Assessment of serum granulysin and cathepsin-L levels in vitiligo patients.

OBJECTIVE: Cellular and humoral immunity plays a role in the pathogenesis of vitiligo. T lymphocytes and natural killer cells involved in cellular immunity carry out their cytotoxic activities through perforin/granzyme-dependent granule exocytosis, in which granulysin and cathepsin-L are also involved. The aim of this study was to investigate the possible role of serum granulysin and cathepsin-L in the etiopathogenesis of vitiligo and their association with disease activity and severity. METHODS: This randomized, prospective case-control study was conducted with 46 vitiligo patients admitted to the hospital for vitiligo between January and November 2021 and 46 healthy volunteers of similar age and gender. Serum levels of granulysin and cathepsin-L were measured by the enzyme-linked immunosorbent assay method. RESULTS: The mean serum levels of granulysin and cathepsin-L were statistically significantly higher in vitiligo patients compared with the control group (p=0.048 and p=0.024, respectively). There was no statistically significant correlation between serum granulysin and serum cathepsin-L levels and disease severity in the patient group (r=0.30, p=0.062 and r=0.268, p=0.071, respectively). Disease activity also showed no significant association with serum granulysin and cathepsin-L levels (p=0.986 and p=0.962, respectively). CONCLUSION: Although granulysin and cathepsin-L are molecules involved in the pathogenesis of vitiligo, the use of these molecules may not be helpful in assessing disease activity and severity. It may be helpful to conduct comprehensive and prospective studies to find new molecules to fill the gap in this area.

Increased levels of circulating soluble CD226 in multiple sclerosis.

BACKGROUND: The glycoprotein CD226 plays a key role in regulating immune cell function. Soluble CD226 (sCD226) is increased in sera of patients with several chronic inflammatory diseases but its levels in neuroinflammatory diseases such as multiple sclerosis (MS) are unknown. OBJECTIVE: To investigate the presence and functional implications of sCD226 in persons with multiple sclerosis (pwMS) and other neurological diseases. METHODS: The mechanisms of sCD226 production were first investigated by analyzing CD226 surface expression levels and supernatants of CD3/CD226-coactivated T cells. The role of sCD226 on dendritic cell maturation was evaluated. The concentration of sCD226 in the sera from healthy donors (HD), pwMS, neuromyelitis optica (NMO), and Alzheimer's disease (AD) was measured. RESULTS: CD3/CD226-costimulation induced CD226 shedding. Addition of sCD226 to dendritic cells during their maturation led to an increased production of the pro-inflammatory cytokine interleukin (IL)-23. We observed a significant increase in sCD226 in sera from pwMS and NMO compared to HD and AD. In MS, levels were increased in both relapsing-remitting multiple sclerosis (RRMS) and secondary-progressive multiple sclerosis (SPMS) compared to clinically isolated syndrome (CIS). CONCLUSION: Our data suggest that T-cell activation leads to release of sCD226 that could promote inflammation and raises the possibility of using sCD226 as a biomarker for neuroinflammation.

Clinical parameters and biological markers associated with acute severe ocular complications in Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous adverse drug reactions with high mortality rates. Its sequelae, such as blindness, persist even after recovery. Patients with SJS/TEN should be accurately diagnosed and receive appropriate treatment as soon as possible. Therefore, identifying the factors for severity prediction is necessary. We aimed to clarify the clinical parameters and biological markers that can predict acute severe ocular complications (SOCs) in SJS/TEN. This retrospective cross-sectional study enrolled 47 patients with SJS/TEN who were divided into two groups according to ocular severity at acute onset: non-severe ocular complications group (n = 27) and severe ocular complications group (n = 20). Multivariate logistic regression analysis revealed that disease severity (body surface area detachment ≥ 10%) was a predictive factor for acute SOCs, and older age (≥ 60 years) was marginally significantly predictive of SOCs. Serum biomarker levels of S100A8/A9 and granulysin were marginally significant and tended to increase in the SOC group. Therefore, during the early acute stage, focusing on disease severity, patient age, and serum inflammatory biomarkers (S100A8/A9 and granulysin) might help predict SOC progression in patients with SJS/TEN who need prompt and aggressive ocular management to prevent severe ocular sequelae.

