).PDW were numerical gradually increased in control group,pure diabetes group,NPDR group,PDR group,the differences were statistically significant(all P ＜ 0.05);Control group compared with pure diabetes group,NPDR group compared with PDR group,the differences in PDW was not statistical significant (all P ＞ 0.05);But compared with control group or pure diabetes group,PDW of NPDR group and PDR group increased,the differences were statistically significant(all P ＜ 0.05).The differences in PCT among groups was not statistically significant(all P ＞ 0.05).Conclusion The platelet activating factor CD62p,CD63 and parameters of platelet show differences in each diabetic retinopathy period,platelet activating factor in the occurrence and development of diabetic retinopathy have important significance.Monitor the platelet activating factor and parameters of platelet can help surveillance of the disease.

Short-, Intermediate-, and Long-Term Changes in Basophil Reactivity Induced by Venom Immunotherapy

PURPOSE: The basophil activation test (BAT) has been used to monitor venom immunotherapy (VIT) due to its high specificity. A previous study has reported a good correlation between a significant decrease in basophil activation during 5 years of VIT and clinical protection assessed by sting challenge. The following prospective study was performed to examine changes in basophil reactivity over a complete VIT period of 5 years. METHODS: BAT in a dose-response curve was studied prospectively in 10 hymenoptera venom-allergic patients over 5 years of VIT. BAT was performed at the time of diagnosis, 1 month after finishing the VIT build-up phase, and 3, 6, 12, 24, and 60 months after beginning treatment. The repeated measures ANOVA was applied to evaluate basophil activation changes throughout VIT. A cross-sectional study was also performed in 6 patients who received treatment for more than 3 years, and in another 12 patients who followed immunotherapy for at least 5 years. RESULTS: An early activation decrease was observed during the first 3 months of treatment, compared to pre-treatment values. This activation decrease was not maintained 6 to 18 months after treatment, but was observed again after 2 years of treatment, and maintained until the completion of the 5-year immunotherapy period. In cross-sectional analysis, the 6 patients who received treatment for 3 years, and 9 of the 12 patients who received treatment for 5 years, had negative BAT results. Three patients in this last group had positive BAT results and 2 patients had systemic reactions after field stings. CONCLUSIONS: BAT appears to be an optimal non-invasive test for close monitoring of VIT.

The expression and clinical significance of CD62p, CD63 and CD64 in children with sepsis / 中国小儿急救医学

Objective To investigate the expression and clinical significance of platelet glycoprotein CD62p,CD63 and neutrophil surface CD64 in sepsis.Methods Fifty-six children with sepsis from March 2010 to March 2013 in Communicable Disease Department of our hospital were divided into severe sepsis group(n =16) and general sepsis group (n =40),normal control group included 34 subjects from health check.CD62p,CD63 and CD64 were detected by flow cytometry in children with sepsis,and compared with normal control group.Results The levels of CD62p,CD63 and CD64 in severe sepsis group were higher than those of general sepsis group (P ＜ 0.01).The levels of CD62p,CD63 and CD64 in general sepsis group were higher than those of normal control group (P ＜ 0.01).Correlation analysis indicated that CD62p and CD63 were in positive correlation with CD64 in children with sepsis(r =0.817,0.796,P ＜0.001).The positive correlations of CD62p,CD63 and CD64 with pediatric critical illness score were also found(CD62p:r =0.883,P ＜0.001;CD63:r=0.862,P ＜0.001;CD64:r=0.805,P ＜0.001).Conclusion CD62p,CD63 and CD64 are closely related to the severity of infection and diseases,and may be used as immune parameters for the estimation of the clinical severity and the prognosis of acute and severe diseases.

Recent applications of basophil activation tests in the diagnosis of drug hypersensitivity

Immediate-type drug hypersensitivity is an increasingly significant clinical issue; however, the diagnosis is frequently hindered due to lack of safe and precise diagnostic tests. Flow cytometry-assisted basophil activation test is a safe in vitro diagnostic tool for assessing basophil activation upon allergen stimulation. In this review, we have summarized current literature on the diagnostic utilities, new indications, and methodological aspects of the basophil activation test for the diagnosis of drug hypersensitivity.

