Flow cytometry in the diagnosis of mature B-cell lymphoproliferative disorders.

B-lineage lymphoproliferative disorders (LPD) are rather frequent diseases, associated with specific clinical or biological features but also sometimes of fortuitous discovery. Multiparameter flow cytometry plays a major role for a rapid diagnostic indication, on peripheral blood or bone marrow samples in most instances, guiding complementary analyses and allowing for the proper therapeutic management of patients. After describing the important pre-analytical precautions required for an adequate assessment, the immunophenotypic features of small-cell and large-cell lymphomas are described in this review. The ubiquitous expression of CD19 is a first mandatory gating step. A possible clonal proliferation is then suspected by the demonstration of surface immunoglobulin light chain restriction. The aberrant presence of CD5 allows to segregate chronic lymphocytic leukemia and mantle cell lymphoma in most cases. Other LPD exhibit specific immunophenotypic features. A table of useful markers and a decision tree are provided. Of note, immunophenotypic data should as much as possible be interpreted in an integrated manner, involving the patient's clinical and other biological features, and be completed by further chromosomal and/or molecular investigations.

[Characterization of B1-cells during experimental leukomogenesis.]

BACKGROUND: Bovine leukemia causes a significant polyclonal expansion of CD5+, IgM+ B lymphocytes, known as persistent lymphocytosis (PL), in approximately 30% of infected cattle. However, it is not yet clear what happens to this subpopulation of B cells in the early period of infection of animals. PURPOSE: Quantitative characterization of IgM+ and CD5+ B cells during the immune response, which can provide important information on the mechanisms of lymphocyte priming in BLV infection. MATERIAL AND METHODS: The experiment used BLV-negative calves of black-motley breed at the age of 8 months (n = 11). Animals (n = 8) were intravenously injected with blood of a BLV-positive cow. Control calves (n = 3) were injected with saline. Studies were performed before and after infection on days 5, 7, 14, 21, 28 and 65 of the immune response. The determination of the number of B-lymphocytes in the blood was carried out by the method of immunoperoxidase staining based on monoclonal antibodies to IgM, CD5. RESULTS: As a result of the studies, it was found that the level of CD5+ B cells increases on the 14th day of the primary immune response, characterized by polyclonal proliferation of CD5+ B cells, which are the primary target for BLV. Our research data confirm that in the lymphocytes of experimentally infected cattle, surface aggregation of IgM and CD5 molecules on B-lymphocytes is absent. DISCUSSION: It is known that the wave-like nature of IgM synthesis, which was shown in previous studies, depends on a subpopulation of B1 cells. After 7 days of the immune response, IgM+ and CD5+ cells do not correlate, which shows their functional difference. The increase in CD5+ cells is probably not associated with B cells, but with T cells differentiating under the influence of the virus. CONCLUSIONS: A subset of B1 cells is the primary target of cattle leukemia virus. The 65th day of the immune response is characterized by the expansion of IgM+ B cells, a decrease in the number of CD5+ cells and a uniform distribution of receptors around the perimeter of the cells.

Daytime melatonin levels in saliva are associated with inflammatory markers and anxiety disorders.

BACKGROUND: The bidirectional interaction between melatonin and the immune system has largely gone unexplored in a clinical context and especially in a psychiatric population. This study explored the association between melatonin during the day and inflammatory cytokines in young adult patients seeking psychiatric care. METHODS: Samples and data were collected from 108 young adults (mean age 21, SD = 2) at an outpatient clinic for affective disorders. Daytime saliva melatonin levels were analyzed with enzyme-linked immunosorbent assay (ELISA) in relation to normalized serum expression levels of 72 inflammatory markers in a proximity extension assay (PEA). In a post hoc analysis the markers associated with melatonin were tested in a generalized linear model to see whether there is a relationship to anxiety disorder or depression. RESULTS: After Bonferroni correction for multiple testing, melatonin levels at 11:00 were positively correlated with CD5 (p = 4.2e-4). Melatonin levels after lunch were correlated with CCL2/MCP-1 (p = 4.2e-4), CCL3/MPI-1α (p = 6.5e-4) and VEGF-A (p = 5.3e-6). In the generalized linear model, positive associations were found for the presence of any anxiety disorder with melatonin after lunch (p = 0.046), VEGF-A (p = 0.001) and CCL3/MPI-1α (p = 0.001). CONCLUSION: Daytime saliva levels of melatonin were related to several inflammatory markers in young adults with psychiatric disorders. This observation likely reflects the bidirectional relationship between melatonin production and the immune system. These findings may have relevance for the understanding of psychiatric disorders and other conditions associated with low-grade inflammation.

