Shanhua Zou

The Ratio of Treg/Th17 Cells Correlates with the Disease Activity of Primary Immune Thrombocytopenia

Background Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that is characterized by decreased platelet count. Regulatory T (Treg) cells and T helper type 17 (Th17) cells are two subtypes of CD4+ T helper (Th) cells. They play opposite roles in immune tolerance and autoimmune diseases, while they share a common differentiation pathway. The imbalance of Treg/Th17 has been demonstrated in several autoimmune diseases. In this study, we aimed to investigate the ratio of the number of Treg cells to the number of Th17 cells in ITP patients and evaluate the clinical implications of the alterations in this ratio. Methods Thirty adult patients with newly diagnosed ITP enrolled in this study. Twelve patients had been clinically followed up for 12 months. The percentages of CD4+CD25hiFoxp3+ Treg cells and CD3+CD4+IL-17-producing Th17 cells in these patients and healthy controls (n = 17) were longitudinally analyzed by flow cytometry. Results The percentage of Treg cells in ITP patients was significantly lower than that of healthy controls, and the percentage of Th17 cells increased significantly at disease onset. The ratio of Treg/Th17 correlated with the disease activity. Conclusion The ratio of Treg/Th17 might be relevant to the clinical diversity of ITP patients, and this Treg/Th17 ratio might have prognostic role in ITP patients.

High-dose dexamethasone modulates serum cytokine profile in patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder which is characterized by decreased platelet count. Serum cytokines play an important role in the pathogenesis of ITP by initiating and perpetuating various cellular and humoral autoimmune processes. To investigate a broad spectrum of cytokines in ITP patients and the effects of high-dose dexamethasone (HD-DXM) regimen on serum cytokines profile, a multiplex cytokine assay was used to measure the serum levels of 20 circulating cytokines simultaneously in 22 patients before and after oral administration of 40mg/day DXM for 4 consecutive days. A cohort of 10 healthy individuals was served as control. Serum levels of interleukin (IL)-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, growth-related oncogene (GRO), interferen (IFN)-γ and tumor necrosis factor (TNF)-α were significantly decreased in pre-treatment patients, compared with healthy controls (p<0.05). After HD-DXM treatment, IL-4, IL-5, IL-6, IL-12p70, IL-13, GRO, IFN-γ and TNF-α were significantly increased in remission patients as compared with patients before treatment (p<0.05). However, there was no significant difference (except TNF-α) between remission patients and healthy controls (p>0.05). All these cytokines decreased again in relapse patients. Our findings suggest that measuring cytokine levels might help in the clinical assessment of ITP, and the HD-DXM therapy could correct the derangement of serum cytokines.

Aberrant Frequency of IL-10-Producing B Cells and Its Association with Treg/Th17 in Adult Primary Immune Thrombocytopenia Patients

Background. Regulatory B cells (Breg) are a distinct B cell subset with immunoregulatory properties. Pivotal to Breg function is interleukin-10. This study was to investigate the role of IL-10-producing B cell (B10) and its association with Treg and Th17 subsets in immune thrombocytopenia (ITP) patients. Methods. Peripheral blood mononuclear cells from ITP patients and controls were stimulated with PMA, ionomycin, and Brefeldin A. The frequencies of CD19+IL-10+ B cells, CD3+CD4+IL-17+ Th17 cells, and CD4+CD25hiFoxp3+ Treg cells were analyzed by flow cytometry. The mRNA expression of Foxp3 and RORγt was detected by real-time quantitative PCR. Results. The number of B10 cells was elevated in ITP patients. After first-line therapies, it remained at high level in patients who achieved complete or partial response but decreased in those who acquired no response. There was a positive correlation between B10 cells and Tregs in ITP both before and after therapies. The ratio of Treg/Th17 decreased in ITP, and it strongly correlated with B10 cells. Conclusions. The frequency of B10 cells is elevated in ITP and it correlates with both the Tregs counts and the Treg/Th17 ratio. B10 cells to regulate functional T cell subsets might be impaired in patients with ITP.