Association of elevated serum soluble CD226 levels with the disease activity and flares of systemic lupus erythematosus.

CD226 is an activating receptor expressed on the cell surface of natural killer cells and T cells. Although CD226 polymorphism is known to be involved in systemic lupus erythematosus (SLE), the involvement of soluble CD226 (sCD226) in SLE is still unknown. In the present study, we measured serum sCD226 levels using an enzyme-linked immunosorbent assay in 58 SLE patients and 33 healthy controls (HCs) and evaluated their associations with SLE Disease Activity Index 2000 (SLEDAI-2K), clinical manifestations, laboratory data, and the cumulative probability of flare. Serum sCD226 levels showed no significant differences between SLE patients and HCs. However, sCD226 levels were significantly elevated in active SLE patients with a SLEDAI-2K score of ≥ 20 compared with HCs. In SLE patients, sCD226 levels were significantly correlated with SLEDAI-2K scores and anti-dsDNA antibody titers. Moreover, the cumulative probability of flare was markedly higher in patients with high sCD226 than in those with low sCD226. In patients with neuropsychiatric involvement, sCD226 levels were elevated and reflected neuropsychiatric disease activity. These findings indicate that serum sCD226 levels are associated with disease activity and flares of SLE. Thus, it may be a useful biomarker for SLE, and its monitoring allows for more precise SLE management.

Extensive serum biomarker analysis in the prethrombotic state of recurrent spontaneous abortion.

The prethrombotic state (PTS) is a possible cause of recurrent spontaneous abortion (RSA). The aim of this study was to identify serum biomarkers for the detection of RSA with PTS (PSRSA). A Quantibody array 440 was used to screen novel serum-based biomarkers for PSRSA/NRSA (RSA without PTS). Proteins differentially expressed in PSRSA were analysed using bioinformatics methods and subjected to a customized array and enzyme-linked immunosorbent assay (ELISA) validation. We used receiver operating characteristic to calculate diagnostic accuracy, and machine learning methods to establish a biomarker model for evaluation of the identified targets. 20 targets were selected for validation using a customized array, and seven targets via ELISA. The decision tree model showed that IL-24 was the first node and eotaxin-3 was the second node distinguishing the PSRSA and NRSA groups (an accuracy rate of 100% and an AUC of 1). Epidermal growth factor (EGF) as the node distinguished the PSRSA and NC groups (an accuracy rate of 100% and an AUC of 1). EGF as the node distinguished the NRSA and NC groups (an accuracy rate of 96.5% and an AUC of 0.998). Serum DNAM-1, BAFF, CNTF, LAG-3, IL-24, Eotaxin-3 and EGF represent a panel of promising diagnostic biomarkers to detect the PSRSA.

Heat-Inactivation of Human Serum Destroys C1 Inhibitor, Pro-motes Immune Complex Formation, and Improves Human T Cell Function.

Heat-inactivation of sera is used to reduce possible disturbing effects of complement factors in cell-culture experiments, but it is controversially discussed whether this procedure is appropriate or could be neglected. Here, we report a strong impact of heat-inactivation of human sera on the activation and effector functions of human CD4+ T cells. While T cells cultured with native sera were characterized by a higher proliferation rate and higher expression of CD28, heat-inactivated sera shaped T cells towards on-blast formation, higher cytokine secretion (interferon γ, tumor necrosis factor, and interleukin-17), stronger CD69 and PD-1 expression, and increased metabolic activity. Heat-inactivated sera contained reduced amounts of complement factors and regulators like C1 inhibitor, but increased concentrations of circulating immune complexes. Substitution of C1 inhibitor reduced the beneficial effect of heat-inactivation in terms of cytokine release, whereas surface-molecule expression was affected by the addition of complex forming anti-C1q antibody. Our data clearly demonstrate a beneficial effect of heat-inactivation of human sera for T cell experiments but indicate that beside complement regulators and immune complexes other components might be relevant. Beyond that, this study further underpins the strong impact of the complement system on T cell function.

