Effects of Tocilizumab Therapy on Circulating B Cells and T Helper Cells in Patients With Neuromyelitis Optica Spectrum Disorder.

Tocilizumab, a humanized anti-IL-6 receptor monoclonal antibody, showed its therapeutic efficacy on neuromyelitis optica spectrum disorder (NMOSD). To assess the immunological effects of this drug on B cells, follicular T helper (Tfh) cells, and peripheral T helper (Tph) cells in patients with NMOSD, peripheral B cell and Tfh cell phenotypes were evaluated in 26 patients with NMOSD before and after tocilizumab treatment by nine-color flow cytometry, as well as the expression of costimulatory and co-inhibitory molecules on B cells. Results showed that the frequency of CD27+IgD- switched memory B cells, CD27-IgD- double-negative B cells, and CD27highCD38high antibody-secreting cells was increased in patients with NMOSD. Tocilizumab treatment led to a significant shift of B cells to naïve B cells from memory B cells after 3 months. Three markers on B cells associated with T-cell activation (i.e., CD86 CD69, and HLA-DR) were downregulated after tocilizumab treatment. The frequencies of total Tfh and Tph cells were decreased, whereas that of follicular regulatory T cells tended to increase. Intrinsic increased PD-L1 and PD-L2 expression was characteristic of B cells in patients with NMOSD. Tocilizumab selectively restored PD-L1 on B-cell subsets. These results provided evidence that tocilizumab enhanced B- and T-cell homoeostasis by regulating B-cell differentiation and inhibiting lymphocyte activation in patients with NMOSD.

Growth arrest and DNA damage-inducible protein 34 (GADD34) contributes to cerebral ischemic injury and can be detected in plasma exosomes.

Growth arrest and DNA damage-inducible protein 34 (GADD34), one of the key effectors of negative feedback loops, is induced by stress and subsequently attempts to restore homeostasis. It plays a critical role in response to DNA damage and endoplasmic reticulum stress. GADD34 has opposing effects on different stimulus-induced cell apoptosis events in many nervous system diseases, but its role in ischemic stroke is unclear. In this study, we evaluated the role of GADD34 and its distribution in a rat cerebral ischemic model. The results showed that GADD34 was increased in the cortex and contributed to brain injury in ischemic rats. Furthermore, treatment with a GADD34 inhibitor reduced the infarct volume, improved functional outcomes, and inhibited neuronal apoptosis in the cortical penumbra after ischemia. The role of GADD34 in ischemic stroke was associated with the dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and phosphorylation of p53. In addition, the GADD34 level was increased in plasma exosomes of cerebral ischemic rats. These findings indicate that GADD34 could be a potential therapeutic target and biomarker for ischemic stroke.

Functional NK surrogate biomarkers for inflammatory recurrent pregnancy loss and recurrent implantation failure.

PROBLEM: Expansion of circulating NK cells has been related to pregnancy complications. This study aims at investigating several surface NK cell markers to identify a baseline inflammatory profile in women with recurrent pregnancy loss (iRPL) and recurrent implantation failure (iRIF). METHOD OF STUDY: Expression of NKp30, TIGIT, NKp46, and DNAM-1 on total peripheral blood NK subsets, regulatory (CD56bright CD16neg ), and cytotoxic (CD56dim CD16pos/neg ) NK cells was measured. RESULTS: Eighty-three women were recruited and classified into two groups, 58 women with RPL and 25 with RIF. A control group of 31 fertile women was included. Expression of NKp30 on cytNK was significantly higher in RPL (p = .019) and RIF (p < .001) than HC. TIGIT on cytNK cells was also higher in both RPL (p < .001) and RIF (p < .01). An optimal cutoff of 70% for NKp30+ cytNK disclosed a sensitivity of 82%, a specificity of 55%, and 83% PPV for RPL diagnosis. A cutoff level of 83% for TIGIT+ cytNK was chosen to discriminate between healthy controls and RPL women, with PPV of 84%. CONCLUSION: Our preliminary data on this RPL and RIF cohorts suggest a simple diagnostic tool by combining NKp30 and TIGIT on cytNK cells to better identify a subgroup of RPL and RIF patients with a baseline inflammatory profile. A more rigorous selection of these patients through phenotyping peripheral cytNK cells may better define patients that could benefit from an immunomodulatory treatment to prevent further pregnancy losses. The performance of these biomarkers requires further investigation and validation in independent cohorts.

