[Evaluation of arthroscopic anterior talofibular ligament and calcaneofibular ligament repair separately for chronic lateral ankle instability in conjunction with subtalar instability].

Objective: To investigate the clinical efficacy of simultaneous arthroscopic repair of anterior talofibular ligament (ATFL) and calcaneofibular ligament (CFL) for treating chronic lateral ankle instability (CLAI) in conjunction with subtalar instability (STI). Methods: This is a retrospective case series study. The clinical data of 15 patients with ankle arthroscopic in the Department of Hand and Foot Surgery, the Second Affiliated Hospital of Soochow University from January 2019 to December 2022 were analyzed retrospectively. There were 11 male cases and 4 female cases, aged (28.6±1.5) years (range: 19 to 39 years). All the patients were evaluated by manual inversion stress X-ray and MRI before operation. Arthroscopically observing and then repairing the ATFL and CFL separately after further diagnostic confirmation. One year after operation, MRI was performed, and pain visual analogue score(VAS), American Orthopedic Foot and Ankle Society ankle hindfoot scale (AOFAS-AH) and Karlsson ankle functional scale(KAFS) were evaluated. Data were compared using paired sample t test. Results: The follow-up period was (23.6±2.3) months (range: 12 to 30 months). At last follow-up,the VAS decreased from 6.1±1.4 preoperatively to 1.4±1.2(t=9.482, P<0.01).The AOFAS-AH improved from 50.5±11.7 preoperatively to 94.2±6.1(t=-13.132, P<0.01), and the KAFS improved from preoperatively 44.3±10.8 to 90.8±6.4 (t=-12.510, P<0.01). There was no complication such as recurred instability or joint stiffness. Conclusions: Arthroscopically repairing the ATFL and CFL separately can effectively restore the stability of the ankle and subtalar joint with small trauma. Patients can recover quickly after surgery. It provides a new idea for the clinical treatment of CLAI combined with STI.

Therapeutic effects of chimeric antigen receptor T cells (CAR-T) on relapse/refractory diffuse large B-cell lymphoma (R/R DLBCL): a meta-analysis.

OBJECTIVE: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL). This study aimed to systematically evaluate the efficacy of chimeric antigen receptor T cells (CAR-T) in treating relapse/refractory DLBCL (R/R DLBCL) and associated complete-remission rate (CR). MATERIALS AND METHODS: PubMed, Cochrane Library, CNKI, VIP, CBM, and Wanfang databases were searched, and literature was collected up to January 2019. According to inclusion criteria and exclusion criteria, two researchers independently reviewed and screened literature, extracted required data and crossly checked them. This meta-analysis was conducted using RevMan 5.3 software. RESULTS: This study finally included 13 English literatures and 263 cases. There was no heterogeneity among all these studies, therefore, fixed effect model was used. Meta-analysis findings showed that total CR rate of R/R DLBCL treated with CAR-T was 46.8% (95% CI: 0.408-0.533). Subgroup analysis showed that CR rate of CD28 group was slightly higher [52.5%, with 95% confidence interval (CI): 0.441-0.602] compared to that of 4-1BB group (41.5%, with 95% CI: 0.324-0.510). CR rate of CD19 group was slightly higher (49.2%, with 95% CI: 0.429-0.556) compared to that of CD20 group (42.2%, with 95% CI: 0.231-0.639). Funnel chart of total CR rate, co-stimulatory factor, and target antigen demonstrated fundamental symmetry. Moreover, age, HSCT administration, CAR-T cell counts, and drug pre-treatment also affected immunotherapy on CAR-T on R/R DLBCL. CONCLUSIONS: CAR-T treatment for R/R DLBCL demonstrated evident curative effect and high complete remission rate. CAR-T cell immunotherapy would be expected to become mainstream therapy for hematolymph system tumors.

Neuroprotective effects of intravenous transplantation of bone marrow mononuclear cells from 5-fluorouracil pre-treated rats on ischemic stroke.

