Significant association between clinical characteristics and immuno-phenotypes in patients with ANCA-associated vasculitis.

OBJECTIVES: To elucidate the association between clinical characteristics and immuno-phenotypes in patients with ANCA-associated vasculitis (AAV). METHODS: Peripheral blood from 36 patients with active AAV and 18 healthy controls was examined for numbers of circulating T cells, B cells, NK cells, dendritic cells, monocytes and granulocytes using flow cytometry. These immuno-phenotyping data were subjected to cluster analysis and principal components analysis to divide AAV patients into subgroups. Associated organ involvement or therapeutic prognosis were assessed for each subgroup. RESULTS: AAV patients had higher proportions of plasma cells, plasmablasts, activated T cells, CD14++ CD16+ monocytes, eosinophils and neutrophils than healthy controls. Immuno-phenotyping findings were similar between patients with microscopic polyangiitis, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis. Cluster analysis indicated that AAV patients could be divided into three subgroups according to peripheral immune cell numbers: antibody production-related (n = 9), cytotoxic activity-related (n = 4) and neutrocytosis/lymphocytopenia-related (n = 23). The antibody production-related or cytotoxic activity-related group was associated with CNS involvement, and the neutrocytosis/lymphocytopenia-related group was associated with high incidence of kidney involvement. Incidence of severe infection was markedly higher in the neutrocytosis/lymphocytopenia-related group than the other two groups. Incidence of disease relapse was comparable among the three groups. CONCLUSION: Patients with active AAV can be divided into three subgroups based on immuno-phenotyping. These results may provide a hint to understanding disease pathophysiology and prognosis, and determining appropriate treatment.

Transcriptomic characterization and innovative molecular classification of clear cell renal cell carcinoma in the Chinese population.

BACKGROUND: Large-scale initiatives like The Cancer Genome Atlas (TCGA) performed genomics studies on predominantly Caucasian kidney cancer. In this study, we aimed to investigate genomics of Chinese clear cell renal cell carcinoma (ccRCC). METHODS: We performed whole-transcriptomic sequencing on 55 tumor tissues and 11 matched normal tissues from Chinese ccRCC patients. We systematically analyzed the data from our cohort and comprehensively compared with the TCGA ccRCC cohort. RESULTS: It found that PBRM1 mutates with a frequency of 11% in our cohort, much lower than that in TCGA Caucasians (33%). Besides, 31 gene fusions including 5 recurrent ones, that associated with apoptosis, tumor suppression and metastasis were identified. We classified our cohort into three classes by gene expression. Class 1 shows significantly elevated gene expression in the VEGF pathway, while Class 3 has comparably suppressed expression of this pathway. Class 2 is characterized by increased expression of extracellular matrix organization genes and is associated with high-grade tumors. Applying the classification to TCGA ccRCC patients revealed better distinction of tumor prognosis than reported classifications. Class 2 shows worst survival and Class 3 is a rare subtype ccRCC in the TCGA cohort. Furthermore, computational analysis on the immune microenvironment of ccRCC identified immune-active and tolerant tumors with significant increased macrophages and depleted CD4 positive T-cells, thus some patients may benefit from immunotherapies. CONCLUSION: In summary, results presented in this study shed light into distinct genomic expression profiles in Chinese population, modified the stratification patterns by new molecular classification, and gave practical guidelines on clinical treatment of ccRCC patients.

Multicentric experience with interferon gamma therapy in sepsis induced immunosuppression. A case series.