Aberrantly elevated suprabasin in the bone marrow as a candidate biomarker of advanced disease state in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are preleukemic disorders characterized by clonal growth of mutant hematopoietic stem and progenitor cells. MDS are associated with proinflammatory signaling, dysregulated immune response, and cell death in the bone marrow (BM). Aging, autoinflammation and autoimmunity are crucial features of disease progression, concordant with promoting growth of malignant clones and accumulation of mutations. Suprabasin (SBSN), a recently proposed proto-oncogene of unknown function, physiologically expressed in stratified epithelia, is associated with poor prognosis of several human malignancies. Here, we showed that SBSN is expressed in the BM by myeloid cell subpopulations, including myeloid-derived suppressor cells, and is secreted into BM plasma and peripheral blood of MDS patients. The highest expression of SBSN was present in a patient group with poor prognosis. SBSN levels in the BM correlated positively with blast percentage and negatively with CCL2 chemokine levels and lymphocyte count. In vitro treatment of leukemic cells with interferon-gamma and demethylating agent 5-azacytidine (5-AC) induced SBSN expression. This indicated that aberrant cytokine levels in the BM and epigenetic landscape modifications in MDS patients may underlie ectopic expression of SBSN. Our findings suggest SBSN as a candidate biomarker of high-risk MDS with a possible role in disease progression and therapy resistance.

Decreased expression of suprabasin induces aberrant differentiation and apoptosis of epidermal keratinocytes: Possible role for atopic dermatitis.

BACKGROUND: Suprabasin (SBSN), a secreted protein, is expressed in various epithelial tissues. The role of SBSN in epidermal differentiation and atopic dermatitis (AD) pathology remains largely unknown. OBJECTIVE: To evaluate the effects of SBSN on epidermal keratinocytes and its role in AD. METHODS: We examined the SBSN expression levels in the stratum corneum and the epidermis by proteome analysis and immunohistochemistry, respectively. The serum SBSN concentration was measured by ELISA. These values were compared between AD and healthy control. Morphological changes in the epidermis were investigated in SBSN-knockdown three-dimensional human living skin equivalent (LSE) model with or without IL-4/IL-13. RESULTS: Epidermal SBSN expression was decreased in AD lesional skin compared to healthy skin, as assessed by the stratum corneum proteome analysis and immunohistochemistry. The SBSN serum levels were significantly lower in AD patients than in normal subjects (P<0.05). The SBSN-deficient LSE exhibited compact stratum corneum, immature stratum granulosum, and increased keratinocyte apoptosis. Th2 cytokines, IL-4 and IL-13, did not affect SBSN expression in LSE. There were no differentiation-associated makers that were affected by the SBSN knockdown. SBSN deficiency-induced apoptosis of keratinocytes was exaggerated by IL-4/IL-13, and accordingly, the addition of recombinant SBSN induced significant keratinocyte proliferation (P<0.05). CONCLUSION: Our data demonstrated that SBSN regulates normal epidermal barrier. Th2 cytokines unaffect SBSN expression in keratinocytes, but promote SBSN deficiency-induced apoptosis. It is suggested that SBSN has an anti-apoptotic activity, and its deficiency is involved in the pathogenesis of AD.

CD4+/CD45RO+: A Potential Biomarker of the Clinical Response to Glatiramer Acetate.