New horizons in platelets flow cytometry

Platelet flow cytometry is an emerging tool in diagnostic and therapeutic hematology. It is eminently suited to study the expression of platelet surface receptors both qualitatively as well as quantitatively. It can serve as a useful marker for the documentation of in vivo platelet activation, and thus, fore-warn the risk of thromboembolism in patients with diabetes mellitus, coronary syndromes, peripheral vascular diseases, and pre-eclampsia. This technique can also be extended to study and compare the effect of various antiplatelet drugs on the level of activation of platelets and to establish any dose-effect relationship of these drugs. Topographical localization of platelet granules and study of platelet-platelet and platelet-leukocyte interaction is also possible by this procedure. All these parameters serve as pointers towards the presence of activated platelets in the circulation with its thromboembolic consequences. This is a simple reliable and cost effective technique which has a wide application in the diagnosis of various inherited and acquired platelet disorders. Study of platelet cluster of differentiation (CD) markers in various inherited disorders i.e. Bernard Soulier’s disease, von Willebrand disease, Glanzman’s disease, and Grey platelet syndrome may help categories the molecular lesions in these oft under-studied disorders.

CD63 e CD123 expressão, autoanticorpos IgG e acurácia do teste do soro autólogo em pacientes com urticária crônica / CD63 and CD123 expression, IgG autoantibody and accuracy of autologous serum test in patients with chronic urticaria

INTRODUÇÃO: Na urticária crônica (UC), o teste cutâneo do soro autólogo (TCSA) pode sugerir a etiologia autoimune. Recentemente, uma nova técnica laboratorial denominada teste de ativação de basófilos (TAB) vem sendo utilizada para esse diagnóstico. OBJETIVOS: Analisar o TCSA em relação ao TAB, assim como avaliar os receptores da interleucina 3 (IL3) (CD123) e a presença de autoanticorpos da classe de imunoglobulina G (IgG) inespecíficos ligados aos basófilos de pacientes com UC. MÉTODOS: Estudamos 33 adultos com UC espontânea com idade média de 42,5 + 14 anos. Por meio da citometria de fluxo foi feita a análise da expressão das moléculas CD63 em basófilos de um doador atópico após o estímulo pelo soro dos pacientes com UC. Também realizamos a pesquisa da expressão da molécula CD123 e de autoanticorpos IgG inespecíficos. RESULTADOS: O odds ratio (OR) entre o TCSA e o TAB foi de 1 (intervalo de confiança [IC] 95 por cento: 0,22-4,5). O TCSA para o diagnóstico da UC autoimune mostrou acurácia de 54,5 por cento, sensibilidade de 66 por cento, especificidade de 33 por cento, valor preditivo positivo de 63 por cento e valor preditivo negativo de 36 por cento. Não houve diferença estatística entre os grupos estudados quanto à média de expressão dos anticorpos IgG inespecíficos e das moléculas CD123 (para um p < 0,05). DISCUSSÃO: Este estudo demonstrou baixa precisão do TCSA no diagnóstico da UC autoimune; o grupo de pacientes com TCSA positivo não mostrou diferença estatística em relação ao grupo com TCSA negativo nos demais aspectos analisados. CONCLUSÃO: Pelos poucos estudos existentes e pela relevância do assunto, acreditamos na necessidade de mais estudos abordando esses aspectos.

INTRODUCTION: The autologous serum skin test (ASST) may suggest an autoimmune etiology in chronic urticaria (CU). A new laboratory technique called basophil activation test (BAT) has been currently employed for its diagnosis. OBJECTIVE: To analyze ASST in relation to BAT as well as to evaluate interleukin 3 (IL3) receptors (CD123) and non-specific immunoglobulin G (IgG) autoantibodies bound to basophils in patients with chronic urticaria. METHODS: We studied 33 adults with CU and mean age of 42.5 + 14 years. After stimulation by serum from patients with CU, CD63 expression on basophils from one atopic donor was analyzed by flow cytometry. Furthermore, we investigated CD123 and IgG autoantibody expressions. RESULTS: The odds ratio (OR) between ASST and BAT was 1.00 (95 percent confidence interval [CI]: 0.22 to 4.5). The ASST for autoimmune CU diagnosis showed an accuracy of 54.5 percent, sensitivity of 66 percent, specificity of 33 percent, positive predictive value of 63 percent, and negative predictive value of 36 percent. There was no statistical difference between the studied groups as to mean non-specific IgG and CD123 expressions (for a p < 0.05). DISCUSSION: This study demonstrated that ASST has low accuracy in the diagnosis of autoimmune CU. Concerning other analyzed aspects, there was no statistical difference between positive ASST and negative ASST. CONCLUSIONS: Due to insufficient studies in this area and the relevance of this issue, further investigation is required.