Evaluation of the frequency of CD5+ B cells as natural immunoglobulin M producers and circulating soluble CD5 in patients with bladder cancer.

Bladder cancer is the most common malignancy of the genitourinary tract. It is the fourth most common malignancy in men and the fifth most common malignancy in the general population, with a high recurrence rate. CD5+ B lymphocytes are a subset of B lymphocytes, which contribute to innate immune responses. These cells are involved in the spontaneous production of self-reactive natural antibodies. On the other hand, natural antibodies can recognize tumor-associated antigens, including proteins or carbohydrates, and eliminate these cells in a complement-dependent manner or via induction of apoptosis. Besides surface CD5, the soluble form of this molecule is involved in the regulation of immune system. Considering the role of CD5+ B cells in the production of natural immunoglobulin M (IgM) and role of these antibodies in antitumor responses, in this study, we aimed to investigate the frequency of CD5 in B cells and to evaluate the diagnostic potential of these cells and also soluble CD5 (sCD5) in patients with bladder cancer. Blood specimens were collected from 40 patients with bladder cancer, who were referred to Sina Hospital in Tehran, IRAN. The levels of CD5+ and CD5- B lymphocytes were measured in the peripheral blood via flow cytometry, and the levels of sCD5 and total IgM were investigated in the serum by ELISA and nephlometry techniques, respectively. The frequency of CD5+ and CD5- B cells was significantly lower in patients, compared with the healthy controls. Detectable levels of sCD5 were found in two patients (5%), while total IgM showed no significant difference between the patient and control groups. The present results suggest that B cell subsets may be affected by malignancy. Therefore, further research is needed to identify B cells and their soluble markers for diagnosis of patients with bladder cancer.

Role of Regulatory B Cells in the Progression of Cervical Cancer.

This study is to investigate the role of regulatory B (Breg) cells in cervical cancer. In total, 70 cases of cervical cancer, 52 cases of cervical intraepithelial neoplasia (CIN), and 40 normal controls were enrolled. The percentage of Breg cells was detected by flow cytometry. Serum levels of IL-10 were measured by ELISA. The correlation between Breg cells and the clinical characterizations of cervical cancer was analyzed. The inhibition effect of Breg cells on CD8+ T cells was tested by blocking IL-10 in vitro. The percentage of CD19+CD5+CD1d+ Breg cells and the level of IL-10 of patients with cervical cancer or CIN were significantly higher than those in the control group (P < 0.05). And the postoperative levels of Breg cells and IL-10 were significantly lower than the preoperative levels (P < 0.05). Breg cells and the IL-10 level were positively correlated in cervical cancer patients (r = 0.516). In addition, the Breg cell percentage was closely related to the FIGO stages, lymph node metastasis, tumor differentiation, HPV infection, and the tumor metastasis of cervical cancer (P < 0.05). The Breg cell percentage was negatively correlated with CD8+ T cells of cervical cancer patients (r = -0.669). The level of IL-10 in the culture supernatant of Bregs treated with CpG was significantly higher than that of non-Bregs (P < 0.05). After coculture with Bregs, the quantity of CD8+ T cells to secrete perforin and Granzyme B was significantly decreased, and this effect was reversed after blocking IL-10 by a specific antibody. Breg cells are elevated in cervical cancer and associated with disease progression and metastasis. Moreover, they can inhibit the cytotoxicity of CD8+ T cells.

CD5+ B lymphocytes secrete IL-10 rather than TGF-ß1 which control the immune response in autoimmune haemolytic anaemia/Evans syndrome.