Our previous findings showed bone marrow mononuclear cells (BMMNCs) from 5- fluorouracil (5-FU) pre-treated rats (named BMRMNCs) had a better therapeutic efficacy in ischemia/reperfusion rats as compared to BMMNCs from untreated rats. This study was undertaken to explore the potential mechanisms underlying the neuroprotective effects of BMRMNCs in middle cerebral artery occlusion (MCAO) rat model. Rats were intravenously pre-treated with 5-FU and BMRMNCs were collected at different time points. The contents of growth factors in the supernatant and CXCR4 expression were detected by ELISA and flow cytometry, respectively. MCAO was introduced to rats, and BMMNCs and BMRMNCs collected at 7 days after 5-FU pre-treatment were independently transplanted via the tail vein 24h later. The neurological function was evaluated before cell transplantation and at 24h, 7d and 14d after cell transplantation. Rats were sacrificed at 14d after cell transplantation, the brains were collected for TTC staining, infarct volume detection, NISSL staining, counting of viable cells in the CA1 region, and observation of transplanted cells. BMRMNCs had elevated expressions of growth factors as well as CXCR4 expression. Our results confirmed the better therapeutic effects of BMRMNCs in MCAO rats, demonstrated by reduction in infarct volume, improvement of neurological function and more viable cells in the hippocampus. In addition, more transplanted cells were found after BMRMNCs transplantation at 7 days and 14 days although there was no marked difference at 14 days. These findings indicate that BMRMNCs transplantation may protect ischemic stroke, at least partially, via increasing the secretion of growth factors and migration to the injured site.

Elevated plasma endothelial microparticles in multiple sclerosis.

OBJECTIVE: To assess endothelial dysfunction in patients with MS and to investigate whether plasma from patients with MS induces endothelial cell dysfunction in vitro. BACKGROUND: Endothelial cell dysfunction may contribute to the pathogenesis of MS. Elevations of soluble adhesion molecules intracellular adhesion molecule, vascular cell adhesion molecule, and platelet-endothelial cell adhesion molecule-1 (CD31) have been reported as markers of blood-brain barrier (BBB) damage in MS, but direct assay of endothelium has been difficult. Endothelial cells release microparticles < approximately 1.5 microm (EMP) during activation or apoptosis. The authors developed a flow cytometric assay of EMP and studied EMP as markers of endothelial damage in MS. METHODS: Platelet-poor plasma (PPP) from 50 patients with MS (30 in exacerbation and 20 in remission) and 48 controls were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD31 and anti-CD51 (vitronectin receptor) antibodies, and two classes of EMP (CD31+ and CD51+) were assayed by flow cytometry. For in vitro studies, patients' plasma was added to the microvascular endothelial cell (MVEC) culture and release of CD31+ and CD51+ EMP were measured in the supernatant. RESULTS: Plasma from patients in exacerbation had 2.85-fold elevation of CD31+ EMP as compared with healthy controls, returning to near control value during remission. The CD31+ EMP concentration showed a positive association with gadolinium enhancement in patients with MS. In contrast, CD51+ EMP remained elevated in both exacerbation and remission. This suggests that CD31+ EMP is a marker of acute injury, whereas CD51+ EMP reflects chronic injury of endothelium. MS plasma induced release of both CD31+ and CD51+ EMP from MVEC culture in vitro. CONCLUSION: Endothelial dysfunction is evident during exacerbation of MS, evidenced by shedding of EMP expressing PECAM-1 (CD31). The in vitro data indicate contribution of one or more plasma factors in endothelial dysfunction of MS.

A flow cytometric assay of platelet activation marker P-selectin (CD62P) distinguishes heparin-induced thrombocytopenia (HIT) from HIT with thrombosis (HITT).

Heparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios approximately 2.5 vs. HIT ratios approximately 1.2; p <0.001). Eleven of 12 HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p <0.0005). In a case of HIT with silent thrombosis, this assay gave a positive results prior to clinically evident thrombosis. In conclusion, this method distinguishes HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.

Anti-CD36 autoantibodies in thrombotic thrombocytopenic purpura and other thrombotic disorders: identification of an 85 kD form of CD36 as a target antigen.

