A case of Alemtuzumab-induced neutropenia in multiple sclerosis in association with the expansion of large granular lymphocytes.

BACKGROUND: Alemtuzumab has been demonstrated to reduce the risks of relapse and accumulation of sustained disability in Multiple Sclerosis (MS) patients compared to ß-interferon. It acts against CD52, leading primarily to lymphopenia. Recent data have shown that mild neutropenia is observed in 16% of treated MS-patients whereas severe neutropenia occurred in 0.6%. CASE PRESENTATION: Herein, we present the case of a 34-year-old woman with relapsing-remitting MS, with a history of treatment with glatiramer acetate and natalizumab, who subsequently received Alemtuzumab (12 mg / 24 h × 5 days). 70-days after the last Alemtuzumab administration, the patient displayed neutropenia (500 neutrophils/µL) with virtual absence of B-cells (0.6% of total lymphocytes), low values of CD4-T-cells (6.6%) and predominance of CD8-T-cells (48%) and NK-cells (47%); while large granular lymphocytes (LGL) predominated in the blood-smear examination. Due to prolonged neutropenia (5-days) the patient was placed on low-dose corticosteroids leading to sustained remission. CONCLUSION: This is the first case of a patient with relapsing-remitting MS with neutropenia two months post-Alemtuzumab, with simultaneous presence of LGL cells in the blood and a robust therapeutic response to prednisolone. We recommend testing with a complete blood count every 15 days in the first 3 months after the 1st Alemtuzumab administration and searching for large granular lymphocytes cell expansion on microscopic examination of the peripheral blood if neutropenia develops.

Early response roles for prolactin cortisol and circulating and cellular levels of heat shock proteins 72 and 90α in severe sepsis and SIRS.

OBJECTIVE: To evaluate the early heat shock protein (HSP) and hormonal stress response of intensive care unit (ICU) patients with severe sepsis/septic shock (SS) or systemic inflammatory response syndrome (SIRS) compared to healthy subjects (H). METHODS: Patients with early (first 48 hrs) SS (n = 29) or SIRS (n = 29) admitted to a university ICU and 16 H were enrolled in the study. Serum prolactin, cortisol, and plasma ACTH were determined using immunoassay analyzers. ELISA was used to evaluate extracellular HSPs (eHSP90α, eHSP72) and interleukins. Mean fluorescence intensity (MFI) values for intracellular HSPs (iHSP72, iHSP90α) were measured using 4-colour flow-cytometry. RESULTS: Prolactin, cortisol, and eHSP90α levels were significantly increased in SS patients compared to SIRS and H (P < 0.003). ACTH and eHSP72 were significantly higher in SS and SIRS compared to H (P < 0.005). SS monocytes expressed lower iHSP72 MFI levels compared to H (P = 0.03). Prolactin was related with SAPS III and APACHE II scores and cortisol with eHSP90α, IL-6, and lactate (P < 0.05). In SS and SIRS eHSP90α was related with eHSP72, IL-6, and IL-10. CONCLUSION: Prolactin, apart from cortisol, may have a role in the acute stress response in severe sepsis. In this early-onset inflammatory process, cortisol relates to eHSP90α, monocytes suppress iHSP72, and plasma eHSP72 increases.

Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS.

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group.

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Visfatin, TNF-alpha and IL-6 mRNA expression is increased in mononuclear cells from type 2 diabetic women.

Visfatin, is a new adipokine, highly expressed in the visceral fat of both mice and humans. To examine whether visfatin is expressed in human peripheral monocyte-enriched mononuclear cells and whether its expression is altered in type 2 diabetes (DM2), we compared 24 DM2 women [17 overweight (BMI >25) and 7 lean (BMI<25)] to 26 healthy women (14 overweight and 12 lean), all premenopausal. Relative visfatin mRNA levels were significantly higher (approximately 3-fold) in DM2 compared to healthy control women (p<0.02), independently of the presence of overweight/obesity. Mononuclear TNF-alpha and IL-6 mRNA expression was also elevated in DM2 compared to control women (p=0.001 and p=0.004, respectively), an increase observed in both lean and overweight DM2 women. By contrast, circulating visfatin, TNF-alpha, and IL-6 levels showed no difference between DM2 and control women, while adiponectin plasma levels were significantly decreased in the DM2 women (p<0.001). Circulating visfatin and TNF-alpha levels did not differ either between the lean and the overweight subgroups of DM2 and control women, while IL-6 plasma levels were significantly higher in both overweight subgroups compared to their lean counterparts. In conclusion, visfatin, TNF-alpha, and IL-6 mRNA expressions are increased in peripheral mononuclear-monocytic cells from women with type 2 diabetes, independent of their BMI, which may enhance the effects of their adipose-derived levels and may contribute to the increased insulin resistance and atherogenic risk of these patients.