BACKGROUND: The sepsis-induced immunodepression contributes to impaired clinical outcomes of various stress conditions. This syndrome is well documented and characterized by attenuated function of innate and adaptive immune cells. Several pharmacological interventions aimed to restore the immune response are emerging of which interferon-gamma (IFNγ) is one. It is of paramount relevance to obtain clinical information on optimal timing of the IFNγ-treatment, -tolerance, -effectiveness and outcome before performing a RCT. We describe the effects of IFNγ in a cohort of 18 adult and 2 pediatric sepsis patients. METHODS: In this open-label prospective multi-center case-series, IFNγ treatment was initiated in patients selected on clinical and immunological criteria early (< 4 days) or late (> 7 days) following the onset of sepsis. The data collected in 18 adults and 2 liver transplanted pediatric patients were: clinical scores, monocyte expression of HLA-DR (flow cytometry), lymphocyte immune-phenotyping (flow cytometry), IL-6 and IL-10 plasma levels (ELISA), bacterial cultures, disease severity, and mortality. RESULTS: In 15 out of 18 patients IFNγ treatment was associated with an increase of median HLA-DR expression from 2666 [IQ 1547; 4991] to 12,451 [IQ 4166; 19,707], while the absolute number of lymphocyte subpopulations were not affected, except for the decrease number of NK cells 94.5 [23; 136] to 32.5 [13; 90.8] (0.0625)]. Plasma levels of IL-6 464 [201-770] to 108 (89-140) ng/mL (p = 0.04) and IL-10 from IL-10 from 29 [12-59] to 9 [1-15] pg/mL decreased significantly. Three patients who received IFNγ early after ICU admission (<4 days) died. The other patients had a rapid clinical improvement assessed by the SOFA score and bacterial cultures that were repeatedly positive became negative. The 2 pediatric cases improved rapidly, but 1 died for hemorrhagic complication. CONCLUSION: Guided by clinical and immunological monitoring, adjunctive immunotherapy with IFNγ appears well-tolerated in our cases and improves immune host defense in sepsis induced immuno suppression. Randomized clinical studies to assess its potential clinical benefit are warranted.

Bone Marrow Mast Cell Antibody-Targetable Cell Surface Protein Expression Profiles in Systemic Mastocytosis.

Despite recent therapeutic advances, systemic mastocytosis (SM) remains an incurable disease due to limited complete remission (CR) rates even after novel therapies. To date, no study has evaluated the expression on SM bone marrow mast cells (BMMC) of large panel of cell surface suitable for antibody-targeted therapy. In this study, we analyzed the expression profile of six cell-surface proteins for which antibody-based therapies are available, on BMMC from 166 SM patients vs. 40 controls. Overall, variable patterns of expression for the markers evaluated were observed among SM BMMC. Thus, CD22, CD30, and CD123, while expressed on BMMC from patients within every subtype of SM, showed highly variable patterns with a significant fraction of negative cases among advanced SM (aggressive SM (ASM), ASM with an associated clonal non-MC lineage disease (ASM-AHN) and MC leukemia (MCL)), 36%, 46%, and 39%, respectively. In turn, CD25 and FcεRI were found to be expressed in most cases (89% and 92%) in virtually all BMMC (median: 92% and 95%) from both indolent and advanced SM, but with lower/absent levels in a significant fraction of MC leukemia (MCL) and both in MCL and well-differentiated SM (WDSM) patients, respectively. In contrast, CD33 was the only marker expressed on all BMMC from every SM patient. Thus, CD33 emerges as the best potentially targetable cell-surface membrane marker in SM, particularly in advanced SM.

Phenotypic characterization of Peripheral B cells in Mycobacterium tuberculosis infection and disease in Addis Ababa, Ethiopia.

BACKGROUND: Mortality and morbidity from tuberculosis (TB) remain one of the most important public health issues. Although cell-mediated immunity is the main immune response against Mycobacterium tuberculosis (MTB), the role of B-cells during MTB infection and disease is unclear. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from treatment naïve Pulmonary TB patients (TB, n = 16), latent TB-infected participants (LTBI, n = 17), and healthy controls (HC, n = 19). PBMCs were stained with various fluorescently labeled antibodies to define B-cell subsets using multicolor flow cytometry. RESULTS: Atypical memory B cells (CD19+CD27-CD21-) and circulating marginal zone B-cells (CD19+CD27+CD21+IgM+IgD+CD23-) were significantly higher in active TB when compared to LTBI and HC. CD5+ regulatory B cells (Breg, CD19+CD24hiCD38hiCD5+) and resting B-cells (CD19+CD27+CD21+) in Active TB patients were significantly lower compared to HC and LTBI. Overall, there were no differences in B cell percentages (CD19+), naïve B cells (CD19+CD27-CD21+), Breg (CD19+CD24hiCD38hi), and activated memory B cells (CD19+CD27+CD21-) among the three study groups. CONCLUSIONS: These results indicated that multiple subsets of B cells were associated with TB infection and disease. It will be useful to examine these cell populations for their potential use as biomarkers for TB disease and LTBI.