OBJECTIVE: To investigate the quantity and secretion function of cytokines-secreted CD5+ B lymphocytes in Autoimmune Haemolytic Anaemia (AIHA)/Evans syndrome (ES) patients. METHODS: Twenty-five untreated AIHA/ES patients, 28 remission AIHA/ES patients and 25 healthy controls (HCs) were enrolled in this study. The quantity of CD5+B lymphocytes which produce interleukin-10 (IL-10) (CD5+IL-10+) and transforming growth factor (TGF-ß1) (CD5+TGF-ß1+) were detected by flow cytometry (FCM). CD5+ B lymphocytes were sorted from peripheral blood (PB) by FCM and the expression of IL-10 and TGF-ß1 mRNA in CD5+ B cells were measured by real-time PCR (RT-PCR). RESULTS: The percentage of CD5+IL-10+B cells in CD5+ B lymphocytes in newly diagnosed patients was 82.18 ± 14.78%, which being significantly higher than that of remission AIHA/ES patients (56.68 ± 24.39%) and HCs (51.90 ± 22.95%) (p < .05). The percentage of CD5+IL-10+ B cells in CD5+ B lymphocytes in newly diagnosed patients was negatively correlated with haemoglobin (Hb), complement 3 (C3) (p < .05) and positively correlated with lactate dehydrogenase (LDH), total bilirubin (TBIL) and indirect unconjugated bilirubin (IBIL) (p < .05). The expression level of IL-10 mRNA in CD5+ B lymphocytes of newly diagnosed patients (49.34 ± 22.84) was higher than that of remission patients (3.97 ± 3.83) and HCs (1.78 ± 1.66) (p < .05). There was no significant difference among three groups with the proportion of CD5+TGF-ß1+ B lymphocytes and the expression level of TGF-ß1 mRNA in CD5+B lymphocytes (p > .05). CONCLUSIONS: CD5+ B lymphocytes could secrete IL-10 rather than TGF- ß1 which control the immune response in AIHA/ES.

TLR2 Expression on Leukemic B Cells from Patients with Chronic Lymphocytic Leukemia.

Antigenic stimulation is considered as a possible trigger of neoplastic transformation in chronic lymphocytic leukemia (CLL). B-cell receptor plays a key role in the interactions between the microenvironment and leukemic cells; however, an important role has also been attributed to Toll-like receptors (TLRs). It is believed that disorders of TLR expression may play a part in the pathogenesis of CLL. In this study, we investigated the potential role of TLR2 in CLL by analyzing its expression on leukemic B cells in correlation with clinical and laboratory parameters characterizing disease activity and patients' immune status. We assessed the frequencies of TLR2+/CD19+ cells by the flow cytometry method in peripheral blood of 119 patients with CLL. The percentage of TLR2+/CD19+ cells was significantly lower in patients with CLL as compared to the healthy volunteers. There was also a lower percentage of TLR2+/CD19+ cells in CLL patients with poor prognostic factors, such as ZAP70 and/or CD38 expression, 17p and/or 11q deletion. On the other hand, among patients with del(13q14) associated with favorable prognosis, the percentage of TLR2+/CD19+ cells was higher than among those with del(11q22) and/or del(17p13) as well as in the control group. We found an association between low percentage of CD19+/CD5+/TLR2+ cells and shorter time to treatment. We also demonstrated the relationship between low percentage of CD19+/CD5+ TLR2-positive and overall survival (OS) of CLL patients. CLL patients with a proportion of 1.6% TLR2-positive B CD5+ cells (according to the receiver operating characteristic curve analysis) or more had a longer time to treatment and longer OS than the group with a lower percentage of TLR2 positive cells. To sum up, the results of the study suggest that low TLR2 expression is associated with poor prognosis in CLL patients. The monitoring of CD19+/CD5+/TLR2+ cells number may provide useful information on disease activity. Level of TLR2 expression on leukemic B cells may be an important factor of immunological dysfunction for patients with CLL. Our study suggests that TLR2 could becomes potential biological markers for the clinical outcome in patients with CLL.

Increased Circulating Th17 but Decreased CD4+Foxp3+ Treg and CD19+CD1dhiCD5+ Breg Subsets in New-Onset Graves' Disease.

Th17 and regulatory lymphocyte subsets such as Tregs and Bregs have been reported to play important roles in autoimmune diseases. The aim of this work was to perform quantitative studies of circulating Th17, Tregs, and Bregs in patients with new-onset Graves' disease (GD). Twenty GD patients and 20 healthy controls were involved in this study. Blood samples were taken for flow cytometry detection of CD4+IL-17+ Th17, CD4+Foxp3+ Tregs, and CD19+CD1dhiCD5+ Bregs and meanwhile, for real-time PCR measurement of gene expressions of RORγt, IL-17 and IL-10. The proportions of Tregs and Bregs as well as the Foxp3 gene expression but not IL-10 were significantly decreased in GD group compared with the healthy controls. The frequency of Th17 together with the gene expressions of RORγt and IL-17 were significantly increased in the GD group. Furthermore, the Th17/Treg ratio was also significantly higher in GD group. A significant positive correlation between Th17 and TSAb (r = 0.656, p < 0.001) but significant negative correlations between Treg/Breg and TSAb (r = -0.339, p = 0.032; r = -0.759, p < 0.001) were identified among the participants. This study indicated that increased Th17 and impaired Treg responses, along with a decreased number of CD19+CD1dhiCD5+ Breg cells, were involved in GD pathogenesis.