The presence of anti-CD36 antibodies in plasma of patients with thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), and heparin-induced thrombocytopenia without/with thrombosis (HIT/HITT) has been examined by immunoblots, and a monoclonal antibody capture assay, the platelet-associated IgG characterization assay (PAICA). Results with PAICA showed that 73% (8/11) of patients with TTP were positive, and 71% (10/14) by immunoblots. With ITP, 20% (6/30) were positive by PAICA and 19% (3/16) by immunoblots; HIT, 30% (3/10) were positive by PAICA and 60% (6/10) by immunoblot; HITT, 50% (2/4) by PAICA and 100% (4/4) by immunoblot. Purification of CD36 by fast protein liquid chromatography (FPLC) from Triton X-100 extracts of normal platelet membranes resulted in the isolation of two different forms: the classic 88 kD form, and a second, lighter 85 kD form. Our data indicated that the patients' plasma autoantibodies reacted strongly with the 85 kD form. Conventional monoclonal and polyclonal antisera produced to the 88 kD form reacted strongly with the 88 kD form but weakly with the 85 kD form. These results confirm the possible importance of anti-CD36 antibodies in the pathophysiology of TTP and other thrombocytopenias and demonstrate the presence of a previously unrecognized target antigen for these antibodies.

Platelet activation in Alzheimer disease.

BACKGROUND: In light of recent reports of diminished platelet serotonin concentration and increased plasma serotonin levels in patients with Alzheimer disease (AD), we hypothesized that a state of heightened platelet activation might be present in AD. OBJECTIVE: To compare baseline activation of unstimulated platelets in patients with AD with that in control subjects. PATIENTS AND METHODS: Flow cytometry was used to measure platelet activation in 91 patients with probable AD and 40 age-matched control subjects. Groups were compared for percentage of circulating platelet aggregates, expression of CD62p, formation of leukocyte-platelet complexes, and presence of circulating platelet microparticles, controlling for effects of demographic, clinical, physiological, and logistical factors. RESULTS: Multiple analysis of covariance on ranked data revealed a 39.5% increase in percentage of platelet aggregates (P=.0001), a 59.3% increase in expression of CD62p (P=.001), and a 53.3% increase in leukocyte-platelet complexes (P=.0001) in the group with AD but no differences in the number of platelet microparticles, overall platelet count, plasma fibrinogen level, or plasma platelet factor 3. Activation was weakly correlated with sex, but was independent of age, severity of disease, duration of disease, depression, agitation, and family history of dementia. CONCLUSIONS: Platelets of patients with AD exhibit greater unstimulated activation than those of controls. Potential causes of such activation include possible stimulation of platelets by damaged cerebral endothelial cells or platelet activation induced by membrane abnormalities previously reported to be present in platelets of patients with AD. In light of recent evidence that platelets are the principal source of both amyloid precursor protein and beta-amyloid peptide in human blood, it is possible that AD platelet activation may reflect or even contribute to the pathogenesis of the disease.

Thrombotic thrombocytopenic purpura plasma enhances platelet-leucocyte interaction in vitro.

In thrombotic thrombocytopenic purpura (TTP), intravascular platelet aggregation and formation of platelet-rich thrombi impair the microcirculation. TTP plasma has been shown to induce aggregation of normal platelets in vitro. The present study investigates the formation of activated platelet aggregates (aPAg) induced by TTP plasma, with particular attention to their binding to leucocytes (LPAg). Results were compared with the effects of plasmas from normal controls (CTL) and from patients with immune thrombocytopenic purpura (ITP) or thrombosis (THR). Following addition of test plasma to normal whole blood (WB), aPAg and LPAg were assayed by flow cytometry using mAbs against CD41 (platelet marker), CD62p (platelet activation marker) and CD45 (pan-leucocyte marker), Compared to control plasma, TTP plasma was more potent than ITP or THR plasma in increasing aPAg: only TTP plasma significantly promoted leucocyte binding to give increased LPAg. Prior removal of neutrophils (PMN) from WB by beads coated with anti-CD15 mAb largely prevented formation of aPAg and LPAg. However, TTP plasma added to normal platelet-rich plasma significantly increased aPAg, which suggested possible hindrance of aPAg formation by erythrocytes and other leucocytes in PMN-depleted blood. We concluded that TTP plasma was most potent in the induction of aPAg and unique in promoting LPAg formation in WB. Neutrophils, and not other leucocytes, appear to be essential for LPAg formation. Enhanced PMN-platelet interaction in the microcirculation may facilitate platelet adhesion to vessel walls and promote the formation of platelet-rich microthrombi in TTP.

Platelet aggregates as markers of platelet activation: characterization of flow cytometric method suitable for clinical applications.