The Role of IL-13, IL-15 and Granulysin in the Pathogenesis of Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis.

Stevens-Johnson Syndrome (SJS) and toxic epidermal necrolysis (TEN) are Severe Cutaneous Adverse Reactions (SCARS) characterized by fever and mucocutaneous lesions leading to necrosis and sloughing of the epidermis. Conjunctival lesions are reported in 85% of patients. The pathogenesis of SJS/TEN/SCARS is not completely understood. It is hypothesized that IL-13, IL-15 and Granulysin expressed in plasma and skin may play a role. We measured the circulating levels of these cytokines in the plasma using ELISA and their expression in the skin using immunofluorescence microscopy. A total of 12 SJS/TEN skin biopsy samples (8 SJS, 2 SJS/TEN overlap and 2 TEN) were analyzed. Biopsy samples from patients with Lichen Planus (an inflammatory condition of the skin and mucous membranes) served as controls. Studies were also performed in human corneal epithelial cells where expression of these cytokines were measured following a challenge with TNF-α (0, 1, 10 and 100 ng/ml). The intensity of immunofluorescence was measured Using Imaris® software. The results showed significantly increased expression of these cytokines in the skin biopsy samples as measured by the average intensities of IL-13 (6.1 x 133.0 ± 4.231 x 10^8), and Granulysin (4.2 x 123.0 ± 4.231 x 10^8) compared to Lichen planus control (3.0 x 123.0 ±1.62 x 10^5). Increased expression of IL-13 and IL-15 were noted in cell culture studies and in the plasma samples when compared to Normal Human Plasma as controls. It is concluded that IL-13, IL-15 and Granulysin play a role in the pathogenesis of SJS/TEN.

Evaluating the Timeliness and Specificity of CD69, CD64, and CD25 as Biomarkers of Sepsis in Mice.

ABSTRACT: Sepsis occurs when an infection induces a dysregulated immune response, and is most commonly bacterial in origin. This condition requires rapid treatment for successful patient outcomes. However, the current method to confirm infection (blood culture) requires up to 48 h for a positive result and many true cases remain culture-negative. Therefore, new diagnostic tests are urgently needed. Recent clinical studies suggest that CD69, CD64, and CD25 may serve as useful biomarkers of sepsis. In this study, we evaluated the cecal ligation and puncture and cecal slurry mouse models as tools to study these biomarkers in young and aged mice, and elucidate the timeliness and specificity of sepsis diagnosis. Fluorescence-activated cell sorting analysis revealed that all three biomarkers were elevated on blood leukocytes during sepsis. CD69 was specifically upregulated during sepsis, while CD64 and CD25 were also transiently upregulated in response to sham surgery. The optimal biomarker, or combination of biomarkers, depended on the timing of detection, mouse age, and presence of surgery. CD69 demonstrated an excellent capacity to distinguish sepsis, and in some scenarios the diagnostic performance was enhanced by combining CD69 with CD64. We also analyzed biomarker expression levels on specific cell populations (lymphocytes, monocytes, and neutrophils) and determined the cell types that upregulate each biomarker. Elevations in blood biomarkers were also detected via microfluidic analyses; in this case CD64 distinguished septic mice from naive controls. Our results suggest that CD69 and CD64 are valuable biomarkers to rapidly detect sepsis, and that mouse models are useful to study and validate sepsis biomarkers.

Phenotypical and functional alteration of unconventional T cells in severe COVID-19 patients.