Diagnosis of CLL revisited: increased specificity by a modified five-marker scoring system including CD200.

The modified Matutes score has been the basis for the diagnosis of chronic lymphocytic leukaemia (CLL) by flow cytometry for the past 15 years. To increase the specificity of the current score we systematically evaluated the diagnostic value of established as well as novel markers, such as CD200, in a large cohort of patients with untreated B-cell malignancies (n = 370). Double positivity for CD5 and CD23 was of very high value to differentiate between CLL and non-CLL cases. In addition, lack of FMC7 expression as well as CD79b expression intensity showed high sensitivity (90·4% and 92·3%) with acceptable specificity (74·4% and 76·9%). For surface IgM, low or absent expression displayed poor specificity in distinguishing CLL from non-CLL cases (51,3%; sensitivity 83,7%). Finally, CD200 positivity showed high sensitivity and specificity. Therefore, CD5/CD23, FMC7, CD79b and CD200 were included in our new CLLflow score, which retained high sensitivity (97·1% vs. 98·6% for the Matutes score, P = 0·38), but showed markedly increased specificity (87·2% vs. 53·8%, P < 0·001). These results were confirmed in our validation cohort (sensitivity 97·0% vs. 100%, P = not applicable; specificity 86·4% vs. 59·1%, P = 0·03). Our data support the use of our new CLLflow score for the diagnosis of CLL with significantly higher specificity.

Clinical and Laboratory Features of CD5-Negative Chronic Lymphocytic Leukemia.

BACKGROUND Chronic lymphocytic leukemia (CLL) usually expresses CD5 antigen. However, 7-20% of patients are CD5 negative. We report here a series of 19 CD5-negative B-CLL cases. MATERIAL AND METHODS We reviewed 19 consecutive CD5-negative B-CLL cases seen in our medical center from 2009 to 2015 and compared them with 105 CD5-positive B-CLL cases. The two groups were compared in terms of clinical parameters, laboratory parameters, and survival characteristics. RESULTS Lymphadenopathy was present in 31.5% of the CD5-negative group and 51.4% of the CD5-positive group (p=0.029). Splenomegaly was present in 42.1% of the CD5-negative group and 16.1% of the CD5-positive group (p=0.029). There was no difference between the groups in terms of Binet A, B, and C stages (p=0.118, p=0.051, and p=0.882, respectively). The median thrombocyte count was 144×109/L and 160×109/L in the CD5-negative and CD5-positive groups, respectively (p=0.044). There was no difference between the two groups in terms of median neutrophil count (p=0.169). The mean lymphocyte count was 43.2±4.0×10^9/L and 36.7±3.2×10^9/L in the CD5-negative and CD5-positive groups, respectively (p=0.001). There was no difference between the groups in terms of autoimmune hemolytic anemia and autoimmune thrombocytopenia. In five-year follow-up, 84.2% of CD5-negative patients and 90.5% of CD5-positive patients were alive (p=0.393). CONCLUSIONS We found more isolated splenomegaly, less lymphadenopathy, a higher lymphocyte count, and a lower thrombocyte count in the CD5-negative group. There was no difference between the groups in terms of clinical stage, autoimmune phenomena, hemoglobin and neutrophil count, and survival.

Cluster of Differentiation 5 (CD5) Levels in the Plasma of Children with Autism Spectrum Disorder (ASD).