The present paper describes a flow cytometric method for assay of platelet aggregates (PAg) in blood. This method combines and simplifies previously reported techniques, simultaneously enumerating PAg formed upon platelet activation, their expression of activation marker CD62P (P-selectin), and their content of bound leukocytes (LPAg). The sensitivity of this method to low levels of agonists (ADP, collagen) is compared to conventional aggregometry and some features of platelet-leukocyte interaction are explored. The results were: (1) ADP or collagen induced a dose-dependent increase in PAg number and corresponding decline in free platelets. The ED50 for ADP (0.15 microM) and for collagen (0.2 microg/mL) was about 1/20 the ED50 found by aggregometry, indicating 20-fold greater sensitivity. (2) At higher concentrations, the fraction of PAg with bound leukocytes (LPAg) increased to 60-70%. This rise correlated with PAg size and CD62P expression, but not with the number of PAg formed. (3) The response of whole blood (WBD) to agonists was qualitatively different from that of platelet-rich plasma (PRP): in WBD the population of CD62P+ PAg was much higher than in PRP and the population of CD62P+ free platelets was much lower. This implies that leukocytes rapidly recruit activated platelets. (4) Desmopressin (DDAVP) at 5 nmol/L induced a significant rise in activated (CD62P+) PAg and platelets, even though no effect of DDAVP could be detected by conventional aggregometry; this further confirms that DDAVP acts directly on platelets. (5) Plasma samples from TTP patients induced a rise in PAg when added to normal PRP, though little or no effect could be detected by aggregometry. In summary, the flow cytometric method described here appears useful for detecting low levels of platelet activation and provides information on platelet leukocyte interaction, potentially important in identifying and differentiating thrombogenic states. Since it is rapid and economical, it is well suited for clinical implementation.

Activated platelet aggregates in thrombotic thromboctyopenic purpura: decrease with plasma infusions and normalization in remission.

Circulating activated platelet aggregates (aPA) were assayed by flow cytometry employing mAb alpha-CD62p in eight patients with thrombotic thrombocytopenic purpura (TTP). Elevation of aPA was observed in all patients in active stages of TTP; aPA normalized in remission. Plasma infusions with plasmapheresis decreased aPA in responding patients. The rise and fall of aPA preceded relapses and improvements, respectively. These changes were seen prior to the traditional indicators, LDH, haematocrit, and platelet count. Incubation of plasma from TTP patients with normal whole blood induced formation of aPA; this effect was significantly greater than that of plasmas from ITP patient controls (P < 0.01), suggesting the presence of an aPA-promoting factor in TTP plasma. Parallel experiments using a platelet aggregometer failed to detect effect of TTP plasma on normal blood. In summary, aPA appear to be a marker of disease activity, rising with relapse, falling with plasma therapy, and normalizing in remission. The flow cytometric assay of aPA is more sensitive than aggregometry in detecting the putative aPA-promoting factor in TTP.

Platelet microparticles bind, activate and aggregate neutrophils in vitro.

The interaction of activated platelets with leukocytes are believed to play an important role in ischemic reperfusion injury and other thrombotic conditions. Upon activation, platelets shed platelet microparticles (PMP) and express activation markers CD62P expressed on activated platelets mediates adhesion of platelets to leukocytes, chiefly neutrophils, but little is known of the interaction of PMP isolated from stored platelets or thrombin activated platelets was incubated with leukocytes and binding assessed by flow cytometry. FITC-labeled alpha-CD41 was used to assess platelet material associated with WBC. Like platelets PMP bound preferentially to neutrophils rather than lymphocytes, and exhibited an absolute dependence on the presence of Ca2+. Binding was time-and concentration-dependent, reaching a plateau at 10 min at a ratio of PMP to neutrophils of 150:1. Fluorescence microscopy showed that most of the neutrophils were aggregated into clusters of 5-20 cells. Clustering of neutrophils was not observed to result form interaction with platelets. In these clusters the adherent PMP appeared to serve as bridges between the neutrophil. Addition of EGTA after brief incubation (5-10 min) released most of the bound PMP but if added after > 10 min, only approximately 60% of bound PMP were released. In contrast, nearly all bound platelets were released by EGTA at the same time of incubation. Incubation of neutrophils with PMP gave significantly higher percentage of CD41a(+)neutrophils than did platelets incubated at the same numerical ratio. PMP association with neutrophils was less markedly inhibited by alpha-CD62P (AC1.2) than platelets, but binding of both PMP and activated platelets was inhibited approximately 90% by antisialyl Lewis X. PMP binding to neutrophils induced a significant increase in both CD11b expression and phagocytic activity in a concentration-dependent manner. These findings suggest a possible role for PMP in addition to providing platelet factor 3, specifically, as an activator and mediator of neutrophils in ischemic injury, thrombosis, and inflammation.