Longitudinal monitoring of circulating immune cell phenotypes in anti-neutrophil cytoplasmic antibody-associated vasculitis.

Anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) is a necrotizing multiorgan autoimmune disease that affects small- to medium-sized blood vessels. Despite the improvements in treatments, half of the patients with AAV still experience disease relapses. In this review, we focus on peripheral leukocyte properties and phenotypes in patients with AAV. In particular, we explore longitudinal changes in circulating immune cell phenotypes during the active phase of the disease and treatment. The numbers and phenotypes of leukocytes in peripheral blood were differs between AAV and healthy controls, AAV in active versus inactive phase, AAV in treatment responders versus non-responders, and AAV with and without severe infection. Therefore, biomarkers detected in peripheral blood immune cells may be useful for longitudinal monitoring of disease activity in AAV.

Concomitant Expression of CD39, CD69, and CD103 Identifies Antitumor CD8+ T cells in Breast Cancer Implications for Adoptive Cell Therapy.

BACKGROUND: In cancer, an effective immune response involves the action of several different cell types, among which CD8 T cells play a major role as they can specifically recognize and kill cancer cells via the release of cytotoxic molecules and cytokines, being of major importance for adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). The inflammation resulting from the tumor growth attracts both activated and bystander T cells. For an effective antitumor response, the T cell must express a specific group of chemokine receptors and integrins which include CD103, CD39, CD69, and CD25. These markers had already been analyzed in various cancers, not including breast cancer and their subsequent subtypes, until now. To analyze, the key receptors on ex vivo expanded tumor-infiltrating lymphocytes in luminal A and luminal B breast cancer (BC) subtypes. MATERIALS AND METHODS: We were successful in expanding TILs ex vivo using a standard TIL culture condition from a cohort study of 15 primary luminal A and luminal B breast cancer patients. Furthermore, we examined the expression of CD103, CD39, CD69, and CD25 biomarkers after the expansion by flow cytometry. RESULTS: We found that the information about the percentage of TILs obtainable after the ex vivo expansion is not associated to nor it is dependent on the heterogeneity of the TIL population before the expansion and does not differ by the molecular subtype (p>0.05). We also found that there is a major population of memory-resident antitumor CD8+CD103+CD39+ and CD8+CD103+CD69+ TILs present in the stroma after the expansion when compared to CD4 immunosubtypes (p<0.0001). Only the CD8+CD103+CD39+ subpopulation was related to BC subtype (0.0009). CONCLUSION: Evidence from our study suggests that CD8 TILs present in the stroma of luminal A and luminal B breast cancer patients can be quantified and phenotyped by flow cytometry and be further expanded ex vivo. The immuno-phenotyping of these markers may be targeted to improve the success of immunotherapeutic approaches, such as adoptive cellular therapy (ACT) in patients with BC.

Longitudinal monitoring of circulating immune cell phenotypes in large vessel vasculitis.

Giant cell arteritis (GCA) and Takayasu arteritis (TAK) are two types of primary large vessel vasculitis (LVV). LVV is an intractable, rare disease with a high relapse rate. Disease progression in asymptomatic patients is an important issue in the clinical management of LVV. Useful biomarkers associated with clinical phenotypes, disease activity, and prognosis may be present in peripheral blood. In this review, we focused on peripheral leukocyte counts, surface markers, functions, and gene expression in LVV patients. In particular, we explored longitudinal changes in circulating immune cell phenotypes during the active phase of the disease and during treatment. The numbers and phenotypes of leukocytes in the peripheral blood were different between LVV and healthy controls, GCA and TAK, LVV in active versus treatment phases, and LVV in treatment responders versus non-responders. Therefore, biomarkers obtained from peripheral blood immune cells may be useful for longitudinal monitoring of disease activity in LVV.