In vitro Biphasic Effect of Honey Bee Venom on Basophils from Screened Healthy Blood Donors

Apis mellifera L. bee venom is the most studied hymenoptera allergen, but many aspects of its action on human basophils remain unclear. Allergologists seek evidence of the effectiveness of bee venom immunotherapy as this approach is the chosen treatment for systemic allergic reactions. The effect of bee venom on human basophils in vitro has not been studied in detail for many reasons, including the paucity of basophils in peripheral blood, inter-individual basophil response variability, and the reliability and predictability of basophil activation tests. We conducted a brief preliminary survey of the effect of Apis bee venom on healthy asymptomatic (non-allergic) subjects. A dose of an aqueous commercial extract of Apis bee venom as high as 10 microg/mL activated resting basophils (CD63=+80-90%, CD203c=+30%), while it inhibited the expression of CD63 (-50%) following basophil stimulation by the soluble agonists formyl-Met-Leu-Phe or anti-IgE. The activation of resting basophils appeared to be dose-related. Only when basophils were activated with an IgE-mediated agonist, did bee venom extract exhibit a possible priming mechanism at the lowest doses used only via CD63, while it was ineffective via CD203c. Autocrine interleukin-3 may play a role in the observed biphasic behavior.

Laboratory Diagnosis and Clinic Significance of Drug Allergies / 中华医院感染学杂志

OBJECTIVE The diagnosis of drug allergy is difficult and is usually based on clinical history,skin tests(for some drugs) and,in a few specialized allergy centers,provocation tests.To establish the method for analyzing basophil activation test(BAT) by flow cytometry(FCM) and evaluate its clinical significance in the diagnosis of drug allergy.METHODS The protocol for FCM analysis of basophil degranulation in allergy dustmite by CD63,CD203c and CD45 combination was established.The clinical significance of activated basophil by FCM was evaluated by comparing with the results of sIgE by fluorescence enzyme-linked absorbent assay(FELISA),regarding the skin prick test as the gold standard.RESULTS Pure basophils were got by CD45 and CD203c gating,CD63 was the best marker for activated basophil;Spearman′s correlation coefficients indicated a moderate positive correlation between SIgE class categorized by Unicap class and activated basophil;there was no significant difference between activated basophil by FCM and sIgE by FELISA in the diagnosis of allergic reaction,but the former was better than the latter in specificity and positive likelihood ratio.CONCLUSIONS Quantification of activated basophil by CD63 expression with FCM is a valuable new and safe method in vitro for diagnosis of immediate type hypersensitization.

Changes in the expressions of CD63 on basophils in patients with penicillin allergy / 中国药理学通报

Aim To evaluate the value of expressions of CD63 on basophils in diagnosing allergy to penicillins.Methods The expressions of CD63 on basophils activated with 9 kinds of antigens(PG:penicillin G,PV:phenoxomethylpenicillin,AMP:ampicillin,AX:amoxicilloyl,6-APA:6-aminopenicillanic acid,PHA:phenylacetic acid,PHOA:phenoxacetic acid,PHPG:N-(?-hydroxyphenyl)glycine,NPG:N-phenylglycine) were detected by FAST(Flow cytometric allergen stimulation test) in 43 patients with penicillins allergy.Eight types of specific IgE and IgG antibodies to penicillins were determinated by RAST and ELISA,respectively.Results The sensitivity and specificity of CD63 used as a marker in patients with penicillins allergy were 65.12% and 93.33%,respectively.Moreover,the sensitivities of CD63 and specific IgE were higher than those of specific IgG in patients with positive skin test(P