Abnormal platelet kinetics are detected before the occurrence of thrombocytopaenia in HBV-related liver disease

Thrombocytopaenia is a frequent feature in patients with HBV‐related liver disease. Its underlying mechanism is not fully understood. Multiple factors might contribute to the development of thrombocytopaenia. In this study, we investigated the reticulated platelets (RP), glycocalicin (GC), serum thrombopoietin (TPO) and platelet glycoprotein (GP) in different stages of the disease.

The expression profile of toll-like receptor signaling molecules in CD19+ B cells from patients with primary immune thrombocytopenia

BACKGROUND B cells play a critical role in the pathogenesis of immune thrombocytopenia (ITP), and toll-like receptor (TLR) signaling is essential for the activation of autoreactive B cells. The objective of this study was to investigate the expression profile of TLR signaling molecules in circulating and splenic CD19(+) B cells isolated from ITP patients. METHODS CD19(+) B cells were magnetically isolated from peripheral blood and splenocytes. Human Toll-Like Receptor Signaling Pathway RT(2) Profiler™ PCR Array was used to determine the differences in mRNA expression of 84 TLR signaling pathway genes between ITP patients and controls. Flow cytometry was used to investigate intracellular expression of cytokines (IL-1β and IL-10). RESULTS A total of 31 genes involving TLR signaling pathways were differentially transcribed in circulating CD19(+) B cells, among which 27 were up-regulated in ITP. By comparison, differentially transcribed genes amounted to 39 in splenic B cells in ITP, among which only two were down-regulated. Up to 18 TLR signaling molecules exhibited up-regulated transcriptional levels both in splenic B cells and in circulating B cells in ITP. However, Only IL-10 and IL-1β were significantly upregulated in both the circulating and splenic B cells of patients with ITP. Intracellular staining of IL-10 and IL-1β confirmed the results of PCR Array. CONCLUSIONS The expression of TLRs and downstream cytokines, including IL-10 and IL-1β, is up-regulated in circulating and splenic B cells in ITP patients, suggesting that activated TLR signaling pathways in B cells may play dual roles in the pathophysiology of primary ITP.

Membranoproliferative glomerulonephritis associated with IgA-λ monoclonal gammopathy.

Dear Editor, From September 2008 to April 2013, 4 patients were found to have amonoclonal IgA-λ component by serum immunofixation electrophoresis among 29 membranoproliferative glomerulonephritis (MPGN) patients, pathologically confirmed via renal biopsy during the same period in our facility (Table 1). All patients were male, and the median age was 53.0(45–65) years. The severity of proteinuria and renal insufficiency varied among patients. The extent of plasma cell dyscrasia was categorized as monoclonal gammopathy of undetermined significance (MGUS) in three patients and POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes) in the other. The diagnosis of MPGN was made via light microscopic observations, and interestingly, no IgA or λ light chain deposit was detected by immunofluorescence surveys. Electron microscopy revealed thickening of basement membrane (inner layer in particular), cellular interposition, diffuse effacement of the foot processes of podocytes, and scattered deposits in subendothelial and mesangial regions (Fig. 1). Patient 1 was treated with prednisone and cyclophosphamide, and proteinuria decreased from 1.86 to 0.34 g/24 h at 3year follow-up. Patient 2 was treated with dexamethasone and thalidomide, but proteinuria increased from 0.24 to 0.81 g/24 h after 1 year. Patients 3 and 4 did not get any specific treatment. The serum creatinine level was stable in all patients during follow-up (0.72– 1.22 mg/dl). The spectrum of renal involvement secondary to plasma cell diseases have been drastically expanded recently. Aside from previously acknowledged entities, including myeloma cast nephropathy, AL amyloidosis, monoclonal immunoglobulin deposition disease, and light chain proximal tubulopathy, the territory of primary glomerulonephritis has also been shattered [1, 2]. Proliferative glomerulonephritis secondary to monoclonal IgG, IgA, or immunoglobulin light chain deposits has been well-described since 2004 [3–5]. The most recent candidate for this heterogeneous group was proposed by Sethi et al. in 2010. In a single-center study, they identified 28 monoclonal/biclonal Igs among total 126 MPGN patients, in which 68 cases were hepatitis negative and evaluated for gammopathy [6]. IgM-κ and IgG-κ were most commonly encountered, and more than half of the patients were categorized as MGUS. Under these circumstances, a recent perspective article in Blood proposed the terminology “monoclonal gammopathy of renal significance (MGRS)” [2]. Here, we presented the first report of MPGN associated with IgA-λ monoclonal gammopathy. Along with the other two patients with IgM-κ and IgG-κ paraproteins, the overall occurrence of monoclonal gammopathy among MPGN B. Wu :W. Wang : F. Li (*) : S. Zou :Y. Cheng Department of Hematology, Zhongshan Hospital Fudan University, 180 Fenglin Road, Shanghai 200032, China e-mail: li.feng@zs-hospital.sh.cn