Diagnosis of gastrointestinal tuberculosis: Using cytomorphological, microbiological, immunological and molecular techniques - A study from Central India.

The present study included three groups: (A) age and gender matched control (n=24) with no previous signs of M. tuberculosis complex (MTBC) infection, (B) patients (n=28) diagnosed with gastro-intestinal TB (GITB), (C) patients (n=50) with clinical and histo-pathological signs of GITB, but were culture and AFB negative. Real time assay performed using fluorescence resonance energy transfer hybridization probes showed a positivity index of 36 % in group C, i.e. 18 were found reactive from the total 50 cases studied. In addition, immune characterization of these 18 cases showed depleted CD(4) (+) count and increased levels of IFN-γ and TNF-α cytokines. No positive case was found in group A, while in group B, out of total 28 cases studied 27 were found positive. A combinatorial diagnostic approach for rapid detection and characterization of GITB might provide specific therapeutic strategies for prevention and treatment of the infection in future.

Longitudinal immune cell monitoring identified CD14++ CD16+ intermediate monocyte as a marker of relapse in patients with ANCA-associated vasculitis.

BACKGROUND: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease that affects small- to medium-sized blood vessels. Despite treatments having been improved, patients often experience disease relapses. It remains unclear how the immune cells involve in the development of vasculitis and how they fluctuate over the course of treatment. In this study, we aimed to identify the immune subsets and serum cytokines associated with disease relapse by comprehensive immuno-phenotyping in AAV patients. METHODS: We reviewed consecutive patients (n = 29) from Keio University Hospital who had been newly diagnosed with AAV from January 2015 to February 2019 and chronologically followed until 52 weeks. Numbers of circulating T cells, B cells, monocytes, and granulocytes were analyzed by flow cytometry (FACS). Serum levels of cytokines were measured by electrochemiluminescence enzyme immunoassay. Clinical information was obtained from patients' records and association with time-course changes in immuno-phenotypes and serum levels of cytokines were assessed. RESULTS: Comprehensive immuno-phenotyping data from 161 samples from 29 AAV patients at diagnosis; at weeks 4, 12, 24, and 52 of treatment; and at time of major relapse were examined. FACS analysis from patients with relapse revealed that CD14++ CD16+ intermediate monocytes and plasma cells concomitantly changed associated with disease relapse, which were independent from treatment regimen, ANCA status, or disease phenotype. In particular, the number of CD14++ CD16+ intermediate monocytes at relapse was significantly higher than that in remission or in healthy controls. Serum cytokine measurement revealed that changes of monocyte-derived proinflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNF-α were associated with disease status. CONCLUSIONS: Chronological changes in CD14++ CD16+ intermediate monocyte counts can be a marker of disease relapse in AAV patients.

Efficacy of Bifidobacterium longum, B. infantis and Lactobacillus acidophilus probiotics to prevent gut dysbiosis in preterm infants of 28+0-32+6 weeks of gestation: a randomised, placebo-controlled, double-blind, multicentre trial: the PRIMAL Clinical Study protocol.