Changes of Platelet Activation and Clinical Outcome in Acute Atherosclerotic Cerebral Infarction Treated with Clopidogrel Loading Dose (300 mg)

BACKGROUND: Platelet activation has an important role in progression of atherosclerotic vascular events. To know the beneficial effects of clopidogrel loading dose about changes of platelet activation and clinical outcome in acute stage of atherosclerotic infarction, we performed a prospective randominized study. METHODS: Patients with large artery atherosclerotic infarction were randomized to clopidogrel loading dose (n=24) or intravenous heparin with low dose aspirin (n=28) during 7 days. We measured the surface expression of CD63 on platelets and the platelet aggregability for 7 days. The National Institute of Health Stroke Scale (NIHSS) score was recorded at 24 hours, 72 hours, and 7 days after stroke. Three-month outcome was recorded by the modified Barthel index (BI). RESULTS: As compared with intravenous heparin, the loading dose of clopidogrel was associated with significant reduction of the surface expression of CD63 on platelets, platelet aggregability, and NIHSS score. The clopidogrel loading dose-treated patients were more likely to have a very favorable outcome on BI index at 3 months. CONCLUSIONS: Our results showed that the clopidogrel loading dose has beneficial effects of clinical outcome of acute stage of large artery atherosclerotic infarction may be mediated by the platelet hyperactivity in these patients. Thus, this loading dose trial deserves further evaluation in clinical trial for treatment in large artery atherosclerotic infarction.

A decrease in the expression of CD63 tetraspanin protein elevates invasive potential of human melanoma cells

CD63, which belongs to the tetraspanin membrane proteins, has been proposed to play an important role in inhibiting melanoma metastasis. To determine whether reduction of CD63 expression, which frequently occurs in the malignant progression of human melanoma, is responsible for metastasis promotion, we transfected the antisense CD63 cDNA into MelJuso melanoma cells having endogenous CD63 expression. The antisense CD63 transfectant clones showing decreased CD63 expression displayed increased cell motility, matrix-degrading activity, and invasiveness in vitro when compared with the control transfectant cells. The antisense CD63 cDNA-transfected cells also exhibited altered adhesiveness to extracellular matrix. The results suggest that reduced CD63 expression contributes to the invasive and metastatic ability of human melanoma cells.

Effects and its efficacy of Ozagrel on CD62p and CD63 expressions in platelets of patients with acute cerebral infarction / 临床神经病学杂志

Objective To observe the effects and its efficacy of Ozagrel on CD62p and CD63 expressions in platelets of patients with acute cerebral infarction(ACI).Methods 64 patients with ACI were devided into two groups:Ozagrel group and Xuesaitong group(control group).The expression levels of CD62p and CD63 in 64 patients with ACI(before and after treating)and that in normal people(normal group)were measured with whole blood flow cytometry.The clinical effect of treatment in the Ozagrel group and the control group were observed and compared.Results The expression levels of CD62p and CD63 in patients with ACI were higher than those in the normal group(all P

Changes and clinical significance of P selectin , lysosome protein expression in patients with ischemic stroke / 临床神经病学杂志

Objective To study the changes and clinical significance of P selectin (CD 62P), lysosome protein (CD 63) expression in patients with ischemic stroke.Methods 168 cases of acute ischemic stroke (both acute and convalescence) and 40 normal controls were tested for CD 62P and CD 63 expression using flow cytometry. The correlation between the expression and neurological functional deficit scale was analyzed.Results (1) The expression of CD 62p and CD 63 in acute phase (9.48%, 8.36%) of ischemic stroke was markedly higher than that in convalescence phase (5.73%, 4.21%)( P0.05). (3) CD 62P and CD 63 expressions were positively related with the scores of neurological functional deficit scale( r=0.84 and r=0.817, P

Measurement of activated blood platelets in peripheral blood of tumor patients and its clinical significance / 重庆医科大学学报

Objective:To compare the measures of activated blood platelets in different disease conditions and the effects of aspirin on blood clots.Methods:The expression of activated platelet CK62p and CD63 and its changing after taking aspirin in 126 tumor patients and 60 non-tumor patients was detected with flow cytometry.Result:The expression of activated platelet CK62p and CD63 in tumor patients was significantly higher than that in non-tumor patients(P