HLA antigens as predictors of disease progression in HIV-infected haemophilia patients (a 22 years' follow up).

Our objective was to assess the influence of genetic factors such as HLA classes I and II antigens and other clinical and laboratory variables on the progression of HIV disease in a cohort of 118 HIV infected haemophilic subjects of Greek origin who had been typed for HLA antigens and were followed up prospectively for 22 years since seroconversion. At the end of the follow up we compared two groups of patients: 22 patients who had a fast progression to AIDS (median 6 years since seroconversion) vs. 33 patients who remained asymptomatic in stage A2 for up to 22 years (median 15 years). The results showed that the two groups did not differ significantly in age at seroconversion or baseline CD4+ T cell count. However there was a difference in the frequencies of certain HLA antigens in the two groups. The fast progressors had a higher frequency of HLA-A28, B21 and DR3, which was statistically significant (P = 0.02, 0.04, 0.05, respectively) compared to the slow progressors. These findings based on classical HLA typing techniques confirm other published observations and support the effect of genetic background in the progression of HIV infection in haemophilics.

TCRgammadelta + T lymphocytes in unexplained recurrent spontaneous abortions.

PROBLEM: It is generally accepted that the immune system and cellular immunity in particular are involved in the mechanisms affecting the outcome of gestation. In order to evaluate a putative role of lymphocytes in the immunological mechanisms of unexplained recurrent spontaneous abortions (URSA), we studied peripheral blood lymphocyte subpopulations in 244 women with URSA and 44 controls. METHOD OF STUDY: Direct immunofluorescence in whole blood with the appropriate combinations of monoclonal antibodies and flow cytometry was used. RESULTS: The study showed: a) a statistically significant increase of the mean CD4/CD8 ratio (2.12+/-0.84 vs 1.85+/-0.63, P = 0,039); b) a statistically significant decrease of the mean value of the percentage of CD5+ CD19+ lymphocytes (0.4+/-0.6 vs 1.4+/-0.78, P < 0.0001); and c) a statistically significant increase of the percentage of T lymphocytes expressing TCRgammadelta (4.68+/-3.19 vs 2.61+/-1.14, P < 0.0001). It should be noted that a statistically significant high number of women with URSA (72/195, 36.9%) showed an increased percentage of TCRgammadelta T cells (> or = 5%, where 5 equals the mean value + 2 standard deviations (SD) of the mean value of controls), whereas such a high percentage was not found in any control subject. CONCLUSIONS: It seems that women who experienced URSA comprise a heterogeneous population, as far as immunological parameters are concerned. At least in a subgroup of them, TCRgammadelta + T cells could be considered to play a role in the immune pathogenesis of fetal loss.

Simultaneous appearance of mycosis fungoides and chronic lymphocytic leukemia in the same patient.

A patient is presented having simultaneously chronic lymphocytic leukemia with a monoclonal B-lymphocyte population and mycosis fungoides with atypical T-cell population in the skin lesion and in the enlarged lymphoid nodes confirmed by detailed phenotyping.

Effects of cyclosporin A on immune activation markers in patients with active psoriasis.

BACKGROUND: Psoriasis is a T-cell mediated autoimmune disease. The objective of this work was to investigate the presence of cellular and soluble activation molecules in the blood of patients with psoriasis, not responding to local treatment and to study the effect of cyclosporin A (CsA) on these markers. METHODS: Twenty-seven patients and 30 healthy controls were included in the study. The results were evaluated at baseline and at 15 days, 3, 6 and 12 months following initiation of treatment. RESULTS: We found increased baseline values of lymphocytes and cells expressing the marker CD3+CD25+, CD54+ (ICAM-1) and CD58+ (LFA-3). Following CsA treatment, a significant decrease in the percentage of activated T cells expressing CD3+CD25+ and CD3+HLA-DR+ was noted at 6 and 12 months. Among the soluble factors studied, increased baseline serum levels of sIL-2R, sCD23 and neopterin were observed. CsA significantly reduced the levels of sIL-2R and IL-12. CONCLUSION: Although there is evidence for systemic immune activation in psoriasis, sIL-2R is the most consistently increased activation marker, related to the Th1 immune response, that may be used as a marker for monitoring disease activity and response to treatment with CsA in psoriatic patients.