Background: Glatiramer acetate (GA) is an effective treatment for the earliest stages of multiple sclerosis (MS)-clinically isolated syndrome (CIS) or clinically definite MS (CDMS). Objective: This study aims to determine the differences in the lymphocyte population (at baseline and the course of five years) between confirmed sustained progression (CSP) and non-CSP groups and to identify potential biomarkers among these parameters that can predict a positive response to the treatment. Methods: Twelve male and 60 female patients were included in the study. Peripheral blood samples were collected before and five years after treatment with GA. The authors compared lymphocyte parameters between the CSP and non-CSP groups by statistical analyses. Univariate and penalized logistic regression models were fitted to identify the best lymphocyte parameters at baseline and their combination for potential biomarkers. Subsequently, the ROC analysis was used to identify cut-offs for selected parameters. Results: The parameter CD4+/CD45RO+ was identified as the best single potential biomarker, demonstrating the ability to identify patients with CSP. Moreover, a combination of four lymphocyte parameters at baseline, relative lymphocyte counts, CD3+/CD69+, CD4+/CD45RO+, and CD4+/CD45RA+ab, was identified as a potential composite biomarker. This combination explains 23% of the variability in CSP, which is better than the best univariate parameter when compared to CD4+/CD45RO+ at baseline. Conclusions: The results suggest that other biomarkers can help monitor the conditions of patients and predict a favourable outcome.

Neuroendocrine differentiation markers guide treatment sequence selection in metastatic castration-resistant prostate cancer.

BACKGROUND: To evaluate the value of the serum neuroendocrine differentiation (NED) markers in helping to select the best treatment sequence of abiraterone acetate (AA) and docetaxel-prednisone (DP) in mCRPC. METHODS: Eighty-eight mCRPC patients were identified (42 in the DP-to-AA group and 46 in the AA-to-DP group). The serum levels of NED markers were measured before the first-line treatment in 88 patients and also before and after DP therapy in 38 patients. We determined their impact on OS, radiographic progression-free survival (rPFS), and PSA-PFS. RESULTS: In men with an elevation of at least one NED marker (n = 46) before the first-line treatment, those who received AA and then DP had significantly better worse OS (21.7 months [95% CI 21.0-22.4] vs 19.9 months (95% CI 15.3-24.5); P = 0.023. In a multivariate Cox regression analysis, treatment sequencing selection (selecting DP-AA rather than AA-DP) independently predicted OS (HR 0.4, 95% CI 0.2-0.9, P = 0.035) in patients with an elevation of at least one NED marker. However, in the subgroup without NED marker elevation, there was no significant difference in clinical outcomes between AA-DP and DP-AA groups (all P > 0.05). In the group with continued NED marker evaluation during DP treatment, patients with higher baseline NED markers and obtaining PSA response to DP were more inclined to experience NED markers decline. CONCLUSIONS: Elevated pretreatment serum NED markers might indicate mCRPC patients would get better clinical outcomes from DP-AA than AA-DP. In contrast, those without NED marker elevation had similar outcomes regardless of which agent was chosen first. mCRPC patients with elevated NED markers and chemotherapy response were more inclined to obtain NED markers decline during DP therapy, which could account for this phenomenon.

CyTOF Measurement of Immunocompetence Across Major Immune Cell Types.

The central role of the immune system is becoming appreciated in a wide variety of diseases. Cancer immunotherapy is one area that has yielded much recent success, although not all patients benefit equally. At the same time, recent studies have highlighted the heterogeneity of the human immune system. Despite this heterogeneity, we do not routinely measure immune competence in clinical practice, and there are no consensus assays of healthy immune function. Using mass cytometry (CyTOF), we can simultaneously detect ∼40 markers to identify various cell subsets and determine their function by the expression of cytokines, cytotoxicity, and activation markers. This can help assess 'immunocompetence' and facilitate better implementation of immunotherapies, both in specific disease settings and perhaps eventually as a prognostic tool in healthy subjects. Here we introduce the concepts behind this assay and provide a protocol that we have successfully implemented to identify possible predictive biomarkers of immunotherapy outcome. © 2017 by John Wiley & Sons, Inc.

Clinicopathological Analysis of 320 Cases of Diffuse Large B-cell Lymphoma Using the Hans Classifier.