COVID-19 includes lung infection ranging from mild pneumonia to life-threatening acute respiratory distress syndrome (ARDS). Dysregulated host immune response in the lung is a key feature in ARDS pathophysiology. However, cellular actors involved in COVID-19-driven ARDS are poorly understood. Here, in blood and airways of severe COVID-19 patients, we serially analyzed unconventional T cells, a heterogeneous class of T lymphocytes (MAIT, γδT, and iNKT cells) with potent antimicrobial and regulatory functions. Circulating unconventional T cells of COVID-19 patients presented with a profound and persistent phenotypic alteration. In the airways, highly activated unconventional T cells were detected, suggesting a potential contribution in the regulation of local inflammation. Finally, expression of the CD69 activation marker on blood iNKT and MAIT cells of COVID-19 patients on admission was predictive of clinical course and disease severity. Thus, COVID-19 patients present with an altered unconventional T cell biology, and further investigations will be required to precisely assess their functions during SARS-CoV-2-driven ARDS.

Deficiency of platelet adhesion molecule CD226 causes megakaryocyte development and platelet hyperactivity.

This study used constitutive CD226 gene knockout (KO) mice as a model to investigate the functions and mechanisms of CD226 in megakaryocyte (MK) maturation and platelet activation. Although CD226 deficiency did not cause MK polyploidization or platelet granule abnormalities, increased MK counts were detected in the femora bone marrow (BM) and spleen of CD226 KO mice. Particularly, CD226 KO mice have a more extensive membrane system in MKs and platelets than wild-type (WT) mice. We also demonstrated that CD226 KO mice displayed increased platelet counts, shortened bleeding time, and enhanced platelet aggregation. CD226 KO platelets had an increased mature platelet ratio compared to the control platelets. In addition, the observed reduction in bleeding time may be due to decreased nitric oxide (NO) production in the platelets. Platelet-specific CD226-deficient mice showed similar increased MK counts, shortened bleeding time, enhanced platelet aggregation, and decreased NO production in platelets. Furthermore, we performed middle cerebral artery occlusion-reperfusion surgery on WT and CD226 KO mice to explore the potential effect of CD226 on acute ischemia-reperfusion injury; the results revealed that CD226 deficiency led to significantly increased infarct area. Thus, CD226 is a promising candidate for the treatment of thrombotic disorders.

Multifaceted effects of soluble human CD6 in experimental cancer models.

BACKGROUND: CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. METHODS: High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEµTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. RESULTS: Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. CONCLUSIONS: Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer.

A mathematical model for dynamics of soluble form of DNAM-1 as a biomarker for graft-versus-host disease.

DNAM-1 (CD226) is an activating immunoreceptor expressed on T cells and NK cells and involved in the pathogenesis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We previously reported that a soluble form of DNAM-1 (sDNAM-1) is generated by shedding from activated T cells. Moreover, higher serum levels of sDNAM-1 in patients before allo-HSCT is a predictive biomarker for the development of aGVHD based on the retrospective univariate and multivariate analyses in allo-HSCT patients. However, it remains unclear how the serum levels of sDNAM-1 are regulated after allo-HSCT and whether they are associated with the development of aGVHD. Here, we constructed a mathematical model to assess the dynamics of sDNAM-1 after allo-HSCT by assuming that there are three types of sDNAM-1 (the first and the second were from alloreactive and non-alloreactive donor lymphocytes, respectively, and the third from recipient lymphocytes). Our mathematical model fitted well to the data set of sDNAM-1 in patients (n = 67) who had undergone allo-HSCT and suggest that the high proportion of the first type of sDNAM-1 to the total of the first and second types is associated with high risk of the development of severe aGVHD. Thus, sDNAM-1 after allo-HSCT can be a biomarker for the development of aGVHD.

Expression and function of DNAM-1 on human B-lineage cells.