INTRODUCTION: The healthy 'eubiosis' microbiome in infancy is regarded as the microbiome derived from term, vaginally delivered, antibiotic free, breastfed infants at 4-6 months. Dysbiosis is regarded as a deviation from a healthy state with reduced microbial diversity and deficient capacity to control drug-resistant organisms. Preterm infants are highly sensitive to early gut dysbiosis. Latter has been associated with sepsis and necrotising enterocolitis, but may also contribute to long-term health problems. Probiotics hold promise to reduce the risk for adverse short-term outcomes but the evidence from clinical trials remains inconclusive and none has directly assessed the effects of probiotics on the microbiome at high resolution. METHODS AND ANALYSIS: A randomised, double blind, placebo-controlled study has been designed to assess the safety and efficacy of the probiotic mix of Bifidobacterium longum and infantis and Lactobacillus acidophilus in the prevention of gut dysbiosis in preterm infants between 28+0 and 32+6 weeks of gestation. The study is conducted in 18 German neonatal intensive care units. Between April 2018 and March 2020, 654 preterm infants of 28+0-32+6 weeks of gestation will be randomised in the first 48 hours of life to 28 days of once daily treatment with either probiotics or placebo. The efficacy endpoint is the prevention of gut dysbiosis at day 30 of life. A compound definition of gut dysbosis is used: (1) colonisation with multidrug-resistant organisms or gram-negative bacteria with high epidemic potential or (2) a significant deviation of the gut microbiota composition as compared with healthy term infants. Dysbiosis is determined by (1) conventional microbiological culture and (2) phylogenetic microbiome analysis by high-throughput 16S rRNA and metagenome sequencing. Persistence of dysbiosis will be assessed at 12-month follow-up visits. Side effects and adverse events related to the intervention will be recorded. Key secondary endpoint(s) are putative consequences of dysbiosis. A subgroup of infants will be thoroughly phenotyped for immune parameters using chipcytometry. ETHICS AND DISSEMINATION: Ethics approval was obtained in all participating sites. Results of the trial will be published in peer-review journals, at scientific meetings, on the website (www.primal-study.de) and via social media of parent organisations. TRIAL REGISTRATION NUMBER: DRKS00013197; Pre-results.

Significant association between clinical characteristics and changes in peripheral immuno-phenotype in large vessel vasculitis.

BACKGROUND: Large vessel vasculitis (LVV) is a type of vasculitis characterized by granulomatous inflammation of medium- and large-sized arteries. Clinical assessment of acute phase reactants has been conventionally used to diagnose and monitor diseases; however, accurate assessment of vascular disease activity status can be difficult. In this study, we investigated comprehensive immuno-phenotyping to explore useful biomarkers associated with clinical characteristics. METHODS: Consecutive patients with newly diagnosed LVV who visited our institution between May 2016 and May 2019 were enrolled. The number of circulating T cells, B cells, natural killer cells, dendritic cells, monocytes, and granulocytes was examined and chronologically followed. Baseline and time-course changes in immuno-phenotyping associated with disease activity were assessed. RESULTS: Comprehensive immuno-phenotyping data from 90 samples from each of 20 patients with LVV were compared with those from healthy controls (HCs). The number of helper T (Th), follicular helper T (Tfh), CD8+ T, CD14++ CD16+ monocytes, and neutrophils were higher in patients with giant cell arteritis (GCA) and/or Takayasu arteritis (TAK) than in HCs. Among them, the number of CD8+ T and CD8+ Tem were higher in patients with TAK than in GCA. Notably, memory CD4+ and CD8+ T cells in patients with TAK remained high even in the remission phase. Further analysis revealed that the number of Th1, Th17, and Tfh cells was associated with disease relapse in GCA and TAK and that the number of CD8+ T cells was associated with relapse in TAK. Th1, Th17, and Tfh cells decreased after treatment with biologic agents, while CD8+ T cells did not. CONCLUSIONS: Our results from peripheral immuno-phenotyping analysis indicate that the numbers of Th and Tfh cells changed along with the disease condition in both GCA and TAK, while that of CD8+ T cells did not, especially in TAK. Treatment with biologic agents decreased the proportion of Th and Tfh cells, but not CD8+ T cells, in the patients. Chronological immuno-phenotyping data explained the difference in therapeutic response, such as reactivities against biologics, between GCA and TAK.

CD8 expression in B chronic lymphocytic leukemia: A case of immuno-phenotypic aberrancy.