IgA monoclonal gammopathy accompanying extranodal B cell lymphomas.

Dear Editor, From January 2008 to April 2013, seven patients with pathologically confirmed B cell lymphomas (three diffuse large B cell lymphomas, two mucosa-associated lymphoid tissue lymphomas, one follicular lymphoma, and one mixed cellularity classic Hodgkin’s lymphoma) were found to have monoclonal IgA component by serum immunofixation electrophoresis (SIFE) in our facility (Table 1); meanwhile, 33 monoclonal IgGs and 88 IgMs were detected by SIFE among all B cell lymphomas during the same time period. All patients were male, and the median age was 57.0 (48–71) years. A preference for λ light chains was observed, but the two patients with κ light chain seemed to have much higher serum IgA level [1,170 (681–1,660) mg/dl vs. 380 (294– 552) mg/dl]. Both serum IgG [845 (270–1,424) mg/dl] and IgM [63 (4–182) mg/dl] levels were decreased to variable extents. No overt plasmacytosis or plasma cell dyscrasia was found by bone marrow smear or biopsy. After R-CHOPbased chemotherapeutic regimens, the monoclonal IgA disappeared in six patients who achieved remission. Aside from two patients (patients 1 and 2) with relapsed diseases, all the newly onset cases presented as focal lesions of extranodal origin (stomach, jejunum, colon, lung, and prostate). Monoclonal gammopathy is not a rare phenomenon among B cell lymphomas. Patients with chronic lymphocytic leukemia and lymphoplasmacytoid lymphoma demonstrate higher incidence of accompanying monoclonal gammopathy, and an IgM component is predominant [1, 2]. On the other hand, monoclonal IgG seems to be more frequently found among FL and DLBCL patients, while the preference of monoclonal Ig type varies among studies concerning MALT lymphomas [3–5]. However, IgA monoclonal gammopathy is the rarest under all these circumstances and comprises lesser than 10 % of all cases with monoclonal gammopathy [3, 4]. In the case of MALT lymphomas, monoclonal gammopathy was believed to correlate with advanced stage, marrow involvement, and poor clinical outcome [5]. Unexpectedly, the above correlation was not observed among MALT lymphoma and DLBCL cases in the present study, which unexceptionally were assessed to be stages I–II extranodal diseases without overt bone marrow involvement. The most intriguing part of our study was the astounding correlation between IgA monoclonal gammopathy and extranodal lesions. Gastrointestinal B cell lymphomas, especially MALT cases, have been argued to have a chronic mucosa inflammation stage which allows lymphocyte infiltration, proliferation, and malignant conversion [6]. Since secretory IgA is crucial in the humoral defense against microorganism in digestive, respiratory, and genitourinary tract [7], it is logical to expect a higher incidence of lymphoid malignancies originating from IgA-secreting lymphocytes in associated tissues including stomach, small intestine, colon, lung, and prostate in our study. Certain microbes as Helicobacter pylori are strongly related to the pathogenesis of gastric MALT lymphomas, therefore further studies upon extranodal B cell lympohmas, especially IgA-secreting ones, might help to discern other potential pathogenic factors among these clinically distinctive disease entities. B. Wu :W. Wang : F. Li (*) : S. Zou :Y. Cheng Department of Hematology, Zhongshan Hospital Fudan University, 180 Fenglin Road, Shanghai 200032, China e-mail: li.feng@zs-hospital.sh.cn

MLL-AF9 rearrangement in myeloid sarcomas involving the breast.