BACKGROUND: Although DNAM-1 is an activating receptor constitutively expressed on the majority of NK cells, CD8+ T cells, CD4+ T cells, monocytes, and platelets in human, several evidences demonstrated that a small population in B-lineage cells also expressed DNAM-1. However, the expression profile of DNAM-1 on B-lineage cells and its function remain obscure. Previous reports revealed that a considerable number of leukocytes including B cells in the peripheral blood conjugated to platelet. Thus, the proportion of DNAM-1+ B-lineage cells determined by flow cytometry analysis in the previous reports might be overestimated. METHODS: We examined whether platelets conjugate B cells and then analyzed the expression of DNAM-1 on the subpopulations of B-lineage cells according to their maturation stages after exclusion of platelet-conjugated B cells. We also assessed the involvement of DNAM-1 in IL-10 and antibody production from cultured B-lineage cells stimulated with CpG-ODN. RESULTS: Approximately 10% of human DNAM-1+ CD19+ B cells in the peripheral blood conjugated to platelets, resulting in the overestimation of the proportion of DNAM-1+ B cells. After exclusion of platelet-conjugating B cells, we show that DNAM-1 expression was detected on subpopulations of memory B cells, plasmablasts, and plasma cells and upregulated by stimulation with CpG-ODN. Moreover, DNAM-1 was involved in IL-10 and antibody productions by B cells after CpG-ODN stimulation. CONCLUSIONS: DNAM-1 may be involved in B-lineage cell-mediated immune responses.

[Effect of resection of adenoids and/or tonsil on the immune indexes in children with obstructive sleep apnea hypopnea syndrome].

Objective: To study the effect on immune indexes in children with obstructive sleep apnea hypopnea syndrome (OSAHS) before and after resection of adenoid and/or tonsil. Methods: A total of 100 children with OSAHS due to adenoid hypertrophy were enrolled in Department of Otorhinolaryngology Head and Neck Surgery, the Second Hospital of Dalian Medical University from December 2016 to December 2018. Some cases were complicated with tonsil hypertrophy or chronic tonsillitis. 6 ml of fasting peripheral venous blood were collected from all subjects at the 1st day before surgery, 4th day, 1 month, 3 months and 6 months after surgery to detect lymphoid subsets percentage (CD3(+), CD4(+),CD8(+), CD4/CD8, CD19, NK) and level of immunoglobulin (IgG, IgA, IgM). Grouping: group A was a total of 51 cases with adenoid hypertrophy after Adenoid plasma ablation; group B was a total of 27 cases with adenoid hypertrophy and chronic tonsillitis after plasma ablation of adenoid and tonsil; and group C was a total of 22 cases hypertrophy of adenoid and tonsil after plasma ablation of adenoid and tonsil.In the baseline data, age, gender and other variables were analyzed by anova and chi-square test, repeated measurement anova was used for intra-group and inter-group comparison of observation indicators at different time points after operation, and independent sample t-test was used for comparison between the two groups at observation points 3 months after operation. Results: (1) In group A, the percentage of CD19 lymphocytes before surgery was higher than that at 4th day after surgery, and the difference was statistically significant (21.85±6.20 vs.19.18±5.91, P<0.05). The other immune indexes were not statistically different before and after surgery (P>0.05). (2) In group B, the percentage of CD19 lymphocytes, CD3(+)T lymphocytes, CD8(+)T lymphocytes and the level of IgG at 4th day after surgery were significantly different between those before surgery (all P<0.05). At the 1st month after surgery, the percentage of CD3(+)T lymphocytes, CD8(+)T lymphocytes, CD19 lymphocytes and the level of IgG were significantly different between those before surgery (all P<0.05). The other immune indexes were not statistically different before and after operation (P>0.05). (3) In group C, the percentage of CD19 lymphocytes and the CD3(+)T lymphocytes at 4th day after surgery were significantly different between those before surgery (all P<0.05).In the 1st month after surgery, the percentage of CD8(+)T lymphocytes and CD19 lymphocytes were significantly different between those before surgery (all P<0.05). The other immune indexes were not statistically different before and after operation (P>0.05). (4) Among three groups, the percentage of CD4(+)T lymphocytes, the levels of IgG and IgA before surgery between group A and Group B were statistically significant (all P<0.05). At 4th day after surgery, the percentage of CD4(+)T lymphocytes in group B and C were lower than those in group A, and the differences were statistically significant (32.22±6.14, 32.36±6.87 vs. 36.36±5.19, all P<0.05); the other immune indexes were not statistically different among each group before and after surgery (P>0.05). Conclusions: Resection of adenoid has no significant effect on the immune indexes in children with OSAHS. The children with OSAHS complicated with tonsil problems have immune index disorder before surgery. Surgery has a certain effect on the immune indexes of children with OSAHS in a short period of time, and tends to normal level after one month.