Immuno-phenotyping forms an integral part of laboratory work up of Chronic lymphocytic leukemia (CLL). Aberrant antigen expressions are a known phenomenon in leukemic blasts, however cross lineage antigen expression is rarely seen in mature B cell leukemia, the reported percentages varying from 1% to 3% in Europe and North America. CASE DETAILS: We report a case of Rai stage 1 CLL showing aberrant expression of CD8 on flow cytometry. The patient had stable disease at a follow up of 9 months with no requirement of initiation of treatment. DISCUSSION: CD8 expression in CLL is rare and approximately 120 cases have been reported from Western population. To the best of our knowledge, this is the first case to be reported from India. The prognostic significance has been variably reported from favorable to poor. At present, our case reflects the phenotypic heterogeneity of leukemia. Long term follow up and evaluation of more cases is required to predict the prognostic role in our patient population. © 2017 International Clinical Cytometry Society.

Phenotypic analysis of peripheral B cell populations during Mycobacterium tuberculosis infection and disease.

BACKGROUND: Mycobacterium tuberculosis (Mtb) remains an unresolved threat resulting in great annual loss of life. The role of B cells during the protective immunity to Mtb is still unclear. B cells have been described as effector cells in addition to their role as antibody producing cells during disease. Here we aim to identify and characterize the frequency of peripheral B-cell subpopulations during active Tuberculosis and over treatment response. Analysis were done for both class switched (CS) and non-class switched (NCS) phenotypes. METHODS: We recruited participants with active untreated pulmonary Tuberculosis, other lung diseases and healthy community controls. All groups were followed up for one week from recruitment and the TB cases till the end of treatment (month 6). RESULTS: Peripheral blood samples were collected, stained with monoclonal antibodies to CD19(+) cells, Immunoglobulin (Ig) M, plasma cells (CD 138(+)), marker of memory (CD27(+)), immune activation (CD23(+)) and acquired on a flow cytometer. Circulating Marginal zone B cells (CD19(+)IgM(+)CD23(-)CD27(+)) and memory phenotypes are able to distinguish between TB diagnosis and end of treatment. The frequency of mature B cells from TB cases are lower than that of other-lung diseases at diagnosis. A subpopulation of activated memory B cells (CD19(+)IgM(+)CD23(+)CD27(+)) cells are present at the end of TB treatment. CONCLUSIONS: This study identified distinctive B cell subpopulations present during active TB disease and other lung disease conditions. These cell populations warrants further examination in larger studies as it may be informative as cell markers or as effectors/regulators in TB disease or TB treatment response.

Chinese Box turtle (Cuora flavomarginata) with lymphoid leukemia characterized by immunohistochemical and cytochemical phenotyping.

Lymphoid leukemia of T-cell origin was diagnosed in a male Chinese Box turtle, Cuora flavomarginata, of approximately 25 years of age. The turtle presented with a history of anorexia, open-mouth breathing, and lethargy for one week. The CBC findings included a mildly increased PCV, and severe leukocytosis due to high numbers of atypical cells interpreted to be blasts. The blasts were medium-sized cells with round to pleomorphic nuclei, slightly clumped chromatin, indistinct nucleoli, and scant moderate-to-dark blue cytoplasm with occasional red-to-purple cytoplasmic granulation. Cytochemical and immunohistochemical staining indicated that the neoplastic cells were positive for CD3 and α-naphthyl butyrate esterase (ANBE), leading to the diagnosis of T-cell lymphoid leukemia. Histology of tissues collected at necropsy showed multifocal infiltrations of neoplastic round cells in the liver, spleen, kidneys, testicles, pancreas, thyroid, duodenum, bone marrow, epicardium, and myocardium. Transmission electron microscopy failed to identify viral particles within the neoplastic cells. This article describes the hematologic, histologic, and ultrastructural abnormalities associated with lymphoid leukemia in this turtle, and advanced diagnostic methods used for phenotyping the T-cell origin.

Ascitic fluid cytology and flow cytometry in the primary diagnosis of lymphoma - a case report.

Primary diagnosis of lymphomas from ascitic fluid is rare. We report a case in which a patient being worked up as a case of carcinoma head of pancreas turned out to be a lymphoma on routine ascitic fluid examination and was further sub-classified as a CD 10+ B-cell lymphoma on flow cytometric analysis.