Dear Editor, A 29-year-old woman (patient 1) presented to our facility in August 2011 for migrating arthralgia at bilateral elbow and knee joints. CT scan revealed coarse articular surface of bilateral sacroiliac joints with slight irregularity. Serological tests for autoantibodies were all negative. Three months later, palpable masses were sequentially reported at the left and right breast. Fine needle biopsy of the left breast indicated malignancy with hematopoietic origin. Bone marrow aspiration revealed acute myeloid leukemia with FAB AML-M4 morphology. Flow cytometric study showed 21 % malignant cells of marrow sample, which had an expression of surface markers including CD117, CD33, CD38, CD15, CD64, CD4, and CD56. Malignant cells possessed a recurrent cytogenetic aberration shown by G-banding as 47, XX, + 8, t(9;11)(p22;q23). Further molecular surveys confirmed the existence of an MLL-AF9 fusion gene. Patient 2 was a 28year-old woman who presented to our facility in October 2012 because of a palpable mass at left breast for 4 months. Fine needle biopsy indicated malignancy with hematopoietic origin. Bone marrow aspiration revealed acute myeloid leukemia with FAB AML-M4 morphology. Flow cytometric study showed 76 % malignant cells of marrow sample, which had an expression of surface markers including CD117, CD13, CD33, CD34, CD38, MPO, and HLA-DR. No overt cytogenetic aberration was shown by G-band karyotyping, but further molecular surveys disclosed the existence of an MLL-AF9 fusion gene. Mutational surveys in both patients revealed no acute myeloid leukemia (AML)-related mutations involving FLT3, NPM1, CEBPA, or C-KIT. They achieved hematological complete remission after two courses of chemotherapy utilizing idarubicin and cytarabine regimen and underwent consolidation with mid-dose cytarabine. Myeloid sarcoma (MS), previously referred to as granulocytic sarcoma or chloroma, is a rare entity of AML [1]. It represented a mixed group of AMLs with overt extramedullary involvement including the skin, lymph node, bone, testis, central nervous system, intestine, and other soft tissues. Breast involvements, even solitary extramedullary cases without evidence of bone marrow lesion, have been well described [2–4]. The presentation of accompanying AML varied among cases, which could occur prior to, simultaneous with, or months later than the diagnosis of myeloid sarcoma [1–6]. Cytogenetic data of MS are relatively limited, partly due to operational difficulties during sampling and banding procedures. Certain cytogenetic aberrations as t(8:21) were related to pediatric patients, especially those with lesions at orbital level [5]. On the other hand, it was reported by Pileri et al. that MLL rearrangement, trisomy 8, and monosomy 7 occurred more frequently among adult patients [6]. Interestingly, both cases of breast involvement with confirmed cytogenetic aberrations reported by Pileri et al. demonstrated trisomy 8, one of which might also harbored MLLAF9 rearrangement (Table 1). Although chromosomal abnormalities including trisomy 8 and t(9;11) have been associated with MS, the possible link between MLL-AF9 and MS has not been frequently reported [7, 8]. MLL-AF9 rearrangement is found in 2–5 % of AML and up to 25 % in de novo M5a of children, while trisomy 8 appears as additional cytogenetic abnormality in 20% of cases harboring MLL-AF9 [9]. MLL-AF9, as well as additional trisomy 8, seemed to demonstrate a strikingly high prevalence among MS patients with breast involvement, but its prognostic value could not be adequately evaluated in this small cohort. Contemporary molecular surveys might help to B. Wu : F. Li (*) : S. Zou Department of Hematology, Zhongshan Hospital Fudan University, 180 Fenglin Road, Shanghai 200032, China e-mail: li.feng@zs-hospital.sh.cn