Association between HLA antigens and progression of HIV infection in Greek haemophiliacs.

The frequencies of HLA antigens in 33 HIV seronegative and in 88 HIV seropositive haemophiliacs, who have been followed for at least 6 years since seroconversion or first HIV positive test, were evaluated in relation to disease susceptibility and disease progression. A high frequency of HLA-A2 and -DR2 antigens and a low frequency of HLA-A9 were found to characterize HIV seropositive patients (p < 0.05). Progressors to symptomatic CDC stage IV had a higher frequency of HLA-A9 (p < 0.01) and DR3. Rapid decline of CD4+ T cells in these patients was associated with HLA-A9, -DR1 and DR3. Our data suggest that HLA antigens may contribute to susceptibility to HIV infection and disease progression in Greek haemophiliacs.

Immune status of Greek patients with beta-thalassemia major negative for anti-HIV.

Patients with thalassemia who receive multiple blood transfusions are at risk for the acquired immunodeficiency syndrome. Peripheral blood lymphocyte subpopulations were studied in 22 multitransfused thalassemic patients; 10 patients were without splenectomy and 12 were studied after splenectomy. Both groups were negative for anti-HIV. Four additional patients who were found positive for anti-HIV and ten healthy controls were also included in this study. Patients without splenectomy compared to controls and to patients after splenectomy showed a significant decrease of both percentage (p less than 0.001) and absolute numbers (p less than 0.001) of Leu-7+ cells without significant abnormalities of T4/T8 ratio (1.56 +/- 0.4). Patients after splenectomy compared to controls and to patients without splenectomy showed a significant increase of the absolute numbers of lymphocytes and lymphocytes subsets T11+, T3+, T4+, T8+ and SmIg+ cells. In the seropositive patients for HIV only a significant increase of the absolute number of T8+ cells was observed while the T4/T8 ratio was 1.24 +/- 0.73. The decrease in the percentage of Leu-7+ cells in patients without splenectomy correlated inversely to the total amount of blood transfused. In conclusion patients with thalassemia had normal T4/T8 ratio and did not show the abnormal immunologic profile that has been reported in haemophiliacs.

Lymphocyte markers and clinical expression of lymphoproliferative disorders with moderate lymphocytosis.

Lymphoproliferative syndrome with well differentiated lymphocytes and moderate lymphocytosis in the peripheral blood includes a heterogeneous group of disorders, that present often difficulties in classification. We have studied the lymphocyte markers (ER, EMR, sIg and T3, T4, T8 antigens) in 36 cases who had lymphocytic infiltration in the bone marrow and peripheral lymphocyte counts less than 15 X 10(9) l-1. Four cases (11.1%) had the characteristics of T8 lymphocytosis and 31 had a B cell monoclonal proliferation in the peripheral blood. Of these, four were sIg-, EMR+, 19 were sIg+, EMR+ and 8 were sIg+, EMR-. Most patients (17/32) had the clinical picture of stage 0 and I B-CLL. Six cases presented as pure splenomegalic form of CLL, three had the features of immunocytic lymphoma and five had the features of lymphocytic lymphoma. It is concluded that the majority of lymphoproliferative disorders presenting with moderate lymphocytosis represent early forms of B-CLL. Occasionally cases of lymphocytic or immunocytic lymphoma may present problems of differential diagnosis since there may be a dissociation of phenotypic characteristics of lymphocytes between tissues and peripheral blood.

Abnormalities of lymphocyte subsets and anti-LAV/HTLV-III status in Greek hemophiliacs.

Peripheral blood lymphocyte subsets and the incidence of LAV/HTLV-III antibodies were studied in 63 patients with hemophilia A who had been transfused with a low dose regimen of commercial (U.S.A.) factor VIII concentrates. Five patients with hemophilia B were also included in this study. In hemophilia A patients a significant reduction in the percentage and absolute numbers of T4+ cells and of the T4/T8 ratio and a significant increase in the percentage of T8+ and Leu-7+ cells were observed. These abnormalities were independent of the presence of anti-LAV/HTLV-III. In hemophilia B patients a significant increase of T8+ cells and a decrease of T4/T8 ratio was noted while the percentage of Leu-7+ cells was normal. A significant negative correlation of F VIII units transfused and T4/T8 ratio was seen only in LAV/HTLV (-) patients, suggesting that F VIII per se could cause immunodysregulation. Seropositive patients were found to have consumed a larger amount of F VIII units than seronegative patients during the period 1980-1984 (p less than 0.005).