Effect of peripherally inserted central catheter (PICC) parenteral nutrition on immune function and nutritional support after radical gastrectomy for gastric cancer.

Objective of the present study was to investigate the effects of peripherally inserted central catheter (PICC) parenteral nutrition support on immune function and nutritional support in patients undergoing radical gastrectomy for gastric cancer. 140 patients who underwent radical gastrectomy for gastric cancer were selected as participants and were divided into study group and the control group by random number table, with 70 cases in each group. Patients in the two groups underwent standard gastrectomy under general anesthesia by the same group of doctors. The study group received postoperative PICC catheter parenteral nutrition, and the control group received central venous catheter (CVC) nutrition support. Comparative study was done using t test and Chi-square test. The serum levels of ALB, TFN, PA, Hb, CD4+, CD8+, CD4+/CD8+, IgA, IgG, IgM and CD3+ in the two groups were observed before and after treatment, and the postoperative complications of the two groups were compared. After treatment, the levels of ALB, TFN, PA and Hb in the two groups were significantly increased (P<0.05). Levels of CD3+, CD4+, CD4+/CD8+, IgA, IgG and IgM also amplified significantly after treatment in both the groups, while CD8+ decreased significantly (P<0.05). What's more, the improvement degree of the study group was significantly greater than that of the control group (P<0.05). The time of drawing drainage tube, recovering intestinal function, getting off bed and the length of hospital stay in the study group were significantly shorter than those in the control group (P<0.05). The incidence of postoperative complications in the study group and control group were 8.6% (6/70 cases) and 11.4% (8/70 cases) respectively, and there was no significant difference (P>0.05). PICC catheter parenteral nutrition support and improve the nutritional status of patients, it was proved a safe and effective nutritional support which improve the cellular immune function and accelerated the recovery of gastrointestinal function.

Systemic and Local Increase of Granulysin Expression in Cytotoxic Lymphocytes in Severe Psoriasis.

Psoriasis is considered to be a cytokine-driven immune-mediated disease, although the cell cytotoxicity mechanisms involved remain unrecognized. Herein, we analyzed granulysin expression in different lymphocyte subsets of peripheral blood of 40 psoriatic patients (20 with severe and 20 with mild psoriasis) and seven sample of psoriatic skin. The simultaneous detection of intracellular granulysin and cell surface antigens was performed using flow cytometry in peripheral blood and immunohistochemistry in skin lesions. The frequency of granulysin+ cells, mean fluorescence intensity for granulysin, and the frequency of CD8+ T lymphocytes, NK cells, and NKT cells expressing granulysin molecules in peripheral blood were significantly higher in patients with severe psoriasis compared to mild disease and healthy individuals. These were also correlated with disease severity. Furthermore, granulysin+ cells, CD8+granulysin+ T lymphocytes, and CD56+granulysin+ NK cells were present in a higher frequency in the epidermal basal cell layer and in the dermal infiltrate of lesional skin as compared to non-lesional and healthy skin. In conclusion, granulysin+ cytotoxic cells are upregulated in blood and lesions of patients with psoriasis suggesting the involvement of granulysin mediated cytotoxicity in psoriasis pathogenesis.

Group 2 innate lymphoid cells exhibit progressively higher levels of activation during worsening of liver fibrosis.