OBJECTIVE: To investigate the cluster of differentiation 5 (CD5) plasma levels and their association with childhood autism rating scale (CARS) in subjects with autism spectrum disorder (ASD) compared to age and gender matched healthy controls, and to explore the link between CD5, severity, and autoimmunity in autism. STUDY DESIGN: Case-control study. PLACE AND DURATION OF STUDY: Autism Research and Treatment Center, Al-Amodi Autism Research Chair, Department of Physiology, Faculty of Medicine, King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia, from October 2014 to May 2015. METHODOLOGY: CD5 levels were determined in the plasma of thirty-one (31) patients using enzyme-linked immunosorbent assay (ELISA), categorized as mild-moderate and severe as indicated by their Childhood Autism Rating Scale (CARS) score and compared to thirty-three (33) age and gender-matched control samples. RESULTS: The preliminary data indicated that children with severe autism (n=12), exhibited significantly (p=0.02) higher plasma level of CD5 [0.55 (0.14-12) pg/ml {median (interquartile range=IQR)}] than those of normal controls [n=33, 0.29 (0.08-0.79) pg/ml {median (IQR)}] and children with mild to moderate autism [n=19, 0.26 (0.13-1.42) pg/ml, {median (IQR)}, p=0.08]. However, there was no significant difference between the CD5 levels of children with mild to moderate autism and normal controls (p = 0.62). Diagnoses of autistic children based on the CARS score >30. Disease severity and the CARS score, which represent stereotyped patterns of behavior in children with autism, were positively correlated (r = 0.43, p = 0.02). CONCLUSION: The high CD5 plasma levels in patients with severe ASD, probably indicated that CD5 might be implicated in the physiology of autism. However, this finding should be treated with caution until further investigations are performed with larger populations to determine whether the increase in plasma CD5 levels is a mere consequence of autism or it plays a pathogenic role in the disease.

CD5 molecule-like and transthyretin as putative biomarkers of chronic myeloid leukemia - an insight from the proteomic analysis of human plasma.

Better and sensitive biomarkers are needed to help understand the mechanism of disease onset, progression, prognosis and monitoring of the therapeutic response. Aim of this study was to identify the candidate circulating markers of chronic-phase chronic myeloid leukemia (CP-CML) manifestations, having potential to develop into predictive- or monitoring-biomarkers. A proteomic approach, two-dimensional gel electrophoresis in conjunction with mass spectrometry (2DE-MS), was employed for this purpose. Based on the spot intensity measurements, six proteins were found to be consistently dysregulated in CP-CML subjects compared to the healthy controls [false discovery rate (FDR) threshold ≤0.05]. These were identified as α-1-antichymotrypsin, α-1-antitrypsin, CD5 molecule-like, stress-induced phosphoprotein 1, vitamin D binding protein isoform 1 and transthyretin by MS analysis [PMF score ≥79; data accessible via ProteomeXchange with identifier PXD002757]. Quantitative ELISA, used for validation of candidate proteins both in the pre-treated and nilotinib-treated CP-CML cases, demonstrate that CD5 molecule-like, transthyretin and alpha-1-antitrypsin may serve as useful predictive markers and aid in monitoring the response of TKI-based therapy (ANOVA p < 0.0001). Two of the circulating marker proteins, identified in this study, had not previously been associated with chronic- or acute-phase myeloid leukemia. Exploration of their probable association with CP-CML, in a larger study cohort, may add to our understanding of the disease mechanism besides developing clinically useful biomarkers in future.

Serum progranulin irrelated with Breg cell levels, but elevated in RA patients, reflecting high disease activity.

Soluble progranulin (PGRN) is known to directly regulate regulatory T cells; however, whether PGRN levels are elevated in patients with rheumatoid arthritis and affect the regulatory subsets of B cells remain unknown. In this study, a total of 80 RA patients and 60 healthy controls were studied. Serum progranulin levels were determined using enzyme-linked immune-sorbent assay. A receiver operating characteristic (ROC) curve was used to evaluate the feasibility of serum PGRN as a biomarker for distinguishing patients with RA. CD19(+)CD5(+)GrB(+) B cells were analyzed by flow cytometry in peripheral blood mononuclear cells (PBMCs). Serum progranulin levels in RA patients (median, 59.4 ng/mL) and in RA patients DAS28 > 5.1 (median, 71.98 ng/mL) were much higher than those in normal controls (median, 46.3 ng/mL; P < 0.001). The area under the ROC curve for progranulin levels was 0.705 for RA versus normal controls and the area under the ROC curve for progranulin levels in RA patients DAS28 > 5.1 was 0.977 versus normal controls (P < 0.001). Interestingly, serum progranulin and DAS28, CRP, ESR were all positively correlated in RA patients (P < 0.001). The number of CD19(+)CD5(+)GrB(+) B cells was significantly higher in RA patients (P < 0.05); however, the level of Breg cells was not related to PGRN (P > 0.05). Our findings indicated that induction of PGRN expression may play a role in RA immune reaction and PGRN levels could be a useful biomarker in RA inflammatory response, but irrelated with Breg cell levels.