Complement-mediated fragmentation and lysis of opsonized platelets: ender differences in sensitivity.

It was reported that elevated levels of platelet microparticles (PMPs) in patients with immune thrombocytopenic purpura (ITP) were associated with decreased bleeding, and in some cases with small vessel thromboses (J Lab Clin Med 1992; 119:334). To investigate the possible role of complement in PMP production in ITP, an in vitro assay was developed to simulate ITP: platelets were opsonized with well-defined monoclonal antibodies against glycoprotein IIb/IIIa, of immunoglobulin G (alpha-CD41), and of immunoglobulin M (alpha-Plt-1) class, then exposed to serum as a source of complement. PMP generation and lysis were monitored by flow cytometer, by release of lactic dehydrogenase, and by generation of procoagulant activity. These effects were largely abolished by heating the serum (30 minutes, 65 degrees) or by incubation with alpha-C1q, confirming the role of complement. At low concentrations of serum, both monoclonal antibodies promoted PMP shedding in a concentration-dependent manner without loss of platelet population; at higher concentrations, extensive lysis occurred, but marked variations in resistance to lysis were observed in platelets from different individuals. The PMPs produced were associated with increased procoagulant activity, as measured by the Russell's viper venom test. The immunoglobulin M antibody was more potent than the immunoglobulin G antibody in promoting lysis, and the resulting PMPs had greater procoagulant activity. To clarify the variation seen in platelets from different donors, data was sorted on the basis of gender, with the finding that women's platelets are significantly more sensitive to complement-mediated damage than men's. This may explain in part why ITP is three to four times more prevalent in women than in men. We conclude that complement activation is the most likely explanation for the elevated level of PMPs often seen in patients with ITP and sometimes associated with thrombosis and that the determining factors are the concentration and nature of antibody as well as individual differences in sensitivity to complement-mediated damage. Because complement activation can occur without participation of antibody, complement activation may also be the cause of elevated PMP levels seen in other thrombotic disorders not involving platelet-specific antibodies.

A higher blood flow window of reduced thrombogenicity and acceptable fragmentation in a hollow fiber hemodialyzer.

The effect of pulsatile blood flow on platelet thrombogenicity and platelet fragmentation (PF) in a hollow fiber hemodialyzer (HFD) was quantified with 111In labeled platelets and 125I labeled fibrinogen; 150 ml of blood was collected from Beagle dogs, Yorkshire pigs, and a human volunteer (non-smoker). Platelets were labeled with 111In tropolone (300 microCi) and fibrinogen was labeled with 125I. Sham dialysis (SHD) was performed with 120 HFDs (0.9 meter2) at 37 degrees C, with flow-rates of 150, 250, 500, and 950 ml/min.; after SHD, the washed HD radioactivity was measured with an ionization chamber. PF was measured by flow cytometry with GP IIb-IIIa murine monoclonal antibody. Platelet deposition decreased significantly for 3 species at higher flow; fibrinogen deposition (10-12%, 55-65 mg/m2), was not affected by flow. Adherent platelet thrombus decreased from (8.2 +/- 3.4) to (3.1 +/- 1.0) with human blood as flow rate increased from 150 to 950 ml/min; platelet thrombus level also decreased significantly (p < 0.005) from (20.3 +/- 6.2) to (4.5 +/- 1.9) with canine blood. Higher values were obtained for canine than human and porcine platelets. Platelet fragmentation, on the other hand, increased from 2.1-2.2% to 10.2-11.3% with increase of flow. Like platelets, deposition of canine fibrinogen was slightly higher than that of pig and human. The studies of adherent thrombus and platelet fragmentation identified an important flow-window of reduced thrombogenicity and acceptable fragmentation, encouraging extracorporeal circulation at higher blood flow.