The estimation of clinical prognosis for diffuse large B-cell lymphoma (DLBCL) with a quick, cost-efficient method is necessary because of the clinical heterogeneity of this disease, which leads to death, relapsed or refractory disease in approximately 40% of patients. We analyzed 320 cases diagnosed from 2007 to 2013 treated with R-CHOP therapy at Tokai University Hospital and associated institutions. DLBCL was classified according to the cell-of-origin using the Hans algorithm [germinal center B-cell-like (GCB) vs non-GCB subtypes], and into 6 subgroups derived from combinations of CD10, BCL6 and MUM1 markers. The percentage of GCB and non-GCB (NGCB) subtypes was 35% and 65%, respectively. GCB-DLBCL was characterized by lower BCL2 immunohistochemical expression, extranodal sites ＜1, better therapeutic response, and favorable overall survival (OS) and progression free survival (PFS) (P＜0.01). The most frequent subgroup was NGCB-1 (CD10-BCL6+MUM1+, 51%) followed by GCB-1 (CD10+BCL6+or-MUM1+, 21%), NGCB-2 (CD10-BCL6-MUM1+, 13%), GCB-2 (CD10+BCL6+or-MUM1-, 10%), GCB-3 (CD10-BCL6+MUM1-, 4%) and NGCB-3 (CD10-BCL6-MUM1-, 2%). In comparison with GCB-2 and GCB-3 (both MUM1-), the GCB-1 (MUM1+) was characterized by favorable PFS (5-year PFS 84% vs 65%, OR 0.368, P＜0.05), independent of high LDH (associated with unfavorable PFS, OR 7.04, P＜0.01) in the multivariate analysis. This predictive value of MUM1 was independent of CD10. Interestingly, triple-negative NGCB-3 tended to have a more favorable prognosis than the other NGCB subgroups. In conclusion, the Hans classifier is a valid method to evaluate the prognosis of DLBCL NOS. In the GCB subtypes, GCB subtypes, MUM1-positivity is associated with a more favorable outcome (PFS).

Congenital immature pure erythroid leukemia with E-cadherin expression.

Congenital pure erythroid leukemia is exceedingly rare and poses a diagnostic challenge. We report an atypical case of congenital pure erythroid leukemia that did not express typical erythroid markers. The patient presented with a high white blood cell count with blastic cells at birth. Although flow cytometric analyses of peripheral blood and bone marrow showed a large CD45-negative cell population, we did not identify any evidence of monoclonality. While the circulating blasts decreased with only supportive care, hepatomegaly with multiple nodules was accompanied by liver failure, disseminated intravascular coagulation, and development of hemophagocytic lymphohistiocytosis. Pathological examination of the liver biopsy specimen revealed a small round cell tumor that was negative for nearly all hematopoietic cell markers, including classical erythroid cell markers, and positive for CD43, CD71, and E-cadherin, an early erythroid marker epithelial calcium-dependent adhesion protein, suggesting that these tumor cells originated from an immature erythroblast. We found high ß-catenin and c-Myc protein expression, which were not previously described in pure erythroid leukemia. Cytosine arabinoside temporarily alleviated clinical symptoms; however, the patient died of progressive disease at 8 months of age. This case indicates that E-cadherin is useful for diagnosing pure erythroid leukemia, even in immature cases.

Phenotypic Changes and Impaired Function of Peripheral γδ T Cells in Patients With Sepsis.

INTRODUCTION: Recent studies demonstrated the significant loss of gamma delta T (γδ T) cells in patients with sepsis. Given the distinct functions of γδ T cells in human anti-infection immunity, we are interested in evaluating the phenotype and function of peripheral γδ T cells in septic patients and determining their prognostic implication. METHOD: This prospective study has been conducted in three intensive care units of a university hospital. During the period from October 2014 to June 2015, we enrolled 107 patients who were consecutively admitted and diagnosed with severe sepsis or septic shock (excluding previous immunosuppression) and 45 healthy controls. Using flow cytometry, we analyzed the in vivo percentage of γδ T cells in cluster of differentiation (CD)3 cells from peripheral blood mononuclear cells as well as their expression of surface markers (CD69, natural-killer group 2 member D [NKG2D], programmed death receptor 1 [PD-1]) and intracellular cytokines (interferon-γ [IFN-γ], interleukin [IL]-17, IL-10, transforming growth factor-ß [TGF-ß]). Then we further evaluated the different responses of γδ T cells after the antigen stimulation ex vivo by measuring CD69 and IFN-γ expression. Lastly, we conducted the multiple logistic regressions to analyze the risk factor for prognosis. RESULTS: Compared with control group, γδ T cells in septic patients displayed a decrease in percentage, increase in CD69, decrease in NKG2D, and increase in cytokine expression (pro-inflammatory IFN-γ, IL-17, anti-inflammatory IL-10, TGF-ß) in vivo. After the antigen stimulation ex vivo, both CD69 and IFN-γ expression in γδ T cells were significantly lower in septic patients than control group. Importantly, the decrease in CD69 and IFN-γ expression was more pronounced in non-survivors than survivors. Multiple logistic regression analysis revealed that lower expression of IFN-γ after stimulation is a dependent risk factor that associated with patient 28-day death in septic patients (OR: 0.908 [95% CI: 0.853-0.966]). CONCLUSION: Septic patients showed altered phenotype and function of γδ T cells. The impaired IFN-γ expression by γδ T cells after the antigen stimulation is associated with mortality in septic patients.

Upregulation of endothelial gene markers in Wharton's jelly mesenchymal stem cells cultured on polyelectrolyte multilayers.

Designing convenient substrates is a pertinent parameter that can guide stem cell differentiation. Current research is directed toward differentiating mesenchymal stem cells (MSCs) into endothelial cells (ECs). It is generally accepted that MSCs cannot be easily differentiated into ECs without high concentrations of proangiogenic factors. To guide either bone marrow-derived mesenchymal stem cells (BM-MSCs) and Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into ECs-like phenotype, poly(allylamine-hydrochloride)/poly(styrene-sulfonate) multilayers film (PAH/PSS) was used as culture coating and compared to type I collagen (as control coating). After 2 weeks of culture and in absence of angiogenic growth factors, PAH/PSS upregulated KDR, PECAM-1, and CDH5 genes, whereas combining PAH/PSS with endothelial growth media (EGM-2® ) led to the production of respective proteins by WJ-MSCs. In contrast, not fully EC-like phenotype is obtained from the differentiation of BM- or WJ-MSCs cultured on type I collagen. Thus, using PAH/PSS coating in synergy with EGM-2® appears as an ideal condition promoting WJ-MSCs differentiation into ECs-like. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 292-300, 2017.

[THE STANDARD VALUES OF SUB-POPULATIONS OF T-HELPERS OF DIFFERENT LEVEL OF DIFFERENTIATION IN PERIPHERAL BLOOD].

The study was carried out to develop standard indicators of relative and absolute content of main populations of T-helpers in peripheral blood of conditionally healthy donors. The examination was implemented to sampling of 52 healthy individuals (29 males and 23 females) aged 18-65 years (median is 30 years). The multicolor cytofluorimetric analysis was applied using panel of following antibodies: CD45RA-FITC, CD62L-PE, CCR4-PerCP/Cy5.5; CCR6-PE/Cy7, CXCR3-APC, CD3-APC-AF750, CD4-Pacific Blue and CXCR5-Brilliant Violet 510TM. The T-helpers 1 were distributed in populations of cells with phenotypes CXCR5-CXCR3+CCR6-CCR4-, also containing Th9, and CXCR5-CXCR3+CCR6+CCR4- referred as Thl/Thl7. The Th2 were detected an the basis of availability of CCR4 at the absence of all other chemokin receptors. The Thi7, besides Thl/Thi7 mentioned above, were detected in composition of CXCR5-CXCR3-CCR6+CCR4- and CXCR5-CXCR3-CCR6+CCR4+. The last population also contained Th22. The follicular Th which expressed at their surface CXCR5, formed six cellular populations with following phenotypes: CXCR5+CXCR3-CCR6-CCR4- (Tfh/Tfh2), CXCR5+CXCR3-CCR6-CCR4+ (Tfh2), CXCR5+CXCR3-CCR6+CCR4- (Tfh17), CXCR5+CXCR3-CCR6+CCR4+ (Tfh17), CXCR5+CXCR3+CCR6-CCR4- (Tfh1) and CXCR5+CXCR3+CCR6+CCR4- (Tfh1/Tfh17). The relative and absolute content of T-helpers of mentioned phenotypes was established both within the framework of total population CD3+CD4+ of lymphocytes and among "naive" T-helpers (CD45RA-CD62L+), T-helpers of central (CD45RA-CD62L+) and effector (CD45RA- CD62L-) memory and also "terminal-differentiated" CD45RA-positive cells of effector memory with phenotype CD45RA+CD62L-. The study results can be applied as standard indicators under diagnostic of pathologic conditions of immune system.

Avoiding false discovery in biomarker research.

BACKGROUND: Human tyrosine-protein phosphatase non-receptor type substrate 1α (SIRPA) is a surface marker identified in cardiomyocytes differentiated from human embryonic stem cells. Our objective was to determine if circulating SIRPA levels can serve as a biomarker of cardiac injury in children undergoing open heart surgery. RESULTS: Paired pre- and post-operative serum samples from 48 pediatric patients undergoing open heart surgery and from 6 pediatric patients undergoing non-cardiac surgery (controls) were tested for SIRPA protein levels using commercially available SIRPA ELISA kits from two manufacturers. Post-operative SIRPA concentrations were significantly higher in patients after cardiac surgery compared to non-cardiac surgery when tested using SIRPA ELISA kits from both manufacturers. To verify the identity of the protein detected, recombinant human SIRPA protein (rhSIRPA) was tested on both ELISA kits. The calibrator from both ELISA kits was analyzed by Western blot as well as by Mass Spectrometry (MS). Western blot analysis of calibrators from both kits did not identity SIRPA. MS analysis of calibrators from both ELISA kits identified several inflammatory markers and albumin but no SIRPA was detected. CONCLUSIONS: We conclude that commercially available ELISA kits for SIRPA give false-positive results. Verifying protein identity using robust protein characterization is critical to avoid false biomarker discovery when using commercial ELISA kits.

Enhanced Osseointegration of Hierarchical Micro/Nanotopographic Titanium Fabricated by Microarc Oxidation and Electrochemical Treatment.

Rapid osseointegration is recognized as a critical factor in determining the success rate of orthopedic and dental implants. Microarc oxidation (MAO) fabricated titanium oxide coatings with a porous topography have been proven to be a potent approach to enhance osteogenic capacity. Now we report two kinds of new hierarchical coatings with similar micromorphologies but different nanotopographies (i.e., MAO and MAO-AK coatings), and both coatings significantly promote cell attachment and osteogenic differentiation through mediating the integrin ß1 signaling pathway. In this study, titanium with a unique hierarchical micro/nanomorphology surface was fabricated by a novel duplex coating process, that is, the first a titanium oxide layer was coated by MAO, and then the coating was electrochemically reduced in alkaline solution (MAO-AK). A series of in vitro stem cell differentiation and in vivo osseointegration experiments were carried out to evaluate the osteogenic capacity of the resulting coatings. In vitro, the initial adhesion of the canine bone marrow stem cells (BMSCs) seeded on the MAO and MAO-AK coatings was significantly enhanced, and cell proliferation was promoted. In addition, the expression levels of osteogenesis-related genes, osteorix, alkaline phosphates (ALP), osteopontin, and osteocalcin, in the canine BMSCs, were all up-regulated after incubation on these coatings, especially on the MAO-AK coating. Also, the in vitro ALP activity and mineralization capacity of canine BMSC cultured on the MAO-AK group was better than that on the MAO group. Furthermore, 6 weeks after insertion of the titanium implants into canine femurs, both the bone formation speed and the bone-implant contact ratio of the MAO-AK group were significantly higher than those of the MAO group. All these results suggest that this duplex coating process is promising for engineering titanium surfaces to promote osseointegration for dental and orthopedic applications.

Elevated survivin expression in peripheral blood mononuclear cells is central to collateral formation in coronary chronic total occlusion.

Survivin is essential to angiogenesis and revascularization, but its role in coronary collateral formation remains unclear. The role of survivin in peripheral blood mononuclear cells (PBMCs) of coronary chronic total occlusion (CTO) patients was investigated. Coronary CTO patients (n=46; mean age 60.1±8.5, male 54.3%) (CTO group) and normal control patients (n=18; mean age 58.0±10.0, male 55.6%) underwent angiographic collateral vessel grading by Rentrop classification (C0 - C3) and provided peripheral blood between June 2006 and February 2007. Rat hind limb ischemia models were constructed using four equal groups of Sprague-Dawley rats (n=36): normal control, sham operation, operation and granulocyte macrophage colony-stimulating factor (GM-CSF). PBMC numbers and characteristics, collateral vessels, survivin, CD4, CD8, CD44, vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) expression were determined using RT-PCR, flow cytometry, immunocytochemistry and western blot analysis. PBMC survivin mRNA and protein expression levels were higher in patients with good collateral circulation (C2 + C3) than in patients with no collateral flow (C0) (all P<0.05). Survivin single-positive and survivin and CD8, VEGF and ICAM-1 double-positive percentages were elevated in patients with good collateral circulation compared to those with normal and no collateral flow (all P<0.05), consistent with the rat model results, wherein higher survivin levels produced significantly larger and more visible collateral vessels. In conclusion, elevated survivin expression in PBMCs, particularly survivin and CD8, VEGF, and ICAM-1 double-positive PBMCs, may be crucial for good collateral formation in patients with coronary CTO, as confirmed by assessment of a rat model.

The ratio of circulating regulatory T cells (Tregs)/Th17 cells is associated with acute allograft rejection in liver transplantation.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) and Th17 cells are known to be involved in the alloreactive responses in organ transplantation, but little is known about the relationship between Tregs and Th17 cells in the context of liver alloresponse. Here, we investigated whether the circulating Tregs/Th17 ratio is associated with acute allograft rejection in liver transplantation. In present study, thirty-eight patients who received liver transplant were enrolled. The patients were divided into two groups: acute allograft rejection group (Gr-AR) (n = 16) and stable allograft liver function group (Gr-SF) (n = 22). The frequencies of circulating Tregs and circulating Th17 cells, as well as Tregs/Th17 ratio were determined using flow cytometry. The association between Tregs/Th17 ratio and acute allograft rejection was then analyzed. Our results showed that the frequency of circulating Tregs was significantly decreased, whereas the frequency of circulating Th17 cells was significantly increased in liver allograft recipients who developed acute rejection. Tregs/Th17 ratio had a negative correlation with liver damage indices and the score of rejection activity index (RAI) after liver transplantation. In addition, the percentages of CTLA-4(+), HLA-DR(+), Ki67(+), and IL-10(+) Tregs were higher in Gr-SF group than in Gr-AR group. Our results suggested that the ratio of circulating Tregs/Th17 cells is associated with acute allograft rejection, thus the ratio may serve as an alternative marker for the diagnosis of acute rejection.

[The flow cytometry in diagnostic of sepsis in children of first year of life with congenital heart disease and operated in conditions of artificial circulation].

The study was organized to evaluate sensitivity and specificity of CD64 index and relative amount of HLA-DR+ monocytes in diagnostic of sepsis in children of first year of life after surgery correction if congenital heart disease in conditions of artificial circulation. To detect CD64 index the kit Leuko64 (Beckman Coulter USA) was applied. The relative amount of HLA-DR+ monocytes was measured by flow cytofluorimeter Navios (Beckman Coulter, USA) using combination of monoclonal antibodies CD14-APC, HLA-DR-PacificBlue, CD45-KrOr. The results of study established that CD64 index in the group with confirmed or supposed sepsis consisted 2.29 (1.96:3.32) that statistically is reliably higher (p = 0.001) than in group without sepsis. The study established no statistically reliable differences in concentration of C-reactive protein in blood serum (p-0.123), absolute amount of leukocytes in peripheral blood (p = 0.128), relative amount of HLA-DR+ monocytes (p = 0.789). It is demonstrated that value of CD64 index higher than 2.00 increases the risk of development of sepsis up to 9.4 times and can be used as a diagnostic criterion of sepsis (AUC = 0.895) with sensitivity up to 80% and specificity up to 90%. The negative prognostic significance of CD64 index and content of procalcitonin in relation to development of sepsis in children of first year of life operated in conditions of artificial circulation amounted to 74% and 76% and 77% and 64% in case of positive prognostic significance correspondingly.