Proteomic Analysis of Plasma in Adult Active Pulmonary Tuberculosis Patients with Diabetes Mellitus.

BACKGROUND: There is a high burden of both diabetes and tuberculosis in China. Diabetes depresses the immunologic response that facilitates the development of infectious diseases, including infection by Mycobacterium tuberculosis, the agent of tuberculosis. Tuberculosis is the third cause of death among subjects with non-communicable diseases, and among the non-communicable diseases, diabetes is one of the most important. The relationship between diabetes and tuberculosis has already been object of many investigations but the association between these two diseases is not fully understood. The aim of this study was to determine whether relative qualitative and quantitative differences in protein expression of plasma could be related to active pulmonary tuberculosis complicated with diabetes. METHODS: Biological parameters are useful tools for understanding and monitoring complicated disease processes. Our study employed two-dimensional gel electrophoresis and mass spectrometry to analyze the proteins associated with active pulmonary tuberculosis complicated with diabetes. RESULTS: Under the baseline condition, we found that the levels of α-1 antitrypsin precursor, vitamin D-binding protein precursor, CD5 antigen like precursor, clusterin precursor, apolipoprotein A-I precursor, haptoglobin, and fibrinogen γ-chain differed between patients with active pulmonary tuberculosis and active pulmonary tuberculosis complicated with diabetes subjects. Western blotting results confirmed differential expression of clusterin. CONCLUSIONS: We identified active pulmonary tuberculosis complicated with diabetes-associated proteins in plasma. C-terminal haptoglobin is a possible candidate protein of interest, which might be a link between active pulmonary tuberculosis and diabetes. The dynamics of protein expression during disease progression may improve our understanding of the pathogenesis of active pulmonary tuberculosis complicated with diabetes.

Anti-Tumor Necrosis Factor Therapy Restores Peripheral Blood B-cell Subsets and CD40 Expression in Inflammatory Bowel Diseases.

BACKGROUND: Anti-tumor necrosis factor (TNF) therapy has become a standard therapy for severe inflammatory bowel diseases (IBD), but its effect on B lymphocytes is largely unexplored. In this study we investigated peripheral blood B cells, B-cell subsets, and CD40 expression in patients with IBD before and during anti-TNF therapy with infliximab (IFX). METHODS: Blood was taken from healthy controls (n = 52) and patients with active IBD before (n = 46) and/or during anti-TNF therapy (n = 55). B-cell markers were detected by immunofluorescent staining and FACS analysis. Patients were classified as responders or nonresponders to anti-TNF therapy. RESULTS: We found a numerical deficiency of circulating CD19 B cells, a lower activation state (CD40 expression) and lower proportions of CD5 B cells and IgMIgDCD27 preswitched memory cells among B cells in active patients with IBD before IFX therapy compared with healthy controls. IFX treatment increased CD19 B-cell numbers as well as the proportions of named B-cell subsets in responders but not in nonresponders. IFX more effectively upregulated CD40 expression in responders than in nonresponders. Restoration of B cells correlated with the biological response to therapy (C-reactive protein). Trough serum levels of IFX correlated with the number of B cells during therapy. CONCLUSIONS: A lower number of circulating B cells, a low CD40 expression, and a decrease in the proportion of CD5 and in the preswitched memory subset characterize active IBD. Restoration of these abnormalities correlates with the clinical response to anti-TNF therapy. The mechanism for this effect on B cells should be further explored.

Higher B-cell activating factor levels at birth are positively associated with maternal dairy farm exposure and negatively related to allergy development.