INTRODUCTION: The interleukin-33/interleukin-13 pathway is involved in the immunopathology of liver fibrosis and recently characterized group 2 innate lymphoid cells (ILC2) were identified as profibrotic immune cells in the liver of mouse models. Our aim was to elucidate whether ILC2 might be present in human liver tissue and whether ILC2 contribute to liver fibrosis. MATERIALS AND METHODS: To identify ILC2 in liver tissue and blood, we purified mononuclear immune cells from needle biopsies, cirrhotic explant specimen, and paired peripheral blood samples. Cell suspensions were incubated with specific markers for ILC2 and analyzed by flow cytometry. The CD69 marker was included to assess the activation level of ILC2. In addition, we determined the IL-33 plasma level. RESULTS: Results were correlated with the METAVIR fibrotic score of patients enrolled in this study. We detected ILC2 in a higher percentage of CD45+ cells in liver tissue than in paired peripheral blood. The number of ILC2 was significantly increased in fibrotic tissue, but only slightly increased in paired peripheral blood. A higher percentage of CD69+ ILC2 was observed in fibrotic tissue, and this increase correlates positively with aggravation of liver fibrosis measured by fibrotic METAVIR score. A higher level of plasma IL-33 was only detected in samples obtained from cirrhotic patients. CONCLUSION: Our study indicates that ILC2 are present in the human liver and are activated in tissue contributing to the immunopathology of human liver fibrosis, independently of the etiology; which might be a potential new therapeutic target.

Clinical significance of soluble forms of immune checkpoint molecules in advanced esophageal cancer.

Immune checkpoint molecules are expressed on cancer cells and regulate tumor immunity by binding to ligands on immune cells. Although soluble forms of immune checkpoint molecules have been detected in the blood of patients with some types of tumors, their roles have not been fully elucidated. Soluble PD-L1, PD-1, CD155, LAG3, and CD226 (sPD-L1, sPD-1, sCD155, sLAG3, and sCD226, respectively) were measured in the sera of 47 patients with advanced esophageal cancer and compared with those of 24 control subjects. Pretreatment levels of sPD-1 and sCD155 were significantly higher in the patients with cancer than in the control subjects (P = 0.023, P = 0.001). The sPD-1 levels tended to be higher in the patients with lymph node metastasis, a large tumor diameter, and higher levels of serum SCC antigen (P = 0.150, P = 0.189, and P = 0.078, respectively). However, higher levels of sCD155 were associated with a better response to chemotherapy and favorable overall survival (P = 0.111 and P = 0.068, respectively). After 2 courses of chemotherapy, the levels of sCD155 and sCD226 were significantly increased (P < 0.001 and P = 0.002, respectively). Moreover, the increase in sCD226 during chemotherapy was associated with poor treatment response (P = 0.019). sPD-1 levels are possibly dependent on the tumor aggressiveness of the esophageal cancer. Furthermore, the pretreatment levels of sCD155 and kinetic change of sCD226 after chemotherapy may be used as biomarkers of the treatment response and prognosis in patients with esophageal cancer.

Detection of culture-negative sepsis in clinical blood samples using a microfluidic assay for combined CD64 and CD69 cell capture.

Sepsis is a life-threatening disease that affects millions of people every year. Rapid detection of sepsis assists clinicians to initiate timely antibiotic therapy and to reduce mortality. At the same time, accurate point-of-care detection is needed to reduce unnecessary use of antibiotics. One of the principal challenges in sepsis diagnosis is that many sepsis cases do not result in positive blood cultures. These so-called culture-negative cases present a significant health threat. In this work, we present a microfluidic cells separation system for the detection of sepsis in both culture-positive and culture-negative cases. Leukocytes were captured in several affinity separation zones of a microchip based on CD64, CD69, and CD25 expression. To validate this assay 40 septic patients and 10 healthy volunteers were enrolled in this study. Septic patients were divided into culture-positive (n = 12) and culture-negative cases (n = 21). CD64 + cell capture demonstrated excellent accuracy for sepsis detection with an area under the receiver operating characteristic curves (AUC) of 0.962. A combined panel of CD64 + and CD69 + cell counts was constructed, and the new panel outperformed each of these two biomarkers alone with the AUC of 0.978. Our affinity microfluidic devices were validated by conventional flow cytometry analysis. Results showed that the cell capture number of specific affinity region increased along with the increase of its corresponding antigen expression. This clinical validation confirms that CD64 and CD69 cell separations are a powerful sepsis assay with the potential for point-of-care analysis in culture-positive and culture-negative cases.

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).