BACKGROUND: A high proportion of circulating immature/naive CD5(+) B cells during early infancy is a risk factor for allergy development. B-cell activating factor (BAFF) is an important cytokine for B-cell maturation. OBJECTIVE: We sought to investigate whether BAFF levels are related to environmental exposures during pregnancy and early childhood and whether BAFF levels are associated with postnatal B-cell maturation and allergic disease. METHODS: In the FARMFLORA study, including both farming and nonfarming families, we measured BAFF levels in plasma from mothers and their children at birth and at 1, 4, 18, and 36 months of age. Infants' blood samples were also analyzed for B-cell numbers and proportions of CD5(+) and CD27(+) B cells. Allergic disease was clinically evaluated at 18 and 36 months of age. RESULTS: Circulating BAFF levels were maximal at birth, and farmers' children had higher BAFF levels than nonfarmers' children. Higher BAFF levels at birth were positively associated with proportions of CD27(+) memory B cells among farmers' children and inversely related to proportions of CD5(+) immature/naive B cells among nonfarmers' children. Children with allergic disease at 18 months of age had lower cord blood BAFF levels than nonallergic children. At birth, girls had higher BAFF levels and lower proportions of CD5(+) B cells than boys. CONCLUSIONS: Farm exposure during pregnancy appears to induce BAFF production in the newborn child, and high neonatal BAFF levels were associated with more accelerated postnatal B-cell maturation, which lend further strength to the role of B cells in the hygiene hypothesis.

Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production.

CD5 negative mantle cell lymphoma: a different clinical entity?

We report herein a 77-year-old patient with CD5 negative mantle cell lymphoma (MCL). We further review the existing literature on clinicolaboratory features of this rare MCL subtype. Although most of the patients in the literature (including ours) had advanced stage at diagnosis, splenomegaly, and bone marrow involvement, they displayed prompt and durable responses to conventional treatment. We postulate that CD5 surface antigen expression could have prognostic implications in MCL. Further research and a larger number of patients are necessary in order to validate these findings.

Serum proteins in chronic hepatitis B patients treated with peginterferon alfa-2b.

AIM: To study the differential protein profile in serum of hepatitis B patients. METHODS: Serum samples were obtained from patients with chronic hepatitis B who were receiving peginterferon alfa-2b. The serum samples were subjected to albumin depletion and analyzed by two-dimensional gel electrophoresis (2-DE). Differentially expressed protein spots were identified by electrospray ionization-quadrupole time-of-flight mass spectrometry. Alpha-2-HS-glycoprotein, complement component C3c and CD5 antigen were further analyzed by an enzyme-linked immunosorbent assay and immunonephelometry. RESULTS: Nineteen patients with HBeAg-positive chronic hepatitis B (CHB) were studied. These patients were followed for at least 1 year after treatment and were classified according to their treatment response: responders (n = 9) and non-responders (n = 10). 2-DE and MS/MS analysis were performed to compare the serum proteins before initiating peginterferon alfa-2b. From the quantitative analysis of the 2-D gel, 7 proteins were detected between the two groups at different levels before treatment. Among these potential candidates, serum levels of alpha-2-HS-glycoprotein, complement component C3c and CD5 antigen-like precursor were further analyzed. In the validation phase, 23 subjects, 9 sustained responders and 14 non-responders, were recruited. Interestingly, the levels of alpha-2-HS-glycoprotein and complement component C3c were elevated in the serum of the non-responders compared to the responders. CONCLUSION: Serum alpha-2-HS-glycoprotein and complement component C3c may be potential serum biomarkers in predicting the treatment response of peginterferon alfa-2b in patients with CHB prior to treatment.

Chronic lymphocytic leukemia in Korean patients: frequent atypical immunophenotype and relatively aggressive clinical behavior.

Chronic lymphocytic leukemia (CLL) is a mature B-cell neoplasm characterized by the expansion of CD5-positive lymphocytes in peripheral blood. While CLL is the most common type of leukemia in Western populations, the disease is rare in Asians. Hence, clinical and laboratory data and studies of CLL in Asian populations have been limited. In this study, we investigated the clinical and laboratory characteristics of CLL in Korea. A total of 39 patients who had been diagnosed with CLL during the period from January 2000 to October 2010 at a single institution in Korea were examined. Clinically, 67 % of the patients were classified as having advanced Binet stages B or C. Up to 56 % of the patients had an atypical immunophenotype with high frequencies of FMC7 positivity and strong CD22 positivity. Twenty-six patients (67 %) received chemotherapy, and more than half of the treated patients (54 %) expired. The overall survival rate at 5 years was estimated at 71 %, which was lower than previously reported. These findings suggested that CLL in Korea has atypical immunophenotypes and that its clinical behavior may be more aggressive than